Cellulases From Fungi And Bacteria And Their Biotechnological Applications
Table of Contents
1. Introduction pag. 4
2. Structure of cellulose
2.1 Chemical structure pag. 7
2.2 Crystalline structure pag. 8
3. Cellulose hydrolysis pag. 9
4. Cellulases classification pag. 10
5. Reaction mechanism of cellulase enzymes pag. 12
6. The cellulosome concept pag. 14
7. Cellulases of mesophilic origin
7.1 Bacterial cellulases pag. 19
7.2 Fungal cellulases pag. 25
8. Cellulases of thermophilic origin
8.1 Bacterial and archaeal cellulases pag. 33
8.2 Fungal cellulases pag. 43
9. Cloning and expression of cellulase genes
9.1 Heterologous cloning and expression in different microbial hosts pag. 47
9.2 Cloning and expression in plant systems pag. 53
9.3 Cloning and expression in Bombix mori cells and larvae
through the baculovirus expression system pag. 56
10. Biotechnological applications of cellulases
10.1 Cellulases in brewery and wine biotechnology pag. 57
10.1.1 Beer brewing process pag. 57
10.1.2 Wine production pag. 58
10.2 Cellulases in animal feed biotechnology pag. 59
10.3 Cellulases in pulp and paper biotechnology pag. 60
10.3.1 Biomechanical pulping pag. 60
10.3.2 Biomodification of fibers pag. 61
10.3.3 Biodeinking pag. 62
10.4 Cellulases in food biotechnology pag. 62
10.4.1 Fruit and vegetable juices pag. 62
10.4.2 Olive oil pag. 63
10.5 Cellulases in textile and laundry biotechnology pag. 65
10.5.1 Biostoning and biopolishing pag. 65
10.5.2 Laundry pag. 66
10.6 Cellulases in bioethanol production pag. 67
11. References pag. 71
Cellulases from Fungi and Bacteria and their biotechnological applications.
A. Morana1, L. Maurelli1, E. Ionata1, F. La Cara1, M. Rossi2
1Institute of Protein Biochemistry, Italian National Research Council, Via P. Castellino 111, 80131, Naples, Italy. 2Dipartimento di Biologia Strutturale e Funzionale, Complesso Universitario Monte S. Angelo, University of Naples "Federico II", Naples, Italy
Cellulases (EC 3.2.1.4) catalyze the hydrolysis of 1,4-β-D-glucosidic linkages in cellulose, and play a significant role in nature by recycling this polysaccharide which is the main component of plant cell wall. Cellulases work in synergy with other hydrolytic enzymes in order to obtain the full degradation of the polysaccharide to soluble sugars, namely cellobiose and glucose, which are then assimilated by the cell.
The enormous potential that cellulases have in biotechnology is the driving force for continuous basic and applied research on these biocatalysts from Fungi and Bacteria. Nowadays, cellulases found application in many fields, such as animal feeding, brewery and wine, food, textile and laundry, pulp and paper industries. Moreover, the growing interest toward the conversion of lignocellulosic biomass into fermentable sugars has generated an additional request for cellulases and their related enzymes. In fact, bioconversion of biomass has significant advantages over other alternative energy production strategies because lignocellulose is the most abundant and renewable biomaterial on our planet. Bioconversion of lignocellulose is initiated primarily by microorganisms which are capable of degrading lignocellulosic materials. Several Fungi produce large amounts of extracellular cellulolytic enzymes, whereas bacterial and few anaerobic fungal strains mostly produce cellulolytic enzymes in a complex associated with the cell wall which is called “cellulosome”. However, the heterogeneous and recalcitrant nature of lignocellulosic waste represents an obstacle for an efficient saccharification process, and pretreatment techniques are required to make the polysaccharide more accessible to the enzymatic action.
Thermostable enzymes can offer potential benefits in the hydrolysis of pretreated lignocellulosic substrates because the harsh conditions often required by several pretreatments can be harmful for conventional biocatalysts. The enhanced stability of thermostable enzymes to high temperature and extreme operative parameters allows improved hydrolysis performance and increased flexibility compared to process configurations, all leading to enhancement of the overall economy of the process.
The present review gives an outline of several mesophilic and thermophilic cellulases from Fungi and Bacteria that have been characterized in the last years. Moreover, applications of these enzymes in some biotechnological fields, with particular regard to lignocellulosic biomass bioconversion, will be illustrated.
Plant biomass is the only predictable sustainable source of organic fuels, chemicals, and materials. As the primary component of the biosphere, biomass is also an industrial raw material uniquely compatible with human and other forms of life. The complex structure of the plant cell wall consists of lignocellulosic material mainly constituted by cellulose fibers strictly linked with hemicellulose and lignin, thus complicating their hydrolysis and which composition differs considerably according to the source (Table 1) [Deobald et al., 1997].
Cellulose is a linear polymer of β-D-glucose units linked through 1,4-β-linkages with a degree of polymerization ranging from 2,000 to 25,000 [Kuhad et al., 1997]. More in detail, cellulose chains form numerous intra- and intermolecular hydrogen bonds, which account for the formation of rigid, insoluble, crystalline microfibrils. Natural cellulose compounds are structurally heterogeneous and have both amorphous and highly ordered crystalline regions. The degree of crystallinity depends on the source of the cellulose and the highly crystalline regions are more resistant to enzymatic hydrolysis.
Cellulosic materials are particularly attractive because of their relatively low cost and abundant supply. As the most abundant polysaccharide in nature, cellulose decomposition plays not only a key role in the carbon cycle of nature, but also provides a great potential for a number of applications, most notably biofuel and chemical production [Lynd et al., 2002]. The central technological impediment to more widespread utilization of this important resource is the general absence of low-cost technology for overcoming the recalcitrance of cellulosic biomass.
A promising strategy to overcome this impediment involves the production of cellulolytic enzymes, hydrolysis of cellulose, and fermentation of resulting sugars in a single process step via free cellulolytic enzymes or consortium. In general, two systems occur in regard to plant cellulose degradation by microorganisms. In the first, the organism produces a
Table 1 – Lignocellulose composition of several agricultural waste.
Modified from [Jorgensen et al., 2007]
set of free enzymes that work synergistically to degrade plant cell wall. In the second, the degradative enzymes are organized into enzymatic complexes. Aerobic Bacteria and Fungi join weakly or not to cellulose and produce free cellulases, while anaerobic Bacteria and Fungi show high tendency to adhere to the polysaccharide, thus producing cellulases included in enzymatic complexes called “cellulosome”.
However, full degradation of cellulose involves a complex interaction between different cellulolytic enzymes. It has been widely accepted that three types of enzymes including endoglucanases (EC 3.2.1.4), exoglucanases (EC 3.2.1.91) and β-glucosidases (EC 3.2.1.21) act synergistically to convert cellulose into β-glucose [Lynd et al., 2002]. Extensive evidence obtained from aerobic cellulolytic microorganisms supports a hydrolysis mode mediated by the synergistic action of endoglucanases and exoglucanases with cellobiose as the main product [Zhang et al., 2004]. Then, cellobiose is further hydrolyzed by β-glucosidases to glucose.
This review will consider only the enzymes involved in the first step of cellulose degradation, namely the endoglucanases. Interest in these enzymes has grown markedly because of the potential of the substrates for yielding marketable products. Cellulose-hydrolyzing enzymes are widespread in Fungi and Bacteria [Tomme et al., 1995], and they have found application in various biotechnological fields. The most effective enzymes of commercial interest are the cellulases from aerobic cellulolytic Fungi, such as Trichoderma reesei (Hypocrea jecorina), Aspergillus niger and Humicola insolens [Nakari-Seta La and Penttila, 1995; Okada, 1988; Davies et al., 2000]. This is due to the ability of engineered strains of these microorganisms to produce large amounts of crude cellulases which possess high specific activity on crystalline cellulose. In general, cellulases can be used to improve color extraction and the yield of juices. Their presence in detergents causes color brightening and softens and improves particulate soil removal. Cellulases are also used for the “biostoning” of jeans instead of the classical stones in stonewashed jeans. Other applications of cellulases include the pretreatment of forage crops to improve nutritional quality and digestibility, and the production of fine chemicals. In addition, the growing interest in the last years toward the conversion of lignocellulosic biomass to fermentable sugars for bioethanol production has generated an increasing demand for cellulases and related enzymes. In fact, bioconversion of lignocellulosic biomass has significant advantages over other alternative energy production strategies because lignocellulose is the most abundant and renewable biomaterial on our planet, and moreover, it is not in competition with food sources.
The aim of the present review is to give an overview of several cellulases from Bacteria and Fungi that have been characterized in the last years. Thermophilic enzymes will also be considered, as their elevated stability to high temperatures and extreme operative parameters allows improved hydrolysis performance, leading to enhancement of the overall economy of the biotechnological process. Molecular and biotechnological aspects of these enzymes, with particular regard to their application in lignocellulosic biomass saccharification, will be illustrated.
2. STRUCTURE OF CELLULOSE.
2.1 Chemical structure.
Payen first used the term cellulose for this plant constituent which is the most widespread organic compound on Earth [Payen 1938; Guo et al., 2008]. The total amount of this polysaccharide on our planet has been estimated at 7 × 1011 tons [Coughlan, 1985] and constitutes the most abundant and renewable polymer resource available today. Cellulose is an insoluble crystalline substrate, flavorless, odorless, hydrophilic, insoluble in water and in most organic solvents, chiral, and with a wide chemical variability. It is a structural component of the cell wall of green plants accounting for almost 33% of the total biomass. It is also biosynthesized in other living systems such as Bacteria and Algae.
Cellulose produced by plants usually exists within a matrix of other polymers primarily hemicellulose, lignin, pectin and other substances, forming the so-called lignocellulosic biomass, while microbial cellulose is quite pure, has a higher water content, and consists of long chains. It is a carbohydrate polymer with formula (C6H10O5)n , consisting of a linear chain of several hundred to over ten thousand 1,4-β-D-glucose units linked through acetal functions between the equatorial -OH group of C4 and the C1 carbon atom [Jagtap and Rao, 2005]. The high stability of this conformation leads to a reduced flexibility of the polymer, so this is usually described as a real tape.
There are two different types of intra- and one interchain hydrogen bonds in the structure, and it has been considered that the intrachain hydrogen bonds determine the single-chain conformation and the stiffness of cellulose, while the interchain hydrogen bond is responsible for the sheetlike nature of cellulose [Watanabe, H; Tokuda, 2001; Klemm et al., 2002; Klemm et al., 2005]. The chains are arranged parallel to each other and form elementary fibrils that have a diameter between 1.5 and 3.5 nm (microfibrils), the length of the microfibrils is about of several hundred nm.
The chain length of cellulose, expressed as degree of polymerization (DP) in relation to the number of monomers, varies with the origin and treatment of the raw material. In case of wood pulp, the values are typically 300 and 1,700; cotton and other plant fibers have DP values in the 800-10,000 range; bacterial cellulose has a similar DP value.
In relation to the amount of hydrogen bonds into and between cellulose molecules, a model of cellulose structure with two states in plant cell wall has been proposed: amorphous and crystalline.
2.2 Crystalline structure.
The high degree of hydrogen bonds within and between cellulose chains can form a 3-D lattice-like structure, while amorphous cellulose lacks this high degree of hydrogen bonds and the structure is less ordered.
The physical and chemical properties of cellulose are defined by intermolecular interactions, cross-linking reactions, polymer lengths, and distribution of functional groups on the repeating units and along the polymer chains.
Initially, crystalline structure of native cellulose (cellulose I) has been studied by X-ray diffraction and has been defined as monoclinic unit cells with two cellulose chains with a twofold screw axis in a parallel orientation forming slight crystalline microfibrils [Gardner and Blackwell, 2004; Klemm et al., 2005].
Afterwards, it was discovered by using 3C-CP/MAS NMR spectroscopy, that the native cellulose was present in two crystalline forms obtained by modifications of cellulose I: Iα with triclinic unit cells and Iß with monoclinic unit cells. The ratio between Iα and Iß changes in relation to the source of the cellulose [Atalla and Vanderhart, 1984].
Moreover, there are other types of crystal structures: cellulose II, III, and IV [Gardner and Blackwell, 2004]; the cellulose I, result the less stable thermodynamically, while the cellulose II is the most stable structure [Klemm et al., 2005; Fengel and Wegener, 1984]. The cellulose I can turn into other forms using different treatments; for example, by mercerization, using aqueous sodium hydroxide or dissolution followed by precipitation and regeneration (formation of fiber and film) [O'Sullivan, 1997; Nishiyama et al., 2002].
However, additional information on the structure of noncrystalline random cellulose chain segments are needed because it is very important for the accessibility and reactivity of the polymer and the characteristics of cellulose fibers [Paakkari et al., 1989].
3. CELLULOSE HYDROLYSIS.
Cellulases belong to a class of enzymes that catalyze the hydrolysis of cellulose and are produced chiefly by Fungi, Bacteria, and Protozoa, as well as other organisms like plants and animals. The cellulolytic enzymes are inducible since they can be synthesized by microorganisms during their growth on cellulosic materials [Lee and Koo, 2001].
A variety of different kinds of cellulose-degrading enzymes are known, with different structure and mechanism of action. In general, the “cellulolytic enzyme complex” breaks down cellulose to β-glucose, and involves the following types of enzymes: endoglucanases (EC 3.2.1.4), exoglucanases (EC 3.2.1.74), cellobiohydrolases (EC 3.2.1.91), and β-glucosidases (EC 3.2.1.21) [Li et al., 2010].
Endoglucanases (EC 3.2.1.4) hydrolyze randomly internal glycosidic linkages in soluble and amorphous regions of the cellulose, and produce new ends by cutting into long cellulose strands. This action results in a rapid decrease of the polymer length and in a gradual increase of reducing sugars concentration.
Exoglucanases (EC 3.2.1.74) hydrolyze cellulose chain and oligosaccharides with high DP removing successive β-glucose units.
Cellobiohydrolases (EC 3.2.1.91) hydrolyze cellulose chains by removing processively 2 units (cellobiose) either from the non-reducing and reducing ends. This action results in rapid release of reducing sugars but little changes in polymer length occurr. Cellobiohydrolases with specificity for the reducing and the non-reducing end have to work together.
β-Glucosidases (EC 3.2.1.21) convert the resulting oligosaccharide products to glucose [Bhat and Bhat, 1997].
These enzymatic components act sequentially in a synergistic system to facilitate the breakdown of cellulose and the subsequent biological conversion to β-glucose (Figure 1) [Beguin and Aubert, 1994].
All these enzymes hydrolyze the 1,4-β-glycosidic bonds in cellulose, but they are different in their specificities based on the macroscopic features of the substrate. There are progressive (also known as processive) enzymes when they interact with a single polysaccharide strand continuously, and non-progressive types when they interact once and then, the polypeptidic chain disengages to attack another polysaccharide strand.
The enzymatic hydrolysis of cellulose requires a carbohydrate binding module (CBM) that binds and arranges the catalytic components on the surface of the substrate. Cellulases from Fungi have a two-domain structure with one catalytic domain, and one cellulose binding domain, that are connected by a flexible linker. However, there are also cellulases that lack cellulose binding domain.
Figure 1. Enzymatic hydrolysis of cellulose.
4. CELLULASES CLASSIFICATION.
Enzymes are designated according to their substrate specificity, based on the guidelines of the International Union of Biochemistry and Molecular Biology (IUBMB). The cellulases are grouped with many of the hemicellulases and other polysaccharidases as O-glycoside hydrolases (EC 3.2.1.x). Since the substrate specificity classification is sometime little informative, because the complete range of substrates is only rarely determined for individual enzymes, an alternative classification of glycoside hydrolases (GH) into families based on amino acid sequence similarity has been suggested [Henrissat, 1991; Henrissat and Bairoch, 1993; Henrissat and Bairoch, 1996]. In addition, Henrissat et al. [1998] have proposed a new type of nomenclature for glycoside hydrolases in which the first three letters designate the preferred substrate, the number indicates the glycoside hydrolase family, and the following capital letter indicates the order in which the enzymes were first reported. For example, the enzymes CBHI, CBHII, and EGI of Trichoderma reesei are designated Cel7A (CBHI), Cel6A (CBHII), and Cel6B (EGI).
Due to the great increase of identified glycoside hydrolases, Coutinho and Henrissat have created an integrated database which is continuously updated (http://www.cazy.org/) [Coutinho and Henrissat, 1999]. At the latest update (13 July 2010), glycoside hydrolases were grouped into 118 families. In addition, 876 glycoside hydrolases have not yet assigned to a family (Glycoside Hydrolase Family “Non-Classified”) because some of them display weak similarity to established GH families, but they are too distant to allow a reliable assignment. Cellulases are found in several different GH families (5, 6, 7, 8, 9, 12, 44, 45, 48, 51, 61, and 74), suggesting convergent evolution of different folds resulting in the same substrate specificity. GH family 9 contains cellulases from bacteria (aerobic and anaerobic), fungi, plants and animals (protozoa and termites). Other families only group hydrolases from a specific origin, as GH family 7 which contains only fungal hydrolases and GH family 8 which contains only bacterial hydrolases. At last, cellulases from the same microorganism can be found in different families (e.g. the Clostridium thermocellum cellulosome contains endoglucanases and exoglucanases from families 5, 8, 9, 44, and 48) [Shoham et al., 1999].
Where necessary, GH families have been subclassified. It is the case of GH family 9 that has been divided into two subfamilies: E1 and E2. Members of the subfamily E1 show a tight association of an Ig-like domain with a catalytic domain, while members of subfamily E2 are associated with a CBM classified in family 3c [Beguin, 1990].
The study of cellulolytic enzymes at the molecular level has revealed some of the features that contribute to their activity. Within each GH family, available data suggest that the various cellulases share a common folding pattern, the same catalytic residues, and the same reaction mechanism, i.e. either single substitution with inversion of configuration or double substitution resulting in retention of the β-configuration at the anomeric carbon [Beguin and Aubert, 1994]. As observed, cellulases are a composite group of enzymes, and the diversity within the cellulase families could reflect the heterogeneity of cellulose within plant materials and the variety of niches where hydrolysis takes place. The insoluble, recalcitrant nature of cellulose represents a challenge for cellulase systems. In addition to catalytic domains, many cellulolytic enzymes contain domains not involved in catalysis, but participating in substrate binding (cellulose-binding domains, CBDs), or to the attachment to the cell surface. Most probably, these domains facilitate cellulose hydrolysis by bringing the catalytic domain in close proximity to the insoluble cellulose, and assisting in the degradation of crystalline cellulose. CBDs are generally located at the -COOH or -NH2 terminus of the polypeptidic chain, and are often separated from the catalytic domains by glycosylated Pro/Thr/Ser-rich linker segments. CBDs are now better classified as carbohydrate-binding modules (CBMs). The previous definition was based on the early discovery of a number of modules that bound cellulose [Tomme et al., 1995].
The binding efficiency of the cellulase is much enhanced by the presence of the CBM and the enhanced binding correlates with better hydrolytic activity toward insoluble cellulose. For example, the presence of CBM in T. reesei is reported to enhance the enzymatic hydrolysis of insoluble cellulose and chemical pulp [Suurnakki et al., 2000].
However, additional modules are continuously found able to bind other carbohydrates besides cellulose. Hence, the need to reclassify these polypeptides using more inclusive terminology. Until now, carbohydrate-binding modules have been divided into 59 families. Additionally, 35 carbohydrate-binding modules have not yet assigned to a family (Carbohydrate-Binding Module Family "Non Classified”).
5. REACTION MECHANISM OF CELLULASE ENZYMES.
The cellulase catalytic mechanism, that has been optimized by many years of evolution, has by far attracted the interest of many researchers. On the basis of kinetic and chemical modification studies some authors have suggested that several cellulases operate by a lysozyme-type mechanism. These conclusions are strengthened by the finding that specific amino acid sequences in these cellulases were homologous with the active centers of known lysozymes [Coughlan , 1991].
Lysozymes were the first glycoside hydrolases to have their three-dimensional structures solved [Blake et al., 1965]. The two catalytic amino acids of the active site were identified, like in most glycoside hydrolases, as aspartate and glutamate residues.
In particular, the mechanism of the lysozyme catalyzed reaction may be described as a double displacement acid hydrolysis in which a non-ionized glutamic acid and an ionized aspartic acid residues participate as proton donor and acceptor, respectively [Lehninger, 1982].
Bacterial enzymes utilizing a lysozyme-type mechanism were identified in Cellulomonas fimi endo- and exoglucanases [Gilkes et al., 1988] and Clostridium thermocellum endoglucanases A and D [Schwarz et al., 1988] by sequences comparison with the active center of lysozyme. In fact, the homology studies confirmed, for these enzymes, putative active centers containing the glutamic and aspartic acid residues. However, other studies demonstrated that may be an error to assign, on the basis of homology data alone, a catalytic role at any glutamic acid/aspartic acid pair residues [Yablonsky et al., 1988].
Site-directed mutagenesis experiments have been performed to elucidate the question, and an example of chemical modification study on the bacterial cellulases is that of Claeyssens and Tomme who provided evidence for the involvement of an histidyl residue in the reaction catalyzed by the endoglucanase D from C. thermocellum [Claeyssens and Tomme, 1989].
An explanation of the mechanism by which the glycoside hydrolases catalyze the cleavage of the glycosidic linkage was provided already in 1953 by Koshland that proposed two different stereospecific reaction mechanisms namely the inverting and retaining [Koshland, 1953].
The Figure 2 shows a representation of the two types of mechanisms hypothesized for cellulase enzymes. The retaining mechanism (a), in which the first residue acts as an acid catalyst (AH) that protonates the glycosidic oxygen and the nucleophilic assistance to leaving group departure is provided by the second residue, the base B-. The resulting glycosyl-enzyme is hydrolyzed by a water molecule and this second nucleophilic substitution at the anomeric carbon generates a product with the same stereochemistry as the substrate, similarly to the reaction of lysozyme [Kelly et al., 1979]. The inverting mechanism (b), in which there is also a protonation of the glycosidic oxygen by the acid residue and the leaving group departure is accompanied by a concomitant attack of a water molecule activated by the base residue B-. This single nucleophilic substitution yields a product with opposite stereochemistry to the substrate as observed in the case of ß-amylase. A detailed description of the catalytic mechanism for glycoside hydrolase enzymes can also be found in several excellent reviews [Vasella, et al., 2002; Zechel and Withers, 2000; Zechel and Withers, 2001].
The main structural difference that occurs in glycosidase enzymes which perform the hydrolysis of the glycosidic linkage in the retaining or inverting manner consists in the distance separating their respective carboxyl groups. In the enzymes that operate with the inverting mechanism, the base and acid carboxyl residues are separated, on average, by 9-10 Å, whereas in retainers the nucleophile and general acid-base catalyst are only ~ 5-5.5 Å apart. The explanation of the greater span found in inverters is justified by the necessity to accommodate the nucleophilic water molecule.
The knowledge of these structural differences in glycosidases has made possible to perform experiments to convert inverters enzymes into retainers and vice-versa, with the appropriate substitution (i.e. mutation) at the nucleophile position [Vocadlo and Davies, 2008].
Therefore, on the basis of these differences, glycosidases can be classified as enzymes that catalyze the glycosidic linkages hydrolysis with retention or inversion of the anomeric configuration at the hemiacetal center of the newly formed product.
The stereochemical course of hydrolyses catalyzed by cellulolytic enzymes from various sources have been investigated. Characteristic examples of several retention mechanisms are the double-inversion of glycosyl enzyme intermediate utilized by lysozyme [Lehninger, 1982] and the carbohydrate hydrolysis catalyzed by Cex from C. fimi, whereas the reaction catalyzed by C. thermocellum CenA enzyme proceeds by inversion of configuration [Withers et al., 1986].
In 1991, Coughlan [1991] underlined some aspects of cellulases which bear the catalytic domain at the N-terminus and utilize lactosides and cellobiosides as substrates of the hydrolytic reaction that proceedes with the retention of the β-configuration. By contrast, those enzymes in which the catalytic domain is located at the C-terminus cannot utilize lactosides and cellobiosides as substrates, obtaining products with an inversion of the β-configuration.
6. THE CELLULOSOME CONCEPT.
All the microorganisms capable of plant cell wall degradation produce complex cellulase enzymes systems; however, two different types of strategy occur between aerobic and anaerobic groups [Tomme et al., 1995]. Aerobic cellulose degraders, both bacterial and fungal, apart from few exceptions [Wachinger et al., 1989], produce a set of free enzymes which are released in the extracellular environment and work synergistically to degrade the plant cell walls [Schwarz, 2001]. Instead, anaerobic microorganisms degrade cellulosic substrates primarily through the cell-bound multienzyme systems known as the “cellulosomes”. These structures, which are quite stable cellulolytic complexes, show considerable dimensions that can vary from 2.0 to 16.0 MDa and even up to 100.0 MDa in the case of polycellulosomes [Béguin and Lemaire, 1996]. The occurrence of the cellulosome was firstly observed in the thermophilic bacterium Clostridium thermocellum [Bayer et al., 1983]; successively, a range of anaerobic bacteria such as C. cellulovorans [Shoseyov et al., 1992], C. cellulolyticum [Pages et al., 1999], C. acetobutylicum [Sabathe et al., 2002], C. josui [Kakiuchi et al., 1998], C. papyrosolvens [Pohlschröder et al., 1994], Bacteroides cellulosolvens, Acetivibrio cellulolyticus [Pages et al., 1997], Ruminococcus flavefaciens [Rincon et al., 2003] and the anaerobic fungi of the genera Neocallimastix, Piromyces, and Orpinomyces [Bayer et al., 2004] were shown to produce cellulosomal systems. The cellulosomes are characterized by the presence of two general components: a) a large non catalytic scaffoldin protein with enzyme binding sites called cohesins, and b) the catalytic components that contain, at the C-terminus, highly conserved noncatalytic modules, called dockerins, which bind to the cohesin modules.
The cellulosome enzymatic components contain not only cellulases but also a large array of hydrolytic activities such as hemicellulases [Kosugi et al., 2002], pectinases [Tamaru and Doi, 2001], chitinases, lichenases, mannanases and esterases. This extraordinary enzymes diversity reflect the chemical and structural complexity of the cellulosome substrate, the plant cell wall, that can be efficiently attacked and degraded only by the concerted action of different enzymatic activities [Fontes and Gilbert, 2010]. Moreover, in the cellulosome assembly, the cohesin domains are unable to discriminate among the dockerins present in the various catalytic modules due to the high level of conservation in the same species of both cohesins and dockerins domains [Yaron et al., 1995]. This leads, through the induction of specific genes by plant cell wall polymers, to different and temporally evolving cellulosome enzyme combinations, that allow a successful cell wall structures degradation. In addition, to the cohesins, the scaffoldin also bears a cellulose-binding module (CBM) that target the cellulosomal enzymes as well as the entire cell to the cellulosic substrates. In fact this domain, interacting with crystalline cellulose, brings the cellulosome into close proximity with the plant cell wall and concentrates the hydrolytic enzymes to a particular site of the substrate [Gilbert, 2007]. In the simplest cellulosome system, there is a single scaffoldin protein with a CBM and 6 to 9 catalytic components in dependence of the cohesin number that varies with the different species [Lynd et al., 2002]. Moreover, several cellulosome-producing microbes express more than one type of scaffoldin: this is the case of the bacteria with cell-surface anchored cellulosomes, such as C. thermocellum, Acetivibrio cellulolyticus, Bacterioides cellulosolvens and Ruminococcus flavefaciens [Bayer et al., 2008]. After its discovery in the mid-1980s, the first polyscaffoldin cellulosome structure, resolved through a combination of biochemical, immunochemical, ultrastructural, and genetic techniques was that of C. thermocellum [Mayer et al., 1987]. This cellulosome revealed an highly ordered structure with sets of polypeptides arranged in parallel chain-like arrays. The cellulosomal system consists of a large scaffoldin protein (CipA) of 147.0 kDa, whose encoding sequence is part of an operon, called “scaffoldin gene cluster”, containing several other genes coding for the secondary scaffoldins (see below) [Fujino et al., 1993]. CipA, that contain a CBM module and 9 cohesin domain, termed of type I, is defined as primary scaffoldin [Bayer et al., 1998]. Cohesin domains are folded in 9-stranded β-barrel like families II and III CBDs, in spite of the total absence of homology. The catalytic components, bearing at their C-terminus a dockerin domain named of type I, are bound in presence of Ca++ to the type I cohesins onto CipA [Salamitou et al., 1994]. A total of 22 catalytic modules, at least 9 of which exhibiting endoglucanase activity (CelA, CelB, CelD, CelE, CelF, CelG, CelH, CelN, and CelP), 4 exoglucanase activity (CbhA, CelK, CelO and CelS), 5 hemicellulase activity (XynA, XynB, XynV, XynY and XynZ), 1 chitinase activity (ManA), and 1 lichenase activity (LicB), are grafted into the cohesin sites of CipA to form the cellulosome complex.
Three different types of cell surface proteins, named SdbA, Orf2p and Olpb, defined as secondary or anchoring scaffoldins, contain different numbers (1, 2, or 7 respectively) of type II cohesins that, interacting with a type II dockerin located at the C-terminus of the primary scaffoldins, allow their attachment to the cell envelope [Leibovitz and Beguin, 1996]. The type II dockerin-cohesin affinity is further enhanced by the stabilizing effect of an hydrophilic domain, named X module, located immediately upstream the type II dockerin. The anchoring scaffoldins also contain a C-terminal threefold reiterated SLH domains, normally found in the S-layer proteins [Rincon et al., 2003] which mediate the anchoring of these structural proteins to the bacterial cell wall. In the case that seven primary scaffoldins are assembled onto the 7 cohesin II of an OlpB anchoring scaffoldin, a polycellulosome bearing 63 catalytic units may be produced.
The C. thermocellum cellulosome structure has been considered the paradigm for such enzymatic nanomachines and several subsequent studies were aimed to verify if the cellulosomes from other bacteria would follow the C. thermocellum paradigm (Figure 3).
Surprisingly, the results obtained showed very divergent cellulosome structures. This is the case of the aforementioned bacteria R. flavefaciens, A. cellulolyticus, and B. cellulosolvens, which revealed very different and versatile cellulosome architecture and modular arrangement [Bayer et al., 2008]. The versatile nature, composition and arrangement of cellulosome is best elucidated by the complex structure of R. flavescens cellulosome that is characterized by at least four types of scaffoldin with different functions. The whole cellulosomic system is attached to the cell surface by the ScaE scaffoldin through a sortase-mediated transpeptidation reaction [Rincon et al., 2005]. The primary scaffoldin ScaB is bound to ScaE and through its 9 cohesin with different specificity can accommodate up to 4 catalytic modules and up to 5 ScaA adaptor scaffoldins, each endowed with 3 cohesins [Fierobe et al., 2005]. Another adaptor scaffoldin, ScaC, that bear a single cohesin with specificity for an unknown group of dockerins is also accommodated onto the ScaB [Jindou et al., 2008]. In addition to this structural characteristic, the R. flavefaciens system shows also several unique features respect to the other cellulosomes that significantly increase its degradative capabilities in the rumen environment. In fact, in the equipment of the cellulosomal catalytic modules, are also included putative pectate lyase, rhamnogalacturan lyase, mannanase, arabinase, transglutaminase, proteinase and peptidase activities. Moreover, the different R. flavefaciens strains show distinct scaffoldin sequences [Jindou et al., 2008] that implies a strain-specific cellulosome organization. This cellulosome strain variety provides a very useful functional diversity in the degradative capacities that are required by the complexity and the heterogeneity of the lignocellulosic substrates found in the rumen [Bayer et al., 2008].
Several studies have also been conducted on the cellulosomes of mesophilic clostridia such as C. cellulovorans [Doi et al., 1994], C. cellulolyticum [Bagnara-Tardif et al., 1992], C. acetobutylicum [Cornillot et al., 1997], C. josui [Kakiuchi et al., 1998], and C. papyrosolvens [Pohlschröder et al., 1994], which also revealed a structure different from that of C. thermocellum [Doi, 2008]. These microorganisms in fact express cellulosomes characterized by a less complex architecture, where the enzymatic modules are grafted on a single primary scaffoldin and no anchoring scaffoldins have been identified [Bayer et al., 2008]. As opposed to the “scaffoldin gene cluster” of C. thermocellum and the other multiple scaffoldin cellulosome producers, in these species an “enzyme linked gene cluster” has been individuated that comprises a primary scaffoldin gene followed by the genes encoding the dockerin-bearing enzymes [Bayer et al., 2004; Doi, 2008]. It is just this collection of enzymes and their coordinated expression regulation, strictly dependant from the carbon sources [Han et al., 2005], that makes the cellulosome system so versatile and efficient in attacking and degrading the plant cell walls. It is important to underline that even the mesophilic clostridium cellulosomes, which exhibit the lowest complexity levels, result in a more efficient system in deconstructing plant structural polysaccharides respect to the “free” enzymes produced by the aerobic microorganisms. This is particularly apparent in the case of C. thermocellum where the cellulosome is reported to display a specific activity against crystalline cellulose 50-fold higher than the corresponding Trichoderma system [Demain et al., 2005]. The evolutionary drivers that brought to the undoubted cellulosome success are not clear but most probably were just the energetic constraints imposed by the anaerobic environment that led to a necessary improvement of the microorgamisms degradative capabilities. In fact, the grafting of plant cell wall-degrading enzymes onto a macromolecular complex brings to a spatial enzyme proximity that potentiates the synergistic interactions among the cellulosomal catalytic units. The catalytic cellulosome efficiency is further increased by enzyme-substrate targeting that allows a close proximity of the cell to the substrate. In fact, due to the restricted extracellular diffusion rate of the degradation products, these are readily removed by an enhanced cell uptake, leading to an increased cellulose hydrolytic rate [Bayer et al., 1998]. Moreveor, the cellulosomes action in concert with noncellulosomal glycosidic hydrolases, further amplify the whole degradative process yield. This has been clearly demonstrated by the concerted actions of cellulosomal hemicellulase XynA and noncellulosomal hemicellulases ArfA and BgaA [Kosugi et al, 2002] in C. cellulovorans.
7. CELLULASES OF MESOPHILIC ORIGIN.
Microorganisms growing best at moderate temperatures (between 10 and 45°C) are named mesophiles. They represent the majority of microbial species on Earth, and their habitats include the soil, the human body, the animals, etc. There are many mesophilic Bacteria and Fungi that play a significant role in the carbon cycle on Earth, and there is increasing interest in the enzymes from these microorganisms, since they have a key function in the conversion of plant biomass into useful products.
The ability to digest cellulose is widely distributed among Bacteria and Fungi and some of them are listed in Tables 2 and 3.
As already described, the different strategy of degradation between the anaerobic and aerobic groups resides in the production of complex cellulase systems, exemplified by the well-characterized cellulosome from the Clostridium genus [Beguin and Lemaire, 1996; Schwarz, 2001], or the extracellular cellulases freely released in the culture supernatant, respectively [Wachinger et al., 1989].
7.1 Bacterial cellulases.
Active research on cellulases and related polysaccharidases began in the early 1950s, owing to their enormous potential to convert lignocellulose to glucose [Mandels, 1985]. In this review we have limited the description of cellulases of mesophilic origin to the last ten years, because of the considerable amount of literature that has been produced up to now, included reviews. Some representatives of bacterial cellulases described before 2000 are reported in Table 4.
Identification, purification and characterization of cellulases are continuously increasing and always in progress, with incessant research and isolation of new microorganisms able to produce novel cellulolytic activities. As an example, a bacterial strain, TR7-06(T), showing high sequence similarity (98.5 %) to Cellulomonas uda DSM 20107(T), was isolated from compost at a cattle farm near Daejeon, Republic of Korea. The isolated type strain of a novel Cellulomonas species, named Cellulomonas composti sp. nov., possesses endoglucanase and β-glucosidase activities [Kang et al., 2007]. A microorganism capable of hydrolyzing rice hull, one of the major cellulosic waste material in Korea, was isolated from soil and identified as
Table 2 – Some mesophilic cellulolytic Bacteria.
Table 3 – Some mesophilic cellulolytic Fungi.
Table 4 – Properties of some cellulases from mesophilic Bacteria.
Bacillus amyloliquefaciens DL-3. Basing on the characteristics of this novel strain of Bacillus, Lee et al. [2008] aimed to develop an economical process for production of cellulases by using cellulosic waste as inexpensive and widely distributed carbon source. The new isolate produced an extracellular cellulase with an estimated molecular mass of about 54.0 kDa. The deduced amino acid sequence of the cellulase from B. amyloliquefaciens DL-3 showed high identity to cellulases from other Bacillus species, a modular structure containing a catalytic domain of the GH family 5, and a cellulose-binding module type 3 (CBM3). The purified enzyme was optimally active at 50°C and pH 8.0, and showed broad thermal and pH stability ranging from 40 to 80°C and from 4.0 to 9.0, respectively.
Although cellulases have been isolated from many microorganisms, no functional cellulase genes were reported for Vibrio genus until now. Gao et al. [2010] isolated from mangrove soil a new bacterium belonging to the Vibrio genus, Vibrio sp. G21, and a novel endoglucanase gene, cel5A, was cloned. The mature Cel5A enzyme was overexpressed in Escherichia coli and purified to homogeneity. It was stable over a wide range of pHs and retained more than 90% of activity after incubation at pHs 7.5-10.5 for 1 h. Moreover, the enzyme was activated after pretreatment with mild alkali, a novel characteristic that has not been previously reported in other cellulases. The deduced protein contained a catalytic domain of the GH family 5, followed by a cellulose-binding module type 2 (CBM2).
The discovery of alkaline cellulases has generated new industrial applications of cellulases as laundry detergent additives [Ito, 1997]. Many microorganisms belonging to Bacillus sp. are producers of alkaline cellulases even if other microorganisms also possess cellulases active at high pH value. Since the discovery of an alkaline cellulase by Horikoshi et al. [1984], many other alkaline cellulases from alkaliphilic Bacillus strains have been identified. A novel strain of Bacillus sphaericus JS1 was isolated from soil. The strain produced an extracellular carboxymethylcellulase (CMCase) with a molecular mass of 183.0 kDa, and a single band of about 42.0 kDa was estimated by SDS-PAGE. The enzyme was active over a broad range of pH (7.0-10.5), with a half-life of 18 h at pH 8.0 and 4.5 h at pH 10.0 at 60°C [Singh et al., 2004]. Endo et al. [2001] purified to homogeneity from the culture broth of the alkaliphilic Bacillus sp., strain KSM-N252, a highly alkaline endoglucanase (Egl-252) with a molecular mass of approx. 50.0 kDa. The enzyme exhibited reasonable homology to other alkaline endoglucanases belonging to GH family 5. In fact, the deduced amino acid sequence of Egl-252 showed moderate homology to that of NK-1 [Fukumori et al., 1986], and to Cel5A from B. agaradherens (accession no. AF067428) with 75.6% and 64.3% identity, respectively. This suggested that also Egl-252 belongs to GH family 5. The optimal temperature for activity was 55°C, and the optimal pH was 10.0 with more than 80% of the maximal activity retained between pH 8.0 and 11.0. The enzyme was very stable between pH 6.0 and 11.5 at 30°C.
An alkaline endoglucanase with a molecular mass of 43.0 kDa (Egl-257) was purified and crystallized from B. circulans KSM-N257 [Hakamada et al., 2002]. The enzyme, showing 76.3% amino acid identity with a lichenase from B. circulans WL-12 which belongs to GH family 8, hydrolyzed carboxymethylcellulose (CMC) as well as lichenan. Egl-257 showed optimal temperature and pH at 55°C and 8.5, respectively. It was stable over a range of pH between 5.0 and 11.0 after incubation at 30°C for 1 h retaining the nearly total activity.
A novel alkaline cellulase from the alkalophilic Bacillus sp. HSH-810 was purified and characterized by Kim et al. [2005]. The purified enzyme was optimally active at pH 10.0 and showed about 60% activity at pH 12.0. In contrast, enzyme from Bacillus sp. strain KSM-N252 showed similar optimum pH but retained only 35% activity at pH 12.0 [Endo et al., 2001].
As already mentioned, microorganisms different from Bacillus are also capable of producing alkaline cellulases. Marinobacter sp. (MSI032), isolated from the marine sponge Dendrilla nigra, produces an extracellular alkaline cellulase at 27°C and pH 9.0 [Shanmughapriya et al., 2009]. Usually, cellulase production by Bacteria occurs during the late growth phase. Thus, maintenance of the culture conditions for long times causes economic disadvantages for the development of industrial processes. Unexpectedly, the production of cellulase by Marinobacter MSI032 occurs at an earlier stage of growth suggesting the usefulness of the strain in industrial processes. The purified enzyme displayed maximum activity at pH 9.0 and at temperature between 27 and 35°C. In addition, it was stable over a broad range of pH, with residual activity higher than 80% between pH 8.0 and 12.0, indicating that this alkaline cellulase has a very high pH stability. Paenibacillus sp., strains KSM-N115, KSM-N145, KSMN440, and KSM-N659 produces cellulases (Egls) that hydrolyze Avicel, filter paper and amorphous cellulose, to cellotriose, cellobiose, and glucose by endo-fashion cleavage at alkaline pH [Ogawa et al., 2007]. The optimal temperature and pH of one representative recombinant enzyme (Egl-659) for degrading CMC and Avicel were 45-55°C and 6.0-8.5, respectively. Even at pH 9.0 the enzyme showed more than 75% relative activity. Egl-659 was very stable over a pH range between 5.0 and 11.0 after incubation at 50°C for 20 h.
Among the anaerobic cellulase-producing bacteria, the genera Clostridium is without doubt the most studied. It numbers mesophilic and thermophilic representatives and multienzyme complexes having high activity against crystalline cellulose, known as the cellulosome, have been identified and characterized in many of these Bacteria as reported above.
C. phytofermentans was isolated by Warnick et al. from forest soil [2002]. The essential component of the C. phytofermentans cellulolytic system (Cel9) is a processive endoglucanase that shows activities on both soluble CMC and crystalline cellulose. In order to obtain high-purity cellulase and facilitate its production, the cel9 gene was recently expressed in E. coli, and the recombinant protein was purified and characterized [Zhang et al., 2010a]. The pH and temperature optima for activity were 6.5 and 65°C, respectively. The unusual high optimal temperatures for Cel9 and for the noncellulosomal Cel48 (60°C) [Zhang et al., 2010b] are somewhat surprising, but can be explained by possible acquisition of the cel9–cel48 gene cluster from a thermophilic microorganism through horizontal gene transfer.
7.2 Fungal cellulases.
Fungal cellulases are well-studied enzymes used in various industrial processes [Bhat, 2000], and the properties of several of them, not considered in this text, are listed in Table 5. A variety of aerobic and anaerobic Fungi are producers of cellulose-degrading enzymes. The aerobic Fungi play a major role in the degradation of plant materials and are found on the decomposing wood and plants, in the soil, and on the agricultural residues. The cellulase systems of the aerobic Fungi Trichoderma reesei, T. koningii, Penicillium pinophilum, Phanerochaete chrysosporium, Fusarium solani, Talaromyces emersonii, and Rhizopus oryzae are well characterized [Bhat and Bhat, 1997]. Much of the knowledge on enzymatic depolymerization of cellulosic material has come from Trichoderma cellulase system. In particular, the cellulase system of T. reesei (initially called T. viride) has been the focus of research for 50 years [Reese et al., 1959; Reese and Mandels, 1971]. A lot of work on cellulases has been directed toward this fungus since it produces readily, and in large quantities, a complete set of extracellular cellulases, and consequently, it has a high commercial value [Claeyssens et al., 1998; Miettinen-Oinonen and Suominen 2002].
In fact, T. reesei is capable of secreting more than 30 g/L of protein into the extracellular medium [Conesa et al., 2001]. It has been reported that T. reesei possesses two CBH (cellobiohydrolase) genes, cbh1-2, and eight EG (endoglucanase) genes, egl1-8, and that CBH I–II and EG I–VI are secreted proteins [Foreman et al., 2003]. Altough the present review essentially concerns cellulases from the last ten years, the authors like to give short signal about endoglucanases from T. reesei as they represent very attractive biocatalysts for industrial applications [Schuster and Schmoll, 2010 ].
EGI (Cel7B) hydrolyzes both cellulose and xylan and has optimal temperature and pH at 30°C and 5.0, respectively [Biely et al., 1991]. The structure of EGI was resolved to reveal the presence of short loops that create a groove rather than a tunnel.
Table 5 – Properties of some cellulases from mesophilic Fungi.
The catalytic domain resembles an open substrate-binding cleft, thus enabling the enzyme to interact more effectively with the amorphous or disordered crystalline cellulose [Kleywegt et al., 1997]. A similar groove was shown for the structure of EGIII (Cel 12A) that lacks a CBM [Sandgren et al., 2000]. The glycosylation profile of EGI and EGII (Cel5A) was determined by Hui et al. combining enzymatic digestion with mass spectrometry techniques, and the analyses indicated that glycosylation accounted for 12-24% of the molecular mass of the enzymes [Hui et al., 2002]. Saloheimo et al. [1988] isolated and determined the primary structure of the gene egl3 coding for the EGIII endoglucanase from T. reesei. The protein was purified, and its amino acid composition and N-terminal sequence supported the data obtained from the gene sequence. The enzymatic properties of EGIII and EGV (Cel45A) have been investigated by Karlsson et al. [2002]. Adsorption studies on Avicel and phosphoric acid swollen cellulose (PASC) showed that Cel45A and Cel45A catalytic core adsorbed to these substrates. On the contrary, Cel12A adsorbed weakly to both Avicel and PASC. Cel12A showed maximal activity at pH 5.0, while pH 4.0 was the best value for Cel45A maximal activity. The optimal temperature for Cel12A was 50°C. Interestingly, Cel45A showed the highest activity at 70°C. EGIV (Cel61A) was homologously expressed in high amounts with a histidine tag on the C-terminus, purified by metal affinity chromatography and characterized [Karlsson et al., 2001]. The only activity exhibited by Cel61A was the endoglucanase activity toward substrates containing 1,4-β-glycosidic bonds (CMC, hydroxyethylcellulose and β-glucan).
In recent years, research on Trichoderma has been facilitated significantly by sequencing of the genomes of three strains representing the most important applications of this genus. The genome of T. reesei has been fully sequenced and published on the http://genome.jgi-psf.org/ Trire2/Trire2.home.html website [Martinez et al., 2008]. Analyses and annotation of the genomes of T. atroviride and T. virens, (http://genome.jgipsf. org/Triat1/Triat1.home.html; http://genome.jgi-psf.org/ Trive1/Trive1.home.html), are still in progress.
The Fungus Acremonium cellulolyticus, isolated in 1987, is known to be a potent producer of cellulases as T. reesei, even if many cellulases and β-glucosidases have not been as well characterized as those produced by T. reesei [Yamanobe et al., 1987; Ikeda et al., 2007]. Since enzymatic saccharification using cellulases has proven to be a powerful method in the production of bioethanol, a comparison between cellulase activity from the two fungi against three lignocellulosic materials (eucalyptus, Douglas fir wood chip and rice straw) has been performed by Fujii et al. [2009]. Saccharification efficiency of both culture supernatants and commercial preparations (AC derived from A. cellulolyticus and Accellerase 1000 derived from T. reesei) was investigated. The culture supernatant from A. cellulolyticus produced higher glucose yield from lignocellulosic materials than the T. reesei supernatant. In the same way, AC produced a greater amount of glucose from lignocellulosic materials than Accellerase 1000.
In the last years, a great deal of attention has been focused on enzymes capable of degrading biomass for a number of applications, and on their potential to be produced industrially. However, the cost of producing sugars from lignocellulosic waste for fermentation into bioethanol is still high to attract industrial attention, mainly due to low enzyme yields from microorganisms. Cellulases produced by Fungi such as the Aspergillus and Penicillium species have been widely studied by numerous researchers, in addition to cellulases from T. reesei [van Peij et al., 1998; Jun et al., 1992]. Recently, Hassan et al. [2008] demonstrated that six filamentous Fungi, including A. terreus DSM 826, produce big amounts of different enzymes involved in the degradation of cellulose (namely endoglucanase and cellobiohydrolase) when grown on media containing corn cobs, corn stalks, rice straw or sugar cane bagasse as carbon sources. Sugar cane bagasse is a very low-cost substrate for endoglucanases production from different microorganisms. An endoglucanase from A. terreus DSM 826 was purified and characterized after growth on sugar cane bagasse as a carbon source [Elshafei et al., 2009]. The purified enzyme showed a high specific activity toward CMC with its optimal activity at pH 4.8 and 50°C. A similar optimal temperature was reported for enzymes from Melanocarpus sp. MTCC 3922 [Kaur et al., 2007] and Bacillus amyloliquefaciens DL-3 [Lee et al., 2007]. When heated at 50°C for 1 h, the endoglucanase from A. terreus DSM 826 did not show loss of activity, seeming to be more thermostable than endoglucanases from other microorganisms such as that from Sinorhizobium fredii which retained 96% of its activity at 40 °C [Chen et al., 2004].
Aspergillus glaucus XC9, grown on 0.3% sugar cane bagasse as a carbon source, produced an extracellular cellulase with a molecular mass of 31.0 kDa [Tao et al., 2010]. The optimum of pH and temperature for enzyme activity were 4.0 and 50°C, respectively. This enzyme was stable over a wide pH range (3.5-7.5) and at temperatures below 55 °C. It retained only 60% activity after incubation at 60°C for 1 h. The newly isolated endoglucanase from A. glaucus XC9 shares common characteristics with those from industrial cellulase-producing Fungi, such as A. niger and T. reesei suggesting its possible use in industry.
In Brazil, sugar cane bagasse is one of the major residues of first-generation bioethanol production, and this residue has been greatly taken into consideration as a carbon source for low-cost cellulase production by several microorganisms [Barros et al., 2010]. Several substrates were generated after different pretreatment of sugar cane bagasse, and they were used as carbon source for Penicillium funiculosum growth. The best results, in terms of cellulolytic enzymes production, were observed when sugar cane bagasse was treated with acid and subsequently, alkali in order to obtain partially delignified cellulignin. The culture filtrate of P. funiculosum contained several cellulolytic activities. The optimal temperature for cellulase action was comprised between 52 and 58°C and the best pH for maximal activity was 4.9. Cellulases from P. funiculosum grown on sugar cane bagasse were highly stable at 37°C, as they retained more than 85% activity at this temperature [de Castro et al., 2010]. Two new fungal strains from subtropical soils, Penicillium sp. CR-316 and Penicillium sp. CR-313, were identified and selected because they secreted high levels of cellulases [Picart et al., 2007]. The culture filtrate from the two strains, analyzed by SDS-PAGE and zymography, showed several bands, indicating that both strains produced a multisystem of cellulases. Zymograms from Penicillium sp. CR-316 showed four activity bands of 35.0, 37.0, 48.0 and 71.0 kDa, respectively, while zymograms from Penicillium sp. CR-313 showed three activity bands of 35.0, 37.0 and 50.0 kDa. Multiple enzyme systems are frequently produced by cellulose-degrading microorganisms, as the cooperation of different cellulases acting in a coordinated manner enhance the degradation of the cellulose [Lynd et al., 2002]. The activity produced by Penicillium sp. CR-316 was higher than that produced by Penicillium sp. CR-313, and for this reason, this activity was better characterized. Crude cellulase of Penicillium sp. CR-316 exhibited optimum of temperature and pH at 65°C and 4.5, respectively, and the activity remained stable after incubation at 60°C and pH 4.5 for 3 h. The high yield of cellulases from Penicillium sp. CR-316, active and stable at high temperatures, should facilitate their use in biotechnological applications to improve the manufacture of recycled paper, and the transformation of cellulosic materials.
Species of the genus Rhizopus are known to have strong starch-degrading activity and this type of enzyme is extensively studied in this fungus [Li et al., 2010]. Conversely, there are few reports describing the production of cellulases by this filamentous fungus. Two extracellular endoglucanases, named RCE1 and RCE2, produced by Rhizopus oryzae FERM BP-6889 isolated from soil, were identified and purified by Murashima et al. [2002]. The molecular masses of the two enzymes were 41.0 and 61.0 kDa, respectively. The optimal pH for the activity of both enzymes was found to be between 5.0 and 6.0, and the optimum of temperature was 55°C. RCE1 and RCE2 did not hydrolyze hemicelluloses such as xylan, galactan, arabinan, or mannan. The amino acid sequences of some fragments obtained from internal regions of RCE1 and RCE2 were found to be homologous to the catalytic domain of EGV from H. insolens which belongs to GH family 45 [Schulein, 1997]. Thus, these findings supported the assumption that the enzymes belong to GH family 45. A novel gene, encoding for an endoglucanase was isolated from R. stolonifer var. reflexus TP-02, sequenced and expressed in E. coli. The recombinant enzyme exhibited an apparent molecular mass of 40.0 kDa, and the phylogenetically analysis on the sequence demonstrated that it grouped with Aspergillus niger (AJ224451), but they only shared 49% identity [Tang et al., 2009].
The white rot Fungus Phanerochaete chrysosporium has been used as a model organism for lignocellulose degradation since it produces a set of cellulases, hemicellulases, and lignin-degrading enzymes for an efficient degradation of the three major components of plant cell wall [Broda et al., 1996]. Several cellulases have been purified by Eriksson et al. [1975a;1975b] more than 20 years ago. Then, two GH family 5 isozymes (Cel5A, previously indicated as EG44) and Cel5B (previously indicated as EG38) and a 28-kDa endoglucanase (Cel12A) have been reported [Uzcategui et al., 1991; Henriksson et al., 1999]. The endoglucanase gene, cel61A, has been characterized although the corresponding protein has not yet been identified [Vanden Wymelenberg et al., 2002].
The gene that encodes the GH family 45 endoglucanase from the Fungus has been identified, cloned, and heterologously expressed in the yeast Pichia pastoris, and the recombinant protein has been characterized [Igarashi et al., 2008]. The enzyme has not carbohydrate binding module and the analysis of its amino acid sequence has revealed that the protein has low similarity (<22%) to known fungal EGs belonging to the GH family 45, thus suggesting that the protein should be classified into a new subdivision of this family. The recombinant protein shows hydrolytic activity toward amorphous cellulose, lichenan, and glucomannan but not xylan.
Cellulases have also been isolated from several brown rot Fungi as Gloeophyllum trabeum, G. sepiarium, and Serpula incrassata [Mansfield et al., 1998; Kleman-Leyer and Kirk, 1994]. The brown rot Fungus Piptoporus betulinus is a parasite for birch (Betula specie) and exhibits a high rate of wheat straw degradation accompanied by a high production of hydrolytic enzymes (endoglucanase, endoxylanase, endomannanase, β-glucosidase, and additional glycolytic activities) which makes it interesting for potential biotechnological applications. The major glycoside hydrolase produced and purified was an endoglucanase (EG1) with a molecular mass of 62.0 kDa [Valaskova and Baldrian, 2006]. This molecular mass was higher than that of cellulases from the majority of brown rot Fungi with the exception of Cel25 from S. incrassata [Kleman-Leyer and Kirk, 1994] and Cel12a from G. trabeum [Cohen et al., 2005]. In fact, the typical molecular mass ranges between 35.0 and 50.0 kDa [Clausen 1995]. EG1 was active in a broad pH range (2.5-6.0) with maximal activity at pH 3.0 and 70°C. The enzyme exhibited the highest substrate specificity for CMC, and it cleaved also xylan and galactomannan at lower rates. Scarce activity was showed toward crystalline cellulose.
Although a number of filamentous Fungi, such as Trichoderma and Aspergillus, are well known as producers of cellulases and accessory cellulolytic enzymes, the search for new strains and new enzymes has become a priority with the increase in diversity of biomass sources. A series of marine sponge-derived Fungi were isolated and screened for cellulolytic activity by Baker et al. [2010] in order to determine the potential of this environmental niche as a source of novel cellulase activities. Several strains of Fungi isolated from the marine sponge Haliclona simulans produced high levels of extracellular cellulases with significant activity at low temperatures. Moreover, a potent endoglucanase-producing Fungus was isolated and identified as a strain of Penicillium pinophilum (P. pinophilum KMJ601). Maximal production of cellulase (Eng5) was observed when the fungus was grown on rice straw or cellulose as a carbon source [Jeya et al., 2010]. The secreted and purified enzyme, with a molecular mass of 37.0 kDa, showed optimal pH and temperature of 5.0 and 70°C, respectively, and a t1/2 value of 15 h at 70°C. Eng5 showed broad substrate specificity, exhibiting maximum specific activity toward lichenan and also acting on xylan. The partial gene sequence of P. pinophilum Eng5 contained the EG-like domain that is found in the GH family 5. NCBI BLAST analysis confirmed that the endoglucanase belongs to the GH family 5.
Anaerobic Fungi were first isolated by Orpin from the rumen of a sheep [Orpin, 1975]. Since then, they have been recovered from the digestive tracts of many species of herbivores, including both ruminants and non-ruminants, where they are believed to be responsible for the digestion of 50-70% of the ingested plant material [Trinci et al., 1994]. Six genera of anaerobic Fungi are recognized: Anaeromyces, Caecomyces (formerly Sphaeromonas), Cyllamyces, Neocallimastix, Orpinomyces, and Piromyces, and excellent information about them can be found in the book edited by Mountford and Orpin [Barr et al., 1989; Mountford and Orpin, 1994]. Unlike aerobic Fungi, anaerobic Fungi produce large multienzyme complexes similar to bacterial cellulosomes, and these complexes can degrade both amorphous and crystalline cellulose [Dijkerman et al., 1997]. Cellulosome-type complexes with endoglucanase, xylanase, mannanase, and β-glucosidase activities containing at least 10 proteins have been found in Neocallimastix frontalis, Piromyces, and Orpinomyces [Wilson and Wood, 1992; Steenbakkers et al., 2002; Ljungdahl, 2008]. The cellulose/hemicellulose degrading system of Piromyces equi, so called because this Fungus was isolated from the caecum of a pony [Orpin, 1981], consists of a large multienzyme complex, which accounts for up to 90% of the cellulase, mannanase and xylanase activities produced by the Fungus. Eberhardt et al. [2000] described the elucidation of the primary structures and enzyme features of two endoglucanases from P. equi, Cel5A and Cel45A. The two endoglucanase cDNAs, cel5A and cel45A, were isolated from a cDNA library of the Fungus. Sequence analysis revealed that cel5A encodes a 1714 amino acid modular enzyme, Cel5A, with a molecular mass of 195.0 kDa. The cDNA cel45A encodes a 410 amino acid modular enzyme, Cel45A, with a molecular mass of 44.3 kDa. Cel45A represented the first GH family 45 endoglucanase to be isolated from an anaerobic organism. The catalytic domains of Cel5A and Cel45A were hyperexpressed as thioredoxin fusion proteins, (Trx-Cel5A’ and Trx-Cel45A’), and subjected to biochemical analysis. Trx-Cel5A’exhibited optimum pH at 5.0, but it retained about 78% activity at pH 6.4. Trx-Cel45A’ exhibited optimum pH at 6.5, and it retained 65% activity between pH 5.2 and 7.9. The influence of temperature on Trx-Cel5A’ and Trx-Cel45A’ activity showed that the initial rate of hydrolysis of CMC, selected as substrate, by Trx-Cel5A’ was highest at 45°C, while the initial rate of Trx-Cel45A’ activity increased with increasing temperature between 40 and 70°C. Trx-Cel45A’ was more thermostable than Trx-Cel5A’. The enzyme was stable for at least 1 h at 65°C, whereas an almost complete loss of activity was observed with Trx-Cel5A’ after 20 min preincubation at 55°C.
While fungal cellulolytic and hemicellulolytic enzymes have been well studied for Neocallimastix, Orpinomyces, and Piromyces, information regarding cellulose-degrading enzymes from Caecomyces is still limited [Gerbi et al., 1996]. In 2008, Matsui and HBan-Tokuda isolated from bovine rumen a new anaerobic Fungus which was classified, after phylogenetical analysis, as Caecomyces CR4 [Matsui and Ban-Tokuda, 2008]. Elevated levels of cellulase activity were found in the culture supernatant of the CR4 isolate when it was grown in medium added with xylose as carbon source. Zymogram analysis showed that cellulase activity could be associated to three protein bands with molecular masses of 64.0, 89.0, and 95.0 kDa, respectively.
8. CELLULASES OF THERMOPHILIC ORIGIN.
The (hyper)thermophilic microorganisms represent a unique group growing at temperatures that may exceed 100°C. More precisely, thermophilic microorganisms thrive at temperatures from 65 to 85°C, and hyperthermophiles grows at temperatures of above 85°C. Hyperthermophiles are microorganisms within the Archaea domain although some Bacteria are able to tolerate temperatures around 100°C. An extraordinary heat-tolerant hyperthermophile is Methanopyrus kandleri, discovered on the wall of a black smoker from the Gulf of California at a depth of 2000 m, at temperatures of 84-110°C. It can survive and reproduce at 122°C [Takai et al., 2008]. Thermophilic and hyperthermophilic microorganisms have received considerable attention as sources of thermostable cellulolytic enzymes, as the properties of these biocatalysts make them interesting candidates for industrial applications. Running biotechnological processes at elevated temperatures has many advantages. High temperature has a significant influence on the solubility of the substrates (especially if viscous or polymers) and on the reaction rate. Moreover, problems of microbial contamination can be avoided when a reaction is performed at elevated temperature.
Degradation of cellulosic and hemicellulosic substrates among thermophiles is mostly due to Eubacteria species, e.g. Rhodothermus marinus, Thermotoga sp., Caldibacillus cellulovorans, Alicyclobacillus acidocaldarius [Halldórsdóttir et al., 1998; Bronnenmeier et al., 1995], while these activities are present in only a few representatives of Archaea (Pyrococcus sp. and Sulfolobus sp.) with cellulases belonging to GH families 5 and 12 (Table 6). Production of cellulases in a small number of thermophilic Fungi has also been reported (Table 7).
8.1 Bacterial and archaeal cellulases.
Thermostable cellulases are of great biotechnological interest [Hongpattarakere, 2002]. A number of cellulolytic thermophilic Bacteria have been isolated, and many cellulose-degrading enzymes have been identified, characterized, cloned and expressed [Bergquist et al.,
Table 6 – Some (hyper)thermophilic cellulolytic Bacteria and Archaea.
Table 7 – Some (hyper)thermophilic cellulolytic Fungi.
1999]. Conversely, screening of hyperthermophilic Bacteria for cellulose-degrading enzymes has revealed that the presence of such enzymes is rather rare in this group. In addition, among the Archaea, only the genus Pyrococcus and Sulfolobus have been found to process thermoactive cellulases (Table 8).
Table 8 – Properties of some cellulases from (hyper)thermophilic Bacteria and Archaea.
Few aerobic thermophilic microorganisms have been described to produce cellulases in comparison with the anaerobic ones. Acidothermus cellulolyticus, isolated from 55-60°C acidic water and mud samples collected in Yellowstone National Park, produces at least three thermostable endoglucanases [Mohagheghi, 1986]. One of them, E1 belonging to GH family 5, was crystallized, while properties and application of the other enzymes are protected by patents [Sakon et al., 1996].
The aerobic thermophilic bacterium Rhodothermus marinus, isolated from a submarine hot spring at Reykjanes, NW Iceland [Alfredsson et al., 1988], produces one higly thermostable cellulase (Cel12A) which retains 50% activity after 3.5 h at 100°C [Hreggvidsson et al., 1996].
The thermophilic filamentous bacterium Thermobifida fusca (formerly Thermomonospora fusca) is one of the most extensively studied aerobic, thermophilic, cellulose degrading bacterium, and a major cellulose degrader in soil. This Actinomycete secretes three endoglucanases Cel9B, Cel6A, Cel5A (formerly named E1, E2, and E5), two exoglucanases Cel6B and Cel48A (formerly E3 and E6), and an endo/exoglucanase Cel9A (formerly E4) which have been characterized in detail [Ghangas and Wilson, 1988; Lao et al., 1991; Irwin et al., 1993; Irwin et al., 2000]. Deepened studies aimed to understand the catalytic mechanism of the cellulase Cel6A by computational and experimental investigation have been performed by Andrè et al. [2003]. In addition, the crystal structure of this enzyme, in complex with substrate and inhibitor has been solved [Larsson et al., 2005]. Zymogram analysis revealed that additional cellulases are produced by T. fusca. A new endoglucanase gene, Tf cel5B, was identified, and heterologous Cel5B was produced in Streptomyces lividans [Posta et al., 2004]. The novel cellulase has a molecular mass of about 67.0 kDa and optimum of pH and temperature of 8.2 and 77°C, respectively. It retained more than 60% of the maximal activity after 24 h incubation at 60°C. The temperature optimum of 77°C is comprised in the temperature optimum range of the other endoglucanase, endoxylanase and endomannanase enzymes from T. fusca (70-80°C), suggesting that the new endoglucanase has similar advantages for industrial application as the other thermostable hydrolases from this thermophilic microorganism. Alignments of the Cel5B catalytic domain to similar regions of other cellulases revealed 67% identity with CEND from Cellulomonas fimi, 66% identity with Cel5A from C. flavigena and 60% identity with CelB from Caldicellulosiruptor saccharolyticus.
The genus Alicyclobacillus was first established by Wisotzkey et al. [1992], and it is characterized by the presence of alicyclic fatty acids as major components of the membrane lipids. All Alicyclobacillus species are highly thermoacidophilic (defined as optimal growth conditions at 45-60°C and pH 2.0-5.0) and may be a good source of acidic glucanases. Alicyclobacillus acidocaldarius is known to produce four cellulases: CelA, CelB, CelG and CelA4. All of them are highly active at acidic pH and at temperatures between 65 and 80°C. The gene encoding for CelA, belonging to GH family 9, was cloned and sequenced, and the recombinant protein, expressed in E. coli, was characterized [Eckert et al., 2002]. The molecular mass was 59.0 kDa and in agreement with that deduced from the ORF encoding a putative protein of 537 amino acids. CelA exhibited temperature and pH optima of 70°C and 5.5, respectively, and it was active against CMC, lichenan and also oat spelt xylan and cello-oligosaccharides, suggesting a role as a cytoplasmic enzyme for the degradation of short-chain sugars imported from the medium. CelB was a membrane-bound protein of 100.0 kDa with pH and temperature optima of 4.0 and 80°C, respectively [Eckert and Schneider, 2003]. The enzyme was extremely stable at acidic pH and temperatures up to 80°C, and was provided with activity toward CMC and oat spelt xylan. High sequence similarity to arabinofuranosidases belonging to GH family 51 was displayed, whereas, in terms of substrate specificity, CelB was comparable only to an endoglucanase from Fibrobacter succinogenes (EGF), also belonging to the GH family 51. Another membrane-bound cellulase was identified and characterized from A. acidocaldarius when it was grown in liquid medium added with CMC as carbon source [Morana et al., 2008]. The enzyme was demonstrated to be glycosylated (therefore named CelG) with a molecular mass of 56.2 kDa. It was active over a broad range of pH (3.0-7.0) with optimal activity at pH 4.0, and showed activity between 40 and 80°C with optimal temperature at 65°C. Themostability was evaluated at 65 and 75°C, and CelG retained 100% activity after 2 h at 65°C and 1 h at 75°C. This enzyme was more thermostable than CelA, which displayed small loss of activity after 1 h at 60°C and an half-life of 30 minutes at 75°C. Moreover, it was found to be specific for CMC hydrolysis, while CelA and CelB were also active toward other polysaccharides. The most recently identified cellulase in A. acidocaldarius is the secreted 48-kDa protein CelA4. It was isolated, purified and characterized by Bai et al. [2010]. The enzyme was the more acidophilic among the cellualses from A. acidocaldarius, being its optimal pH 2.6. In addition, it was stable under acid conditions, retaining more than 80% activity in the pH range of 1.8-6.6 after incubation for 1 h at 37°C. The optimal temperature was 65°C, but the total activity was lost after incubation at this temperature for 1 h. Purified CelA4 was protease-resistant and exhibited the highest enzymatic activity toward barley β-glucan. The high activity at low pH and protease resistance make CelA4 a good candidate as commercial cellulase able to improve nutrient use in the animal feed industry. In fact, barley β-glucan cannot be digested by monogastric animals and, furthermore, the pig gastrointestinal tract is highly acidic and contains a lot of proteolytic enzymes. As consequence, an ideal glucanase should have high activity in extremely acidic conditions and be protease-resistant to function under the conditions present in the digestive tract of animals [Li et al., 1996].
Aquifex aeolicus, a hyperthermophilic microorganism belonging to the Bacteria domain, was originally isolated in the Aeolic Islands at north of Sicily near underwater volcanic vents. It represents the deepest branch in the bacterial phylogeny and its growth temperature can reach 95°C [Pitulle et al., 1994]. Only one thermostable cellulase has been reported to be produced by this microorganism to date, and the gene cel8Y has been cloned and expressed in E. coli by Kim et al. [2000]. The gene product (Cel8Y) has been purified and characterized. It has a molecular mass of 36.7 kDa and it was able to hydrolyze β-1,4 linkages in CMC but not in Avicel. Optimal pH and temperature for activity were 7.0 and 80°C, respectively. At 90 and 100°C, the activity of the recombinant Cel8Y decreased by 50% after 4 and 2 h, respectively. On the basis of sequence comparisons, the enzyme is very closely related to other cellulases of GH family 8.
Three genes (sso1354, sso1949, and sso2534) encoding three endoglucanases belonging to GH family 12, have been annotated in the fully sequenced genome of the hyperthermophilic and acidophilic crenarchaeote Sulfolobus solfataricus strain P2 [She et al., 2001]. This microorganism, originally isolated from a solfataric field in the area of Naples, Italy, grows at temperatures ranging from 80 to 87°C and at very low pH values (2.0-4.0) [De Rosa et al., 1975]. The gene sso2534 was identified in S. solfataricus, strain MT4, and its product (CelS) showed 100% amino acid sequence identity with CelB from S. solfataricus strain P2 [Limauro et al., 2001]. CelS (38.4 kDa) was detected as an extracellular cellulase. It was active at pH 5.8, but no further indication about optimal conditions for activity or about pH and temperature stability, and substrate specifity were reported. In 2005, the gene sso1949 was cloned in E. coli and expressed by Huang et al. [2005]. The purified recombinant enzyme hydrolyzed CMC as well as cello-oligomers, whereas Avicel, xylan and lichenan, appeared not to be substrates for the enzyme. The failure to detect activity toward Avicel could be ascribed to the lack of a cellulose-binding module. The same feature was observed for the SSO1354 protein. SSO1949 protein exhibited pH and temperature optima of 1.8 and 80°C, respectively, and showed a half-life of approx. 8 h at 80°C and pH 1.8. When incubated at 95°C and pH 1.8, SSO1949 was rapidly inactivated. Analysis by tandem mass spectrometry led to identify the endoglucanase precursor, encoded by the sso1354 gene, as the protein provided with xylanase activity isolated from S. solfataricus strain Oα, a strain capable of growing in minimal medium supplemented with xylan as sole carbon source [Cannio et al., 2004]. SSO1354 protein was membrane-bound and possessed both cellulolytic and xylanolytic activities. It was also active toward arabinan and debranched arabinan in contrast to SSO1949 protein which was inactive toward xylan and others polysaccharides. The enzyme was glycosylated with a molecular mass of 57.0 kDa, and exhibited optimal temperature and pH of 95°C and 3.5, respectively [Maurelli et al., 2008]. Among the characterized cellulases from thermophilic microorganisms, only EglA from P. furiosus [Bauer et al., 1999] and CelB from T. neapolitana [Bok et al., 1998] showed higher optimal temperatures. SSO1354 was highly thermophilic and thermostable, retaining 71% of the initial activity at 100°C and 49% at 110°C, and showing a half-life of 53 min at 95°C.
Clostridium thermocellum is a thermophilic, strictly anaerobic bacterium, and in 1983 the cellulosome concept was established in this microorganism as cellulases were found to be organized into a high molecular weight, cellulolytic complex [Lamed et al., 1983]. C. thermocellum produces a number of cellulases both free and assembled in cellulosome [Fauth et al., 1991; Lemaire and Béguin, 1993; Ashan et al., 1997]. Kurokawa et al. [2002] identified an additional endoglucanase (CelT) in 2002. The mature form of CelT consists of a GH family 9 cellulase domain and a dockerin domain responsible for cellulosome assembly, but has neither an Ig-like domain domain nor a family 3c CBM. However, it is more similar to the family E2 cellulases than the family E1 with respect to sequence similarity, and has a catalytic domain that is homologous with the catalytic domains of T. fusca E4, C. cellulolyticum CelG and C. thermocellum CelQ. The enzyme exhibited optimal pH and temperature at 7.0 and 70°C, respectively, but no data concerning pH and temperature stability have been reported. Immunological analysis indicated that CelT is a catalytic component of the C. thermocellum F1 cellulosome. To identify the predominant catalytic components of C. thermocellum cellulosome, the cellulolytic complexes were purified and the components were separated and identified by MALDI-TOF/TOF [Zverlov et al., 2005]. Ten of the components were already known, and in addition, three hitherto unknown genes, which were named according to their sequences celR, xghA and xynD, were detected. According to the putative function of their catalytic modules, the corresponding gene products were called Cel9R, Xgh73A and Xyn10D, with catalytic modules of GHF9 (endoglucanase), GHF74 (xyloglucanase) and GHF10 (endoxylanase), respectively [Coutinho and Henrissat, 1999]. The new putative endoglucanase Cel9R gave one of the most prominent protein spots of all cellulosomal components.
Highly cellulolytic microorganisms are found among the anaerobic (hyper)thermophiles, e. g. Thermotoga [Liebl, 2001], Caldicellulosiruptor [Rainey et al., 1994], and Pyrococcus [Bauer et al., 1999]. Several highly thermophilic and thermostable cellulases have been isolated and chacracterized from the hyperthermophilic anaerobic bacterium Thermotoga sp. Two thermostable endoglucanases (CelA and CelB) were purified and characterized from T. neapolitana, an extremophilic bacterium firstly isolated and described in 1986 as coming from the vicinity of a black smoker in the bay of Naples, Italy [Jannasch et al., 1988; Bok et al., 1998]. Highly active cellulases were also characterized from T. maritima, which was originally isolated from a hot marine sediment at Vulcano, Italy [Huber et al., 1986]. Its genome sequence indicates the presence of a number of endoglucanases that can be classified according to the GH families to which they belong as Cel5A, Cel5B, Cel12A, Cel12B and Cel74, and their biochemical properties have been reported [Bronnenmeier et al., 1995; Liebl et al., 1996; Liebl, 2001]. The most recently described was the cellulase Cel74, which was expressed in E. coli as a 77.0 kDa protein. The enzyme exhibited optimal pH and temperature of 6.0 and 90°C, and was highly thermostable with a half life of 5 h at 90°C. It was most active toward barley β-glucan and, to a lesser extent, toward CMC, glucomannan and xyloglucan. The endo-mode of action of Cel74 was confirmed by the failure in hydrolyzing oligosaccharides with a degree of polymerization smaller than six units [Chhabra and Kelly, 2002].
Among the three species belonging to the genus Pyrococcus, one endoglucanase showing regions of homology with GH family 12 (EglA or Cel12) has been found in P. furiosus, a hyperthermophilic anaerobic archaeon isolated from geothermally heated marine sediments with temperatures between 90°C and 100°C at the beach of Porto Levante, Vulcano Island, Italy [Fiala and Stetter, 1986]. It has been reported that the purified recombinant Cel12 hydrolyzes β-1,4 but not β-1,3 glucosidic linkages [Bauer et al., 1999]. There has been no report so far on endoglucanases from P. abyssi, while the gene coding for an endoglucanase from P. horikoshii, originally isolated from a hydrothermal vent at a depth of 1395 meters in the Okinawa Trough in the Pacific Ocean, has been cloned and expressed in E. coli [Gonzalez et al., 1998]. The enzymatic characteristics of the gene product were examined. This enzyme (EGPh) was the first endoglucanase belonging to GH family 5 found from Pyrococcus species, and it hydrolyzed CMC, Avicel, and lichenan. The pH optimum was between 5.4 and 6.0, and the temperature optimum was higher than 97°C. The endoglucanase from P. horikoshii was highly thermostable since the residual activity was 80% after 3 h at 97°C [Ando et al., 2002]. The features of thermophilicity and thermostability make this enzyme of potential use for industrial hydrolysis of cellulose at high temperatures, particularly in biopolishing of cotton products. In fact, the desizing, that is the step to remove starch from the fabrics, is performed at temperatures of 70°C or above as amylases active at these temperatures are already available. If a hyperthermostable cellulase will be introduced, it will be possible to combine desizing and biopolishing in a single step. Very recently, the X-ray crystal structure of EGPh has been determined [Kim and Ishikawa, 2010a].
The potential for glucose production from cellulosic materials using hyperthermophilic cellulolytic enzymes from Pyrococcus sp. (endoglucanase from P. horikoshii, EGPh and β-glucosidase from P. furiosus, BGLPf) has been investigated. The findings obtained indicated that the EGPh and BGLPf mixture was a successful enzyme cocktail in totally converting phosphoric acid swollen Avicel into glucose at extremely high temperature [Kim and Ishikawa, 2010b].
A cellulolytic and thermophilic anaerobic bacterium closely related to Moorella thermoacetica (99% identity) was isolated from soil by Karita et al. [2003]. The microorganism, identified as Moorella strain F21, secretes cellulolytic enzymes after growth on ball-milled cellulose or Avicel as carbon source, and ferments cellulosic materials to organic acids.
8.2 Fungal cellulases.
Among the thermophilic Fungi, only a few number is described to be cellulase-producer (Table 9).
Table 9 – Properties of some cellulases from thermophilic Fungi.
The thermophilic filamentous fungus Humicola sp. has been known to produce several cellulases, and some of the genes have been cloned, sequenced and expressed [Takashima et al., 1997]. The cellulase system of the thermophilic fungus Humicola insolens possesses a battery of enzymes that allows the efficient utilization of cellulose. This system, homologous to that of T. reesei, contains five endoglucanases: EGI (Cel7B), EGII (Cel5), EGIII (Cel12), EGV (Cel45A), and EGVI (Cel6B) in addition to two cellobiohydrolases: CBHI (Cel7A), and CBHII (Cel6A) [Schulein, 1997]. All the endoglucanases showed optimal activity between pH 5.5 and 9.0. Cel7B was highly active in a broad pH range, retaining more than 60% activity between pH 5.0 and 10.0. Its optimal pH was 5.5. Cel45A showed optimum of pH at 9.0, whereas the remaining three cellulases had pH optima between 6.0 and 8.0. No data about optimal temperature and stability were reported. The reason why H. insolens, as well as T. reesei and many other cellulolytic microorganisms, produces so many enzymes is not completely clear. The most accredited theory recognizes a different role to each enzyme in relation to the diversity of the substrate (solubility, degree of substitution or polymerization). In order to better understand the various role of several cellulases produced by a same microorganism, the efficiency in hydrolyzing soluble cellulose (CMC) by the endoglucanases Cel5A, Cel7B and Cel45A from H. insolens was investigated by Karlsson et al. [2002], and compared with the catalytic efficiency showed by the endoglucanases Cel7B, Cel12A, and Cel45Acore from T. reesei. The hydrolysates were analyzed for production of substituted and non-substituted oligosaccharides with size exclusion chromatography and with matrix-assisted laser desorption/ionization mass spectrometry. The authors demonstrated that the enzymes clearly differ in their capacity in hydrolyzing CMC and in product formation. Cel5A form H. insolens and Cel7B from T. reesei were the most efficient enzymes able to hydrolyze the substrate significantly, whereas others were more affected by the presence of substituents on the polymeric chain. So, the use of pure cellulases allows the selective cleavage of cellulose derivatives. Besides to the above mentioned endoglucanases, H. insolens produces another cellulase of about 51.2 kDa, Avi2 (avicelase 2), which was purified from the culture medium of H. insolens FERM BP-5977, an industrial cellulase producing strain [Jensen, 2002]. The avi2 gene was cloned and sequenced. The cDNA of avi2 contained an ORF encoding 476 amino acids residues. The catalytic domain of Avi2 (residues 117-476) was very similar to the catalytic domain of cellulases belonging to GH family 6 [Moriya et al., 2003]. No data about the biochemical characterization of the enzyme were depicted.
The thermophilic fungus Chaetomium thermophile var. dissitum, was able to produce in the culture medium all the enzymes involved in cellulose breakdown, namely endoglucanase (41.0 kDa), exoglucanase (67.0 kDa) and β-glucosidase [Eriksen and Goksoyr, 1977]. In 2002, Lu et al. [2002] reported that C. thermophile secreted in the culture medium a glycosylated endocellulase with an apparent molecular weight of 67.8 kDa, as determined by SDS-PAGE. The enzyme was optimally active at pH 4.0-4.5 and 60°C, and it retained 30% activity after 60 min at 70°C.
Melanocarpus albomyces, a rare true thermophilic Ascomycete capable of growing copiously at 50°C, has been documented to produce high levels of endoglucanases under optimized culture conditions [Jatinder et al., 2006]. The endoglucanases from this Fungus have been recognized as potentially important in denim washing. In fact, the supernatant from M. albomyces worked well in biostoning, with low backstaining. Three cellulases were identified and purified to homogeneity, and two of them were endoglucanases with apparent molecular masses of 20.0 kDa (Cel45A) and 50.0 kDa (Cel7A) [Miettinen-Oinonen et al., 2004]. Cel45A was relatively heat stable retaining 70% activity after 1 h at 80°C. The temperature and pH optima were 70°C and 6.0-7.0, respectively. The enzyme had a broad range of pH activity exhibiting 80% or more of its maximum activity throughout the pH interval 4.0-9.0. Cel45A has been crystallized [Hirvonen and Papageorgiou, 2002]. Cel7A was glycosylated and showed optimal temperature between 65 and 70°C and optimal pH at 6.0. Recently, a new strain isolated from composting soils and identified as Melanocarpus sp. MTCC 3922, was demonstrated to secrete two endoglucanases (EGI and EGII). The enzymes were purified and characterized by Kaur et al. [2007]. The molecular mass of EGI was 40.0 kDa, and the enzyme showed optimal temperature and pH at 50°C and 6.0, respectively. It was highly active in the 5.0-7.0 pH range, but loss of activity was observed as the temperature was increased from 50 to 80°C. EGII has a molecular mass of 50.0 kDa, optimum of temperature for activity at 70°C, ad optimum of pH at 5.0. It was active over pHs 4.0-6.0 and 40-80°C. Similar range of pH and temperature optima were reported for endoglucanases from the thermophilic Fungi, Chaetomium thermophile var. coprophile [Ganju et al., 1990] and Myceliophthora thermophila [Roy et al., 1990]. Because of their properties, EGI and EGII could be suitable under different conditions. For example, acidic cellulases found application in the non-ionic surfactant-assisted acidic deinking of old magazines [Xia et al., 1996]. Cellulases active in the range of pH 6.0-10.0 are useful in textile industry in biostoning [Kochavi et al., 1990], and in laundry [Suominen et al., 1993].
The thermophilic Fungus Thermoascus aurantiacus produces high levels of cellulase components when grown on lignocellulosic carbon sources such as corncob and cereal straw [Khandke et al., 1989]. As these enzyme components are remarkably stable over a wide range of pH and temperatures, they appear to have great commercial potential. A major extracellular endoglucanase, with a molecular mass of 34.0 kDa, was purified and characterized [Parry et al., 2002]. It was optimally active at 70-80°C and pHs 4.0-4.4, and it was stable at pH 5.2 and up to 60°C for 48 h. At 70°C and pH 5.2 the enzyme retained 40% of the original activity after 48 h. The cellulase exhibited the highest activity toward CMC; barley β-glucan and lichenan were also hydrolyzed, but the enzyme was inactive on laminarin, confirming that it was an endoglucanase and was specific toward β-1,4 linked polysaccharides. Sequence alignment of the first 33 amino acid suggested that the endoglucanase from T. aurantiacus is a member of the subfamily A6 of the GH family 5 [Lo Leggio et al., 1997]. The gene eg1 encoding for the endoglucanase was cloned and expressed in S. cerevisiae by Hong et al. [2003].
A cellulase complex capable of degrading both soluble and insoluble cellulose has been found in the culture filtrate of the thermophillic fungus Talaromyces emersonii. When grown on media containing cellulose, this microorganism produces a complete extracellular cellulase system containing seven endocellulases, four exocellulases and three β-glucosidases [McHale and Coughlan, 1980; McHale and Coughlan, 1981]. Moloney et al. [1985] isolated and characterized the endoglucanases EGI-EGIV. Successively, McCarthy et al. [2003] identified and characterized three novel endoglucanases, namely EGV (22.9 kDa), EGVI (26.9 kDa) and EGVII (33.8 kDa). These enzymes work in an endoacting mode, exhibiting greatest activity against mixed 1,3;1,4-β-D-glucans. EGVI and EGVII displayed also activity against 1,3-β-glucan (laminarin) and therefore, are likely to belong to EC 3.2.1.6.
Since the production of microbial enzymes has a large impact on the overall microbial process economy, and T. emersonii is capable to produce high level of cellulases in shaken cultures, optimization experiments have been carried out in the last few years to further improve the cellulase production by this Fungus through the addition, to the culture medium, of cheap and readily available substrates as sugar cane bagasse. The aim was to obtain large quantities of enzymes to test their effectiveness in the textile field [Gomes et al., 2007]. Speaking more in general, T. emersonii is a Fungus able to produce an enzyme cocktail attractive for biotechnological applications since it comprises not only cellulolytic, but also amylolytic and hemicellulolytic enzymes. These fungal enzymes work at temperatures 10-20°C higher than the commercially available Trichoderma sp. enzymes, and moreover, like these mesophilic Fungus, T. emersonii also has GRAS (generally regarded as safe) status, making it safe for use in food processing [Waters et al., 2010]. Many enzyme systems from this Fungus have been described, and patents have been developed for key applications [Tuohy et al., 2007].
9. CLONING AND EXPRESSION OF CELLULASE GENES.
Recombinant DNA techniques offer powerful means to solve various problems which arise both in the development of efficient cellulose producers for commercial applications and in the studies of complex cellulolytic microbial systems. For example, cloning and expression in non cellulolytic hosts of a cellulase gene that takes part in a complex aggregate, such as the cellulosome of Clostridium thermocellum, allows to separate that cellulase from all the other components of the system and to examine its catalytic properties [Gilkes et al., 1991]. Moreover, the expression of protein fused with suitable tags permits the separation of the chimeras also when they are expressed in a cellulolytic host.
Cellulase genes have been cloned from different microbial genera into various suitable hosts.
The genes encoding cellulases are chromosomal in both Bacteria and Fungi. In the Fungi, cellulase genes are randomly distributed over the genome and each gene has its own transcription regulatory elements. However, in two exceptional cases such as for Phanerochaete chrysosphorium and Trichoderma reseei was found a genes co-localization. In P. chrysosphorium, a cluster of three cellobiohydrolase genes was found by restriction mapping and sequence analysis of library cosmid clones [Covert et al., 1992]
Recent studies revealed that also in T. reseei, a big number of the genes involved in cellulose and hemicellulose degradation is not randomly distributed over the genome but clustered in several areas located among chromosomal regions of synteny with the other groups of Sordariomycetes. Genes co-localization, that implies a coordinated gene regulation, is probably the reason of the Trichoderma elevated efficiency in cellulosic substrates degradation despite its smaller cellulolytic enzymes repertoire respect to other fungal species [Ouyang et al., 2006]. In Bacteria, cellulase genes are either scattered over the chromosome [Aubert et al., 1988] or clustered on the genome. In the species belonging to the Clostridium genus, the genes coding for the proteins which constitute the cellulosomal complexes are often co-localized in clusters. In C. thermocellum despite the fact that most of the cellulase and xylanase genes are randomly distributed, several clusters have been found, suggesting the presence of operons as units of gene regulations [Miettinen-Oinonen, 2004]. The cellulosomal genes cluster of C. cellulolyticum is composed of 12 genes which are transcribed in two large polycistronic mRNA of 14 and 12 kb [Abdou et al., 2008]. Similar arrangements have been found in C. cellulovorans where 9 genes which constitute the cbpA cellulosomal cluster are transcribed as polycistronic mRNAs of 8 and 12 Kb [Han et al., 2003].
9.1 Heterologous cloning and expression in different microbial hosts.
The cellulase genes isolated from different microbial genera have been initially cloned in E. coli [Beguin et al., 1987]. The expression of catalytically active cellulases in E. coli is generally achieved at low levels due to the absence of post-translational modifications such as the glycosylation and to the intracellular accumulation of the recombinant enzymes. Most of the cellulase genes bear a signal peptide that is not well recognized by the E. coli expression system. This often lowers the production level of the recombinant proteins or even impairs their expression.
The thermostable cellulase Cel12A from Rhodothermus marinus, that contains in its sequence a highly hydrophobic putative signal peptide, when cloned in E. coli, was produced at extremely low levels and was also cytotoxic causing an extensive cell lysis. A successful expression was achieved only cloning a deletion mutant of the cellulase gene lacking of the hydrophobic signal peptide region [Wicher et al., 2001]. A similar approach was utilized for the sso1949 gene coding for an highly acid-stable and thermostable β-endoglucanase in Sulfolobus solfataricus that bears an N-terminal signal peptide linked to the catalytic domain by a serine and threonine-rich region sequence. The expression of the active enzyme was obtained only when the putative signal peptide of 24 amino acids was deleted. The attempt to improve the over-expression, using a N-terminal deletion mutant lacking also the serine and threonine-rich region was unsuccessful [Huang et al., 2005]. In the case of the cellulase gene celB1 from Bacillus sp. (strain 186-1), the expression of the active enzyme and its exportation in the periplasm was obtained only after the substitution of its signal peptide with that of the E. coli periplasmic outer membrane protease (OmpT) [Sànchez-Torres et al., 1996 ].
Since the recombinant cellulases, as described before, are often accumulated in the cytoplasm, this results in improper protein folding, leading to the formation of insoluble aggregates known as inclusion bodies [Villaverde and Carrio, 2003]. To overcome this problem one strategy is to decrease the formation of the inclusion bodies in vivo by fusing the recombinant protein with a domain which can enhance the solubility of the chimera. This approach was adopted for the cellulosomal cellulase EngB from Clostridium cellulovorans, that has been expressed as insoluble proteins in E. coli. The expression of EngB in a soluble form was achieved by fusing its catalytic domain with the proline-threonine rich region (PT-linker) and the cellulose-binding domain (CBD) of the non cellulosomal cellulase EngD [Murashima et al., 2003]. In successive studies the CBDs of different protein from Clostridium species and the PT-linker from C. cellulovorans were studied for their capacities to improve the solubility of various recombinant cellulosomal proteins when fused to their N- or C-terminal ends. The better results were obtained with the EngD PT-linker that, fused at the C-terminal end of the recombinant cohesin domain Coh6, enhances of three folds its solubility [Xu and Foong, 2008].
Another method to avoid the intracellular accumulation of recombinant insoluble cellulases is to obtain their extracellular secretion. E. coli is very limited in its ability to secrete proteins into the extracellular environment [Pugsley, 1993]. Recombinant proteins such as endoglucanases, which are secreted by their source organisms, can be accumulated in the periplasmic space in E. coli [Missiakas and Raina, 1997]. For example, the recombinant cellulase from Fibrobacter succinogenes AR1, was successful expressed in E. coli under its own promoter and due to the signal peptide contained in its sequence, was, in the percentage of 80%, secreted in the periplasm and the remaining part was found in the extracellular medium [Cavicchioli and Watson, 1991].
A novel thermostable1,4-β-endoglucanase CelI15 from Bacillus subtilis strain I15 was expressed in E. coli BL21(DE3) with a production level three times higher than that of the wild-type strain, and it was entirely found in extracellular medium [Yang et al., 2010]. Therefore, the CelI15 extracellular signal peptide could be functional in the heterologous host.
The studies of Zhou et al. [1999] were aimed to obtain the extracellular production in E. coli of the CelZ endoglucanase from Erwinia chrysanthemi. The production of CelZ that was previously expressed in E. coli B as a periplasmic product, was enhanced using a strong promoter derived from the Zymomonas mobilis genome; morever, its extracellular secretion was obtained reconstituting in E. coli the E. chrysanthemi secretion system II encoded by the out genes [He et al., 1991]. Several other recent studies are aimed to improve the ability of E. coli strains to secrete recombinant proteins that is very useful to eliminate the additional process steps for the release of enzymatic activity in the extracellular medium [Mergulhão et al., 2005].
E. coli is generally used as the initial host organism for the isolation and expression of bacterial genes. The choice of alternative hosts is motivated by the ability to secrete proteins into the extracellular medium, by the closer evolutionary kinship that allows more efficient expression, and by the capacity to effect post-translational modification such as glycosylation [Beguin, 1990].
To obtain cellulases that both are produced in high yield and secreted into the medium, several Bacillus species have been used as host cells. Zhang et al. [2010b] studied the expression of the non cellulosomal family 48 cellulase from Clostridium phytofermentans in E. coli and B. subtilis. CpCel 48 was expressed intracellularly in a soluble active form in E. coli with and without a histidine tag fused at its C-terminal end and in a secretory active form in B. subtilis. This was the first report on the expression of a secretory family 48 glycoside hydrolase in B. subtilis obtained by cloning the CpCel 48 gene into the pP43NMK E. coli-B. subtilis shuttle expression vector in-frame with the neutral protease B (NprB) signal peptide-encoding sequence.
In the above and the two successively mentioned reports is underlined the importance of the signal peptide sequences for the secretory production yields. In the case of the Cel9 endoglucanase from Mixobacter spA1, the signal peptide was functional either in E. coli and B. subtilis where the secretory expression of the active enzyme was achieved [Avitia et al., 2000]. Celdc from Pyrocccus horikoshi, instead, was expressed only at low levels in E. coli but a high extracellular production was obtained with the host-vector system of Bacillus brevis. Celdc lacking of the N-terminal 28 aminoacids signal peptide and C-terminal 12 aminoacids was cloned in the expression-secretion vector pNU226 downstream the promoter, the translation initiation region and the modified signal peptide sequences of the middle wall protein (MWP) gene of B. brevis 47 [Sagiya et al., 1994]. Several attempts altering the sequence of the signal peptide inserting different amino acids or short peptides at the cleavage site dramatically increased the cellulase production level [Kashima and Udaka, 2004].
While B. subtilis may suffice for many applications, B. megaterium and B. stearothermophilus are attractive alternative systems which offer the advantages of increased plasmid stability [Chen et al., 2008] and growth at a higher temperature [Soutschek-Bauer and Staudenbauer, 1987], respectively.
Since the impossibility to obtain through the prokaryotic biosynthetic machinery, post-translational modifications, several cellulase genes have been expressed in eukaryotic hosts. Among the several yeast species endowed with a broad range properties useful to express recombinant cellulase genes at the correct level of post-translational maturation, Saccharomyces cerevisiae has received the most attention. Although most of the cellulases that have been successfully produced in S. cerevisiae were of fungal origin, there are reports of successful bacterial cellulase production [van Zyl et al., 2007; Van Rensburg et al., 1998; Parvez et al., 1994]
Most reports regarding the expression of cellulases and hemicellulases in yeast describes strong (or other constitutively expressed) promoters to drive expression of the heterologous genes. The promoter choice undoubtedly has a great influence on the expression levels but also leader sequences strongly affect the recombinant protein yields. In a recent report [Zhu et al., 2010], the expression of the endoglucanase Egl1 cloned from Trichoderma viride CICC3038 in S. cerevisiae was achieved replacing the native signal sequence with the mating factor α prepro-leader sequence (MFα) signal peptide. A 61.5% enhancement of the specific endoglucanase activity was obtained accompanied by a faster substrate consumption and growth rate in presence of CMC as the sole carbon source.
Apart from the production of several saccharolytic enzymes [Oin et al., 2008; Hong et al., 2003], many efforts have been made for enabling S. cerevisiae to directly ferment cellulosic biomass to ethanol. The absence of a suitable technology for bioethanol production is due to the high costs required to obtain large amounts of cellulases for cellulose hydrolysis into fermentable sugars. A good solution to solve this problem is to develop a whole cell biocatalyst able to perform cellulase production, cellulose hydrolysis and sugars fermentation in a single consolidated bioprocessing (CBP) [Wen et al., 2010; Lynd et al., 2002]. In order to realize such CBP technology, engineering ethanologenic microorganisms to heterologously express a functional cellulase system is the most promising strategy. S. cerevisiae is one of the best candidates to realize a CBP mainly due to its superior traits, including high ethanol yield and tolerance, robustness in industrial fermentation, a wide variety of genetic engineering tools and safe status [Van Zyl et al., 2007]. Several research groups have expressed into S. cerevisiae different types of carbohydrate active enzymes in a free form mimicking a rudimentary cellulase system [Den Haan et al., 2007]. However, the best way to overcome the recalcitrant nature of cellulose and obtain the highest conversion yields into ethanol is to express the hydrolytic enzymes in a cellulosome fashion where the different enzymes work together synergistically [Mingardon et al., 2007]. Recent studies exploiting the cellulosomal modular nature are aimed to make a combination of recombinant chimeric components to construct artificial cellulosomes. Chimeric scaffoldins that contain cohesins from different species and an optional CBM are incubated with hybrid cellulases (either cellulosomal or noncellulosomal) from different origins expressed as fusion protein with suitable dockerin domains. In this way it is possible to obtain the most performant cellulosomal configuration for different substrate bioprocessing integrating suitable hydrolytic enzymes at specified positions. The new generation of designer cellulosomes in which three different enzymes has been integrated into the chimeric scaffoldin are found to be considerably more active than the corresponding free enzyme.
To further improve cellulose hydrolysis, also exploiting the effect of enzyme-substrate-microbe complex synergy [Lu et al., 2006], different researchers achieved the co-displaying of single cellulases on the yeast cell surface directly or after their insertion in a minicellulosome. In the first case, Trichoderma reesei endoglucanase II and cellobiohydrolase II, and Aspergillus aculeatus β-glucosidase I were simultaneously co-displayed as individual fusion proteins with the C-terminal-half region of α-agglutinin. The co-displaying of all three genes allowed an ethanol production of 3 g per liter after 40 h directly from the amorphous cellulose and this yield was impaired by the co-displaying of only β-glucosidase I and endoglucanase II [Fujita et al., 2004]. An improvement in both cellulose hydrolysis and ethanol production has been achieved when an entire minicellulosome has been assembled on the yeast cell surface. Tsai et al. [2009] showed the assemblage of an entire functional minicellulosome on the cell surface of S. cerevisiae. A trifunctional miniscaffoldin, consisting of an internal CBD flanked by three divergent cohesin domains from C. thermocellum, C. cellulolyticum, and Ruminococcus flavefaciens, was expressed and displayed on S. cerevisiae cell surface by using the GPI anchor linked at the N-terminal side of the scaffoldin [Boder and Wittrup, 1997]. Subsequently the incubation of the engineered S cerevisiae cells with E. coli lysates containing an endoglucanase (CelA) fused with a dockerin domain from C. thermocellum, an exoglucanase (CelE) from C. cellulolyticum fused with a dockerin domain from the same species and a β-glucosidase (BglA) from C. thermocellum tagged with the dockerin from R. flavefaciens resulted in the assembly of a functional minicellulosome on the yeast cell surface. The minicellulosome showed the synergistic effect for cellulose hydrolysis and the yeast produced ethanol directly from phosphoric acid-swollen cellulose (PASC) at a concentration of 3.5 g/l after 48 h of incubation.
The most recent studies on recombinant minicellulosomes have been made by Fei et al., [2010]. They obtained the co-expresssion of all the cellulosomal components into a recombinant S. cerevisiae host and the in vivo assembling of a functional cellulosome on the cell surface. The cell surface display of the complete trifunctional cellulosome did not require the in vitro loading onto the scaffoldin of the enzymatic components, previously produced in E. coli, but the expression of the miniscaffoldin dictated the formation of the entire complex by the high-affinity interactions between cohesins and dockerins. In this study, were successfully displayed two miniscaffoldins, CipA3 and CipA1, based on the well-characterized scaffoldin protein CipA from C. thermocellum [Gerngross et al., 1993]. CipA3, containing a cellulose-binding domain (CBD) and three cohesin modules, was designed to assemble minicellulosomes with up to three enzymatic activities while CipA1, containing a CBD and one cohesin module was designed to assemble a spatially restricted unifunctional minicellulosomes on the yeast cell surface. The enzyme components used in this study, including T. reesei EGII and CBHII and A. aculeatus BGL1, had fungal origins, and all of them were functionally previously expressed in S. cerevisiae [Fujita et al., 2004]. The capability of synthesizing a trifunctional minicellulosomes gave to the yeast cells the ability to simultaneously break down and ferment PASC to ethanol with a titer of 1.8 g per liter. This yield was higher than those obtained when the single hydrolytic enzymes were displayed on cell surface through cohesin-dockerin interactions and were spatially distributed.
In addition, several cellulase genes have also been expressed efficiently in other microbial systems such as Pichia pastoris, Humicola insolens, Streptomyces, Aspergillus oryzae [Moriya et al., 2003; Rashid et al., 2008; Wonganu et al; 2008].
9.2 Cloning and expression in plant systems.
The expression of cellulolytic enzymes in transgenic plant offers a huge economic advantage over the more traditional production system from recombinant microorganisms. Transgenic plants can express different cellulases at high levels and the production scale up requires reduced capital investments compared to those needed for the purchase and maintenance of large fermentors and associated equipments. Several research groups have investigated on the practicality of producing various cellulases in crop plants [Ziegelhoffer et al., 2009; Jin et al., 2003; Hood et al., 2007]. Cellulolytic enzymes produced directly in biomass crops make possible the utilization of this resource firstly as a bioreactor to accumulate cellulase enzymes and, subsequently, as feedstock for fuel ethanol production. The heterologous enzymes extracted both from fresh or dry transgenic crop or produced in a dedicated crop such as alfalfa [Ullah et al., 2002] can be then added to the pretreated biomass [Sticklen, 2008 Oraby et al., 2007]. As an alternative, the expression of the cellulolytic activities in plant crops, not followed by the enzyme extraction, could potentially yield the biomass more favorable for bioprocessing [Sticklen, 2007] eliminating the pretreatment costs. Cellulose breakdown can be allowed to occur in the field, providing that the temperature optimum of the cellulase is suitable, or, alternatively, the residues can be harvested after the crop, and the sugars fermented off-site, to produce ethanol.
One of the most important requirement for an economically viable utilization of cellulases produced by transgenic plant is the high level of active enzyme expression. To accomplish this task the most important problem to solve is the improvement of plant transformation techniques. Stable nuclear genetic transformation consisting in the insertion of foreign gene(s) in the plant genomes through several transformation methods, such as Agrobacterium tumefaciens or polyethylene glycol-mediated systems or, more recently developed, microprojectile bombardment technique, results in low enzyme yields ranging about 1% of total soluble proteins (TSP) [Bogorad, 2008]. In this regard, chloroplast transformation offers a great advantage with recombinant protein yields over 10% TSP, and exceptional transformants reaching as high as >40%TSP [Gray et al., 2009]. In fact, differently from the nuclear, the plastid transformation results in thousands of transgene copies per cell that are actively transcribed and translated [McKenzie, 2008]. Moreover, the enzyme stability is enhanced by the reduced exposure to proteases. In such way, transplanctomic expression allows to reach the production of high levels of active, recoverable, and intact enzyme, the accumulation of which does not compromise plant growth and development. In this context, relevant studies have been reported on the expression of the highly thermotolerant endoglucanase E1 from Acidothermus cellulolyticus. E1 has a considerable potential for a successful production in plants due to its thermostability and reduced activity at ambient temperature that allows the enzyme accumulation in the cell with minimal effects on plant growth, and its easily recovery in an active form. Direct expression of the E1 protein as holoenzyme or as catalytic domain (CD) alone has been achieved in several plants with significantly varying levels of expression. In recombinant potato lines, the expression under the leaf-specific promoter allowed an accumulation of holoenzyme up to 2.6% of TSP [Sun et al., 2007]. Recent studies are in progress regarding the E1 production in transgenic tobacco. The E1 expression in tobacco chloroplast greatly increased the cellulase production, that was obtained at levels of 12% of chloroplast TSP [Ziegelhoffer et al., 2001]. However, implementation of this system, is not straightforward because it depends on plastid transformation which is not yet possible in most plant species such as the feedstock biomass crop.
A more direct and generally applicable strategy involves expression of a nuclear transgene and targeted secretion of the gene product into the apoplast. The highest levels of accumulation have been achieved when the E1CD was secreted into the apoplast of the leaves of primary Arabidopsis thaliana transformants, reaching levels up to 25.7% of the total soluble protein content [Ziegler et al., 2000]. Attempts to reach the expression of E1 in maize biomass crop are also reported [Biswas et al., 2006].
Two Thermobifida fusca thermostable cellulases, Cel6A and Cel6B, were also expressed in tobacco varieties following chloroplast transformation. In the first attempts Yu et al. [2007] inserted cel6A and cel6B in the chloroplast genome of a nicotine-free and a nicotine-containing tobacco varieties.
Higher accumulation yield was obtained when the cel6A coding region was expressed in chloroplast of Nicotiana tabacum cv. Samsun with its start codon fused to a downstream box (DB) region [Gray et al., 2009]. The best results were obtained with tetanus toxin fragment C (TetC) DB region that allowed level of TetC-Cel6A accumulation of 10% of chloroplast TSP. These values have improved the accumulation of Cel6A over 100-fold respect to the yield of 0.1% TSP obtained upon the nuclear Cel6A expression [Ziegelhoffer et al., 1999].
The expression of the EgI endoglucanase from R. albus is an example of how the expression of cellulase activity could improve some specific characteristic of transgenic plants such as the digestibility level of silage plant. The egI gene that codes for one of the major cellulolytic enzyme from the rumen bacterium R. albus, has been expressed in tobacco cells BY2. These cells expressed an intracellular catalically active EgI at a level 30-folds higher than the wild type cells. Although the expression of egI in BY2 cells did not affect their growth, the enzyme was active toward the host cell wall after cell disruption. Transgenic tobacco plants transformed with the Agrobacterium-mediated method [Sakka et al., 2000] expressed also a strong CMC degrading activity. The transgenic tobacco plants were morphologically similar to the wild type plants grown in the same conditions but EgI was able to enhance the degradation of the plant tissue by macerating the plants. If this type of transgenic grass is fed to cattle, the cellulase released from the cells by mastication and disruption should degrade the cellulosic compounds enhancing the grass digestibility.
9.3 Cloning and expression in Bombix mori cells and larvae through the baculovirus expression system.
The baculovirus vector system for heterologous gene expression in insect cells is the most suitable method to overcome problems such as the poor solubility and the overglycosylation of the recombinant protein produced in E. coli and yeasts hosts, respectively.
In recent reports [Zhou et al., 2010; Li et al., 2010], high cellulase expression levels of the endoglucanase EGII and EGI, from T. reesei and T. viride respectively, have been shown in the silkworm Bombyx mori cells and larvae using a baculovirus expression system. The cellulase genes have been introduced in bacmids, E. coli and Bombyx mori shuttle vectors, that consist in the baculovirus genome containing a bacterial origin of replication, a kanamycin resistance marker, a segment of DNA encoding the lacZ peptide and a targeting site for the bacterial transposon Tn7 (att-Tn7). To obtain the recombinant bacmid, the cellulase gene was firstly introduced in the multiple cloning site, flanked by the left and right bacterial transposon Tn7 sequences of a donor plasmid. After the introduction of the recombinant donor plasmid in the E. coli DH10β strain, that contain the bacmid and the helper plasmid coding for a transposase protein, the cellulose gene transpose into the att-Tn7 site in the bacmid genome. With this novel Bac-to-Bac system, the recombinant baculovirus, easily generated through gene transposition and previously propagated in E. coli, has been transfected in the B. mori BmN cells and larvae that produced high level of recombinant protein. In the case of EGI a further improvement of the cellulase yield was obtained utilizing mutant bacmid lacking the virus-encoded chitinase and cathepsin genes of B. mori nucleopolyhedrovirus. For EGII a putative yield of about 386 g per larva (equal at a concentration of about 150 mg/l) of catalitically active cellulase was reached after the baculovirus infection.
10. BIOTECHNOLOGICAL APPLICATIONS OF CELLULASES.
10.1 Cellulases in brewery and wine biotechnology.
The macerating enzymes, comprising cellulases, hemicellulases and pectinases, hydrolyze the plant cell wall and, consequently, can be used in the brewery and wine biotechnology to improve the quality of finished products and avoid the use of chemicals. Enzyme preparations are used in the brewing and distilling industries to reduce the viscosity of the mash and to improve the overall efficiency of the process. In fact, cellulolytic and hemicellulolytic enzymes allow the conversion of undigestible lignocellulosic biomass into fermentable sugars, with consequent increase of alcohol yield. The quality of the products results improved and, at the same time, the overall costs of production are reduced.
10.1.1 Beer brewing process.
Barley is the most common cereal used for the production of beer although wheat, corn, and rice are also widely used. The main processes involved in beer production include milling to reduce the size of the dry malt in order to increase the availability of the carbohydrates; mashing where water is added to the malt; lautering where spent grains are removed from the wort, boiling of the wort with flavouring hops, fermentation of the wort liquor, maturation, conditioning, filtration and packaging of the final product. The high concentration of β-glucan in the brewing process, resulting from unsuitable brewing process or low quality barley, produces high viscosity of beer, formation of gelatinous precipitate, decrease of the extract yield, and lower run-off of wort [Bamforth, 1994; Guo et al., 2010; Bhat, 2000].
In brewing process, cellulases are used during the mashing stage in order to hydrolyze excess β-glucans and reduce the viscosity, thus improving the separation of the wort from the spent grains. Oksanen et al. [1985] observed that the endoglucanase and the cellobiohydrolase from the Trichoderma cellulase system produced a large reduction of the degree of polymerization of the β-glucans, and wort viscosity. Moreover, the increased addition of enzymes used resulted in improved filtering.
A. niger, T. reesei, and P. funiculosum, which are generally recognized as food grade microorganisms, are the major source of cellulases currently used in the mashing step, as these enzymes provide technological benefit to beer manufacture [de Castro et al., 2010; Karboune et al., 2008].
An alternative solution is that production of cellulolytic enzymes, enzymatic hydrolysis of the polysaccharidic fraction, and fermentation of the resulting sugars are all combined in a single step. S. cerevisiae is a promising candidate, as it produces ethanol at high concentrations, has GRAS status, and can be easily genetically manipulated. Unfortunately, S. cerevisiae completely lacks of a cellulose-degrading enzyme system but it can be employed industrially as host for expression of heterologous celluase genes. As example, the processive endoglucanase Cel9A of T. fusca was recently produced in S. cerevisiae. In addition, to improve the cellulolytic capability of the yeast and to investigate the level of synergy among cellulases produced by a recombinant host, the cel9A gene was co-expressed with cel5A (egII) and cel7B (egI) genes of T. Reesei [van Wyk et al., 2010]. Yeast strains with acquired ability to degrade barley β-glucans and to accumulate sulfite, can improve the quality of beer. In fact, sulfite is an important component of beer because it has antioxidant and antimicrobial activities and can also forms aldehyde adducts that stabilize the flavour of beer.
10.1.2 Wine production.
Wine manufacture is a biotechnological process in which yeast cells and enzymes are indispensable for ensuring a high quality product. The use of cellulases, hemicellulases and pectinases during wine making, allows a better skin maceration, and superior color extraction, particularly important in the production of red wine; in addition, it improves clarification, filtration, and the overall quality and stability of the wine [Galante et al., 1998a]. However, it is also important to recall that studies on the effect of enzymes on wine color and anthocyanin content have led to contradictory results [Sacchi et al., 2005].
The polysaccharidic fraction of wines comes from the pecto-cellulosic cell walls of grape berries [Pellerin et al., 1996; Visal et al., 2003; Ducasse et al., 2010] , and its composition and quantities depends on the wine making process that can be changed by using different enzymes [Ayestaran et al., 2004; Guadalupe et al., 2007].
Pectinase preparations, used in wine making, were lately modified by addition of cellulases and hemicellulases in small quantities to realize a more complete breakdown of the cells with consequent fruit liquefaction in a moderately short time period [Plank and Zent, 1993]. It was demonstrated that the mixture of macerating enzymes worked better than pectinases alone in grape processing [Haight and Gump, 1994].
Since 1980, the use of a β-glucanase from Trichoderma sp. has been proposed for wine making from grapes infected by Botrytis cinerea [Dubordieu et al., 1981; Villetaz et al., 1984]. This microorganism produces a soluble high molecular mass 1,3-β-glucan with short side chains linked through 1,6-β-glycosidic bonds, thus complicating wine filtration and clarification. To overcome this obstacle, a β-glucanase from T. harzianum was identified and patented to hydrolyze glucans for resolving undesirable effects generated by the presence of B. cinerea.
10.2 Cellulases in animal feed biotechnology.
Although cellulose is the main food resource for many animal species, most omnivores and herbivores are unable to produce the cellulases by themselves; contrariwise, the ruminants live in symbiosis with cellulolytic microorganisms (mixtures of highly specialized bacteria and protozoa localized in the digestive tract) that degrade cellulose under anaerobic conditions [Kobayashi et al., 2008]
Low forage digestibility limits the intake of accessible energy for animals, comprised ruminants; in addition, it contributes to increase nutrient excretion by livestock, and prevents the possibility of using low-quality feedstuffs. As plant polysaccharides are degraded relatively slowly and incompletely than other components of feedstuff, an efficient system for the complete enzymatic hydrolysis is required for improving the use of low-quality highly fibrous silage [Graham and Inborr, 1992; Chesson and Forsberg, 1997; Ozkose et al., 2009]. Research aimed to develop animal feed from different kinds of agro-industrial waste, in order to minimize feed costs, is under way. For this purpose, several Fungi, including some species of Pleurotus, are utilized to biodegrade the vegetable residues for their use as animal feed. Pleurotus can colonize different kinds of lignocellulosic residues, such as citric bagasse and rice straw, and increases nutritional values and digestibility of these raw materials thanks to its extracellular cellulolytic and hemicellulolytic enzymes.
The addition of lignocellulolytic digestive enzymes into animal diet is widespread lately, not only for ruminants [Bowman et al., 2002], but also for non-ruminant farm animals [Carneiro et al., 2008 ] and poultry [Woyengo et al., 2008].
In this context, cellulases have a wide range of potential applications in the animal feed industry; these hydrolytic enzymes allow to increase the nutritional quality of feed, through the improvement of cell wall digestion and efficiency of feed utilization, and also contribute to cut down excessive nutrient excretion by livestock.
They can be added to the fodder, also in the early step [Zhu et al., 1999], and several fibrolytic enzyme products, used at present as feed additives in ruminant diets, were originally developed as silage additives [Lewis et al., 1996]. Often, commercial enzymes utilized in the livestock feed industry, are obtained from microbial fermentation, and enzymatic products for animal diets are obtained from both Fungi (mostly T. longibrachiatum, A. niger, A. oryzae) and Bacteria (mostly Bacillus spp.) [Pendleton, 2000; Bhat and Hazlewood, 2001 ].
In the genus Lactococcus, L. lactis is one of the main species to be considered as a dairy-product-associated bacterium [Svec and Sledacek, 2008]. In addition, it could also be used as potential silage inoculant because it is recognized as a safe microorganism and farm environment is a natural
habitat of this species. Moreover, the use of biological additives can control the amount and pattern of fermentation in forage-based silages by decreasing the populations of harmful microorganisms in the ensiled forage.
A gene encoding for a cellulase from the anaerobic rumen fungus Neocallimastix sp. was cloned and successfully expressed into two L. lactis strains (IL403 and MG1363). The transformed strains were then employed as silage additives for pre-biodegradation of the plant biomass to improve the fiber digestibility during the ensiling process [Ozkose et al., 2009 ].
Recently, a novel cellulase (CelA4) from the thermoacidophilic bacterium Alicyclobacillus sp. A4 has been purified and characterized [Bai et al., 2010]. This enzyme, highly acid stable and protease-resistant, hydrolyzes with high efficiency barley β-glucan, and under simulated gastric conditions, decreases the viscosity of barley-soybean feed to a greater extent. These properties make CelA4 a good candidate as a new commercial glucanase to improve the nutrient bioavailability of pig feed.
10.3 Cellulases in pulp and paper biotechnology.
10.3.1 Biomechanical pulping.
Mechanical pulping process is electrical energy intensive and results in low paper strength. Biomechanical pulping, defined as the enzymatic treatment of lignocellulosic materials before the mechanical pulping step, has shown at least 30% savings in electrical energy consumption, and significant improvements in paper strength properties.
The potential of enzymatic treatments has been assessed and the processes have proved successful [Gubitz et al., 1998; Bajpai, 1999]. Since biofibers were stronger than the conventional fibers, it was possible to reduce the amount of bleached softwood kraft pulp by at least 5% in the final product. Utilization of cellulases from fungal sources (T. reesei, Aspergillus sp.) [Buchert et al., 1998; Suurnakki et al., 2000] saves 33% electrical energy and significantly improves paper strength properties compared to the control. Fungal cellulases pretreatment reduced brightness, but brightness was restored to the level of bleached control with 60% more hydrogen peroxide.
A cellulase preparation produced by the ascomycete Fungus Chrysosporium lucknowense for using in the pulp and paper industry represents, at present, an attractive alternative to the well-known cellulases from Fungi like Aspergillus sp. and T. reesei for protein production on a commercial scale [Bukhtojarov et al., 2004; Hinz et al., 2009].
10.3.2 Biomodification of fibers.
In recent years the fiber biomodification has become more and more interesting because this process is environmentally friendly, consumes less energy and makes less damage to fiber than traditional process, also improving drainage, beatability and runnability of paper mills [Pellinen et al., 1989; Henriksson and Gatenholm, 2002; Yang et al., 2008]. For this purpose, cellulases together with other enzymes like hemicellulases can be used [Bhat, 2000; Noe et al., 1986]. As example, an endoglucanase from T. reesei provided with a dual activity on xylan and cellulose was utilized in fiber biomodification [Pere et al., 1995], and it showed a drainage improvement of 30% compared to the endoglucanase from same microorganism specific only for cellulose.
As cellulases used in the fiber biomodification can act on the surface and into the inner layers of cellulose fibers, a careful study on their mechanism of action has been done in the last years [Suurnakki et al., 2003]. The aim was to understand the changes produced on fibers in order to obtain a final product provided with better quality, namely improvement of fiber-fiber bonds with consequently better cohesion between the fibers in the finished product, and to lower the production costs. Particularly, Cadena et al. [2010] studied the endoglucanase cel9B from Paenibacillus barcinonensis in biopulping refining to investigate the ability of this multidomain enzyme to improve the paper strength property and reduce production costs.
10.3.3 Biodeinking.
All over the world people offer more attention to the environment and so, the recycle of waste paper has to be considered also as a necessity for the protection of forest and economy. Paper mill will gain profit from the utilization of recycled fiber, since it is profitable to decrease pollution, cost, and investment.
Conventional deinking technology with alkali is characterized by a low efficiency on laser printed paper and is not retained environmentally friendly. Consequently, researchers have concentrated their attention to new deinking technologies [Moon and Nagarajan, 1998]. The principle of enzymatic deinking is based on the weakening of the connections between toner and fibers due to the enzyme attack with separation of toner particles from fibers [Yingjuan et al., 2005; Shufang et al., 2005]. The enzymatic deinking allows to avoid the use of alkali; moreover, using enzymes at acidic pH it is possible to prevent the yellowing, modify the distribution of the ink particle size, improve fiber brightness strength, pulp freeness and cleanliness, reduce fine particles and reduce environmental pollution.
Until 2000, the use of enzymes to perform biodeinking was only investigated at the laboratory scale [Buchert et al., 1998; Bhat, 2000]. Subsequently, a mixture of cellulase, lipase, and amylase was employed in biodeinking process at industrial level [Morbak and Zimmermann, 1998].
The effect of combined deinking technology with ultrasounds, UV irradiation and enzyme on laser printed paper was investigated. The results confirmed that the dose of alkali can be reduced using biodeinking technology. Cellulases from different microorganisms such as A. niger, T. reesei, Humicola insolens, Myceliophtora fergusii, Chrysosporium lucknowense, Fusarium sp. were used for this purpose [Marques et al., 2003].
10.4 Cellulases in food biotechnology.
10.4.1 Fruit and vegetable juices.
The fruit and vegetable juices consumption in Europe, Australia, New Zealand and the USA has increased in recent years and therefore, have a significant importance from a commercial standpoint. Juices are consumed by a wide range of consumers throughout the year, for the availability of nutritious components from fruit and vegetables, but also for their perceived health benefits [Lampe, 1999; Kurowska et al., 2000]. For example, orange juice is rich in vitamin C, folic acid, potassium, and it is an excellent source of bioavailable antioxidant phytochemicals [Franke et al., 2005]. Juice is the liquid that is naturally contained in fruit or vegetable tissues. It is prepared by extraction, which involves maceration followed by pressing or decanting, to separate the juice from the solid, followed by clarification and stabilization. When fruit industries began to produce juice, the yields were low, and many difficulties were encountered in filtering the juice to an acceptable clarity. Enzymes can play a key role in this process improving yield, clarity and stability of the juice, and the addition of “macerating enzymes” is constantly increasing [Askar, 1998; Bhat, 2000; Dongowski and Sembries, 2001]. This mixture consists of a multi-enzyme system comprising proteases, amylases, pectinases, cellulases, hemicellulases and lysozyme from food-grade microorganisms (A. niger and Trichoderma sp.) useful in breaking the fruit tissues to release more juice. The enzymatic process of fruit juice production is claimed to offer a number of advantages over mechanical-thermal procedure. In particular, the use of cellulases and pectinases is an integral part of modern fruit processing technology involving treatment of fruit mashes as these enzymes not only facilitate easy pressing, but also increase juice recovery. In addition, they ensure the highest possible quality of end products such as aroma, phenolic components content and absence of cloudiness [Buchert et al., 2005; Ramadan and Thomas, 2007]. Kapasakalidis et al. [2009] tried to enhance cell wall degradation from black currant pomace by including a “cellulase-assisted” hydrolysis step as an essential treatment for the production of polyphenol-rich extracts that could be further processed for the manufacture of dietary supplements or food additives. For this purpose, a commercial preparation of cellulase from T. reesei was used. The enzyme treatment significantly increased plant cell wall polysaccharide degradation as well as enhanced the availability of phenols for subsequent methanolic extraction.
10.4.2 Olive oil.
Olive oil production is very important because it is an old tradition dating back a thousand years and represents one of the most interesting fields of Italian agriculture. It is important to note that the virgin olive oil is a healthy fat due to its high content of oleic acid and antioxidants, particularly phenolic compounds [Manna et al., 1999]. Nowadays, the olive oil extraction is carried out with technological industrial processes (continuous or discontinuous), although the quality and the quantity of the obtained oil are still to be improved. A way for trying to solve the problem could be the utilization of biotechnology in olive oil industry, also considering eco-sustainability and lower environmental impact of the enzymes [Voragen et al., 2001; Chiacchierini et al., 2007].
Extraction of olive oil involves: (1) crushing and grinding of olives in a stone or hammer mill; (2) passing the minced olive paste through a series of malaxeurs and horizontal decanters; (3) high-speed centrifugation to recuperate the oil. To obtain a product of high quality it is very important to utilize freshly picked, clean and not fully mature fruit, under cold pressing conditions. However, high amounts of oil have also been obtained with fully ripened fruits processed at temperatures higher than room temperature, but this leads to a worse quality. The oil has high acidity, rancidity and poor aroma [Galante et al., 1998a; Garcia et al., 2001 4, 5]. Specifically, during extraction the content of some components is significantly modified according to the extraction technique employed [Amirante et al., 2001], while new components can be formed, as a result of chemical and/or enzymatic pathways [Ranalli et al., 1999a]. During the last decades, the enzyme preparations are used in the processing of fruits and vegetables to improve the yield and quality of the products. Since 1980, systematic research revealed that for the efficient maceration and extraction of oil from olives no single enzyme was sufficient but pectinases, cellulases and hemicellulases were found to be very important all together for this result. The commercial enzyme preparation Olivex, a pectinase preparation with low levels of cellulase and hemicellulase from A. aculeatus, was initially used for the extraction of olive oil [Fantozzi et al., 1977]. Afterwards, a commercially available combination of enzymes from different microorganisms (Cytolase 0) consisting of pectinases from Aspergillus, cellulases and hemicellulases from Trichoderma, proved to be superior than the enzymes from a single microorganism [Ranalli et al., 1999b]. More recently, the enzymatic complex Bioliva showed to have positive effects on colour pigments and chromatic parameters [Ranalli et al., 2005; Chiacchierini et al., 2007].
10.5 Cellulases in textile and laundry biotechnology.
Since the early part of the last century, enzymes such as the cellulases have been used for a wide range of applications in textile processing in replacement of the traditional methods.
10.5.1 Biostoning and biopolishing.
Jeans manufactured from denim, are one of the world's most popular clothing items. In the late 1970s and early 1980s, industrial laundries developed methods for producing faded jeans by washing the garments with pumice stones, which partially removed the indigo dye revealing the white interior of the yarn, which leads to the faded, worn and aged appearance. This process was designated as “stone-washing”. The use of 1-2 kg stones per kg of jeans for 1 h during stone-washing met the market requirements, but caused several problems including rapid consumption of washing machines, and unsafe working conditions.
As an alternative to the stone-washing, biostoning is by far the most economical and environmental friendly way to treat denim. The cotton fabrics treated with the enzymes loose the indigo, which later is easily removed by mechanical abrasion in the wash cycle [Cavaco-Paulo, 1998; Yamada et al., 2005]. The substitution of pumice stones by an enzymatic treatment includes many advantages: washing machines lower consumption and elevated productivity, short treatment times, less intensive working conditions. Moreover, it is possible to operate in a more safe environment because pumice powder is not produced, and the process can be mechanized controlling, with the use of computer, the dosing devices of liquid cellulase preparations [Bhat, 2000].
Nevertheless, a very important problem during biostoning is the “back-staining”, namely the high propensity of the released dye to redeposit on the clothes. This process masks the overall blue/white contrast of the finished product; therefore, controlling the back-staining is essential.
Much interest of the researchers has been focused on the mechanism of cellulose adsorption of cellulases as the best cellulases to utilize for application in textile processing are those with sites on the surface of protein globule capable of binding indigo with low adsorption ability on cellulose [Galante et al., 1998b].
According to recent research, back-staining is dependent on pH value and type of enzyme [Muntazer and Sadeghian Maryan, 2010]. However, neutral and alkaline cellulases are preferred to acid cellulases due to the decrease of the staining intensity [Sinitsyn et al., 2001]. Among cellulases potentially useful in the textile industry, thermophilic cellulases have received great attention as additives in biostoning and biopolishing. The cellulase from the thermophilic fungus T. emersonii was used as additive in biopolish by treating the jute-cotton union fabrics in order to test its effectiveness [Gomes et al., 2007]. The enzyme enhanced whiteness, brightness and softness of the treated materials, and pilling and fuzziness of the treated samples were remarkably reduced without loss of tensile strength beyond acceptable limits.
In the textile wet processing, the biopolishing is usually carried out with desizing, scouring, bleaching, dyeing and finishing by utilization of cellulases. However, there are not clear indications about the best cellulase mixture to use also if, in general, less quantity of endoglucanases implies reduced loss of tissue weight [Miettinen-Oinonen and Suominen, 2002]. The use of these enzymes allow many improvements such as the removal of short fibers, surface fuzziness smooth, polished appearance, more color uniformity and brigthness, improved finishing, and fashionable effects. At last, due to increasing environmental concerns and constraints being imposed on textile industry, cellulase treatment of cotton fabrics is an environmentally friendly way of improving the property of the fabrics.
In 2007, Anish et al. [2006] isolated an endoglucanase from the alkalothermophilic bacterium Thermomonospora sp. The enzyme, used for denim biofinishing under alkaline conditions, was effective in removing hairiness with negligible weight loss and imparting softness to the fabric. Higher abrasive activity with lower back-staining was a preferred property for denim biofinishing exhibited by the Thermomonospora endoglucanase.
10.5.2 Laundry.
The most important reason to use enzymes in detergents is that they are biodegradable and a very small quantity of these inexhaustible biocatalysts can replace very large quantity of chemicals. Since detergents hold ionic and anionic surfactants, and bleaching agents (oxidizing agents) that can partially or completely denature proteins, the enzymes for laundry must be resistant to anionic surfactants and oxidizing agents.
The accumulation of microfibrils on the surface of the fabrics makes the fabrics look hairy and scatters incident light, thereby lessening the brightness of the original colors. In detergent industry, cellulases are used to remove microfibrils from the surface of cellulosic fabrics, enhancing color brightness, hand feel and dirt removal from cotton garments that during repeated washings can become fluffy and dull. As consequence, the most promising candidates should have high defibrillation capacity, such as the endoglucanases from GH family 45 which are the most used in the detergent industry. Shimonaka et al. [2006] examined the properties of GH family 45 endoglucanases from Mucorales sp. The defibrillation activities of RCE1 and RCE2 from Rhizopus oryzae, MCE1 and MCE2 from Mucor circinelloides, and PCE1 from Phycomyces nitens were much higher than those of the other GH family 45 endoglucanases.
10.6 Cellulases in bioethanol production.
The increasing concerns about environmental protection, the rising cost of fuels, the decrease of the world reserves of fossil energy and global weather change caused by increased carbon dioxide emissions, have directed scientific interest toward the production of bioethanol from renewable resources for a “greener” alternative energy which can respond to the high energy demand of the world [Li et al., 2009]. Yearly, photosynthesis produces more than 1011tons of dry plant material worldwide, and cellulose constitutes almost half of this material [Leschine, 1995].
Ethanol, also called grain alcohol, is a clear colorless liquid, biodegradable, low in toxicity which produces little environmental pollution when burns to produce carbon dioxide and water. Bioethanol is the principle fuel used as a petrol substitute for road transport vehicles, and it is produced using biological renewable resource such as the“lignocellulosic biomass” materials [Hamelinck et al., 2005; Hill et al., 2006]. The advantage over fossil fuels is that bioethanol decrease the greenhouse gas emissions which are mainly produced by the road transport system. Another usefulness of bioethanol is represented by the possibility to further reduce the amount of carbon monoxide produced by the old engines, thus improving air quality without additional costs. Moreover, very important is the ease with which this biofuel can be simply integrated into the existing road transport system since it can be mixed with conventional fuel in quantities up to 5% without the need of engine modifications.
At first, the bioenergy industry was based on the fermentation of glucose derived from food crop using conventional technologies; however, starch raw materials are not sufficient enough to meet increasing demand, and are expensive.
However, it is possible to utilize biomass from different kinds of materials at lower price. These materials, like wood, municipal solid waste, waste paper, agricultural and industrial waste are already available to produce bioethanol and are not in competition with food sources [Kim and Dale, 2004; Lin and Tanaka, 2006].
The yield of fermentable sugars, for low cost fuel production, represents a principal test in global efforts to utilize renewable resources rather than fossil fuels. The lignocellulosic biomass refers to plant biomass which is composed of cellulose, hemicelluloses and lignin. In such kinds of biomass, the chains of cellulose and hemicelluloses are embedded in a lignin matrix, which hinders their efficient degradation. Cellulose and hemicelluloses can be hydrolyzed by enzymes or chemical methods into their sugars that can subsequently be converted into bioethanol by well established fermentation technologies. In general, the production of bioethanol from lignocellulosic biomass consists in three important steps:1) pretreatment that allows delignification of the biomass to release cellulose and hemicellulose from their complex with lignin, making more accessible these polysaccharides to the enzymes so the hydrolysis could be much more effective [Mosier et al., 2005; Alvira et al., 2010];
2) saccharification: conversion of the polysaccharides into fermentable sugars. Enzymatic degradation of biomass has been extensively studied and requires the action of several enzymes acting in cooperation such as endoglucanase, β-glucosidase, xylanase, α-arabinosidase, β-xylosidase, and others;
3) fermentation by yeast or other appropriate microorganisms to obtain ethanol from the resulting mixture of hexose and pentose sugars.
Cellulases are essential for successful bioconversion of lignocellulosic biomass; thus, the search for cellulolytic enzymes is ongoing in last years, and various microorganisms of bacterial as well as fungal origin have been evaluated for their ability to degrade cellulosic substrates into glucose monomers [Kumar et al., 2008].
The primary interest in using Fungi comes from their capacity to produce significant amounts of cellulases which are secreted into the medium with following easy isolation and purification. The genera Aspergillus and Trichoderma are the most used for this purpose among the filamentous fungi genera. Already in 1950, a Trichoderma strain which produced a cellulase complex capable of degrading native cellulose was identified [Dashtban et al., 2009]. T. reesei RutC30 is known as an excellent cellulases producer, but the low content of β-glucosidase in its extract, which is required for total hydrolysis of cellulose to glucose, is pointed out as a disadvantage [Kim et al., 2003]. On the other hand, A. niger strains have been studied due to their ability to produce high levels of β-glucosidase, although the production of endoactive enzymes is deficient. In order to produce well-balanced extracts, mixed cultures from two genera are employed but, the synergism between the different groups of cellulases produced by pure cultures is often better than that observed from co-cultures. The search for new microorganisms and cellulases with potential use in lignocellulosic biomass degradation is incessantly in progress. As example, A. niger isolated from soil sampled from Ejura farms (Gahana) was used to hydrolyze corncobs, the main agrowaste from maize which accounts for 30% of its weight, into simple sugars for subsequent fermentation to bioethanol in a simultaneous saccharification and fermentation process. The highest ethanol concentration of 0.64 g per liter was recorded over the 24 h fermentation period [Zakpaa et al., 2009].
It is reported that in solid state cultivation T. reesei secretes a complex array of degradative enzymes. The production of cellulases by T. reesei F-418, cultivated on alkali treated rice straw, was recently investigated by Abd El-Zahern and Fadel, in order to produce bioethanol from rice straw, an abundant lignocellulosic waste by-product. The solid state fermentation technique was employed. After saccharification of the biomass obtained by cellulases produced from T. reesei, glucose fermentation step was conducted by S. cerevisiae SHF-5 under static condition giving 5.1% (v/v) ethanol after 24 h fermentation period [Abd El-Zaher and Fadel, 2010].
However, the isolation and characterization of glycoside hydrolases from Eubacteria are now becoming widely exploited. Bacteria often have a higher growth rate than Fungi allowing for higher recombinant production of enzymes. Moreover, bacterial glycoside hydrolases are often expressed in multi-enzyme complexes known as “cellulosome” providing better activity and synergy [Bayer et al., 2007].
It must be underlined that the drastic conditions required by many pretreatment methods such as high temperature, pressure, or low pH may generate problems when using mesophilic enzymes. In order to overcome this difficulty, microorganisms thriving in habitats characterized by extreme conditions can be taken under investigation as source of polysaccharide-degrading enzymes, since they allow to perform biotransformation processes at “non-conventional conditions” under which common enzymes are completely denatured.
Several extremophilic microorganisms belonging to Bacteria and Archaea domains produce cellulolytic strains which can be extremely resistant to environmental stresses.
Enzymes from these microorganisms can survive the harsh conditions found in the bioconversion processes, as they are resistant to high temperatures, low or high pH values, organic solvents and all common protein denaturing agents. These features make these biocatalysts a powerful tool in industrial biotransformation processes of lignocellulosic biomass degradation [Maki et al., 2009]. The exploitation of lignocellulosic biomass for the production of biofuel is potentially feasible; however, several biotechnological constraints must be overcome. One of the first requirements is the efficient production of a hydrolyzate rich in fermentable sugars; therefore, to obtain a considerable cellulose degradation, a proficient enzyme blend containing all enzymes required for total hydrolysis of the polysaccharide is required. One example is represented by the enzyme extract from the hyperthermophilic and acidophilic archaeon S. solfataricus, that contains the main glycolytic activities (namely endoglucanase and β-glucosidase) required to hydrolyze cellulose into glucose. This extract was used to hydrolyze at high temperature agro-based raw materials such as brewer’s spent grains after preliminary strong acid pretreatment. The enzyme saccharification produced high conversion of cellulose into fermentable glucose with a yield of 64% [Morana et al., 2009].
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