D. Wade Abbott and Alicia Lammerts van Bueren (eds.), Protein-Carbohydrate Interactions: Methods and Protocols, [605063]

45
D. Wade Abbott and Alicia Lammerts van Bueren (eds.), Protein-Carbohydrate Interactions: Methods and Protocols,
Methods in Molecular Biology, vol. 1588, DOI 10.1007/978-1-4939-6899-2_5, © Springer Science+Business Media LLC 2017Chapter 5
Colorimetric Detection of Acetyl Xylan Esterase Activities
Galina Mai-Gisondi and Emma R. Master
Abstract
Colorimetric detection of reaction products is typically preferred for initial surveys of acetyl xylan esterase
(AcXE) activity. This chapter will describe common colorimetric methods, and variations thereof, for measur –
ing AcXE activities on commercial, synthesized, and natural substrates. Whereas assays using pNP- acetate,
α-naphthyl acetate, and 4-methylumbelliferyl acetate (4MUA) are emphasized, common methods used to
measure AcXE activity towards carbohydrate analogs (e.g., acetylated p -nitrophenyl β-d- xylopyranosides)
and various acetylated xylans are also described. Strengths and limitations of the colorimetric assays are highlighted.
Key words Acetyl xylan esterase, Colorimetric assays, pNP-acetate, α-Naphthyl acetate,
4-Methylumbelliferyl acetate, Acetylated xylooligosaccharides, β-Xylosidase-coupled assay
1 Introduction
Within the carbohydrate-active enzyme (CAZy) database (www.
cazy.org), acetyl xylan esterases are currently classified into carbohy-
drate esterase (CE) families 1–7, and 16, as well as the recently pro-
posed family CE17 [1 –3]. Most of AcXEs employ a Ser-His- Asp
catalytic triad to catalyze the deacylation of substituted sugars [1 ],
although family CE4 includes enzymes that employ a metal- ion
dependent hydrolysis mechanism [4 ]. Given the lack of low cost and
well characterized acetylated xylo-oligosaccharides and xylans, broad screens for AcXE activity often begin by using p -nitrophenyl acetate
(pNP-acetate), α-naphthyl acetate, or 4MUA [5 –7]. Slight variations
to the standard assay are summarized in Table 1 . Whereas continuous
product detection is possible through assays that use p NP-acetate,
α-naphthyl acetate, and 4MUA, main limitations to using p NP-acetate
is instability at alkaline pH [8 ]. Because 4MUA is more stable under
these pH conditions, it is often the preferred substrate for determi-
nation of pH optima [7 , 8].
Certain AcXEs, such as those belonging to CE4 family, exhibit
no activity towards the chromogenic substrates described above [ 1],

46
which has motivated the application of alternative commercial
compounds (Table 2) and synthesis of additional substrate analogs
(Table 3). These substrates have also been used to uncover the
regioselectivity of AcXEs [9–14].
Following initial activity screens and determination of pH
optimum, the specific activity and kinetic parameters of AcXEs can
be determined using natural acetylated oligosaccharides and poly-
saccharides. For example, AcXE activity has been measured using
native, acetylated xylan from birchwood [5, 14, 17], beechwood
[17], and oat spelt [11], whereas oligosaccharides can be prepared through β-xylanase treatment of xylans [38–40]. Alkali extracted
xylans can also be chemically acetylated prior to their use in activity
assays [23, 41]. The release of acetic acids from these substrates is
typically measured by monitoring the drop in pH of reaction mix-
tures either directly [13, 42], or indirectly using a colorimetric pH Table 1
Summary of reported methods for reaction termination and product detection in assays containing pNP-acetate, α-naphthyl acetate, and 4MUA
Substrate Developing reagent Product detection Reference (s)
p-Nitrophenyl acetate
(pNP- acetate)
None p-nitrophenol at 410 nm [5, 9–12]
None p-nitrophenol at 420 nm [6, 13–16]
None p-nitrophenol at 405 nm [17–22]
Sodium carbonate (Na 2CO 3)p-nitrophenol at 405 nm [23]
none Acetic acid detection kit from
Boehringer Mannheim[8]
α-naphthyl acetate
Fast Garnet GBC in sodium
dodecyl sulfate; incubate at room temperature for
15 minα-naphthol in complex with
developing reagent at
560 nm[16–18, 24]
Fast Corinth V salt in sodium
acetate buffer (pH 4.3) containing Tween 20;
incubate at room
temperature for 10 minα-naphthol in complex with
developing reagent at 535 nm[25–27]
none α-naphthol directly at 321 nm [28]
4-methylumbelliferyl
acetate (4MUA)
Citric acid to decrease pH to
2–34-methylumbelliferone
(4-MU) at 354 nm[7]
Galina Mai-Gisondi and Emma R. Master

47
indicator such as bromothymol blue (BTB) [43]. Such measure-
ments have been facilitated by the establishment of commercial kits
that also permit indirect, spectrophotometric detection of acetic
acid release (e.g., acetic acid assay kit from Megazyme (K-ACET)
or the acetate colorimetric assay kit from Sigma (MAK086).
Alternatively, high performance liquid chromatography (HPLC) is widely used for direct, quantitative measurement of acetic acid Table 2
Summary of noncommercial, synthesized carbohydrate analogs used to detect AcXE activity
Substrate Reported approach to product detection Reference(s)
Acetylated methyl β- d-xylopyranosides
(e.g., 2,3,4-tri- O-acetylated
methyl-β-xylopyranoside)
Acetylated xylobiose
• Gas-liquid chromatography (GLC-MS)
• Acetic acid detection with K-ACETRM acetic acid kit from Megazyme
• TLC detection of released sugars
•
1H NMR determination of regioselectivity[2, 9, 29–31]
Deoxy and fluoro derivatives of methyl
β-d- xylopyranoside diacetates (e.g.,
2-deoxy-2-fluoro-3,4-diacetylated methyl β-
d-xylopyranoside)
• TLC detection of released sugars [32, 33]
Monoacetylated p-nitrophenyl β- d-
xylopyranosides (e.g., 2-O-acetyl nitrophenyl β-
d-xylopyranoside)
Addition of Na 2B4O7 (or N 2CO 3) followed
by p-nitrophenol detection at 405 nm[34–36]
Acetyl (Xylan) Esterase Detection

48
release, where an Aminex HPX-87 column and H 2SO 4 eluent are
commonly used [1, 5, 17, 44]. Finally, nuclear magnetic resonance
spectroscopy (NMR), including 1H NMR and 2D-NMR, can be
used to detect deacetylation products [45]. In particular, 1H NMR
has been the method of choice to confirm the regioselectivity of
AcXEs [46, 47], whereby release of acetyl groups can be continu-
ously tracked to distinguish enzyme action from spontaneous acetyl group migration [1, 34]. This chapter describes the main
substrates and methods used to measure AcXE activity, and details
five common methods for their colorimetric detection.
2 Materials
Common buffers can be used unless otherwise specified. The stock buffers described here are prepared as 500 mM solutions.
1. Substrate stock solution: 50 mM p-nitrophenyl acetate (pNP-
acetate) in dimethyl sulfoxide DMSO (see Note 1). Weigh
0.09 g of pNP-acetate (181.15 g/mol) using a digital balance
(0.1 mg readability), transfer to a 10 mL volumetric flask, and add DMSO up to the mark.2.1 Assay Using
pNP-AcetateTable 3
Summary of reported methods for reaction termination and product detection using natural substrates
Substrate Developing reagent Production detection Reference
Acetylated birchwood
xylanAddition of ice-cold ethanol
and incubation on ice to
precipitate the xylan;
removal of precipitate by
centrifugation; freeze and lyophilize supernatantThe residue is suspended in water,
acidified by addition of phosphoric acid, and then
acetate is measured by gas–
liquid chromatography[18]
Acetylated birchwood
xylanNone Acetic acid detection kit from
Boehringer Mannheim[10]
Acetylated beechwood
xylanNone Direct measurement of pH shift
and reference to a standard curve of 2 mM Na
2PO 4 buffer
(pH 7.0) containing 0–2 mM acetic acid[13]
Acetylated oat spelt
xylanNot mentioned Acetic acid detection kit from
Boehringer Mannheim[13]
Acetyl glucuronoxylan
(oligosaccharides)Denaturation of enzyme at
100 °C for 5 minCarbohydrate detection by
MALDI ToF and TLC following GH10 xylanase and esterase treatment[37]
Galina Mai-Gisondi and Emma R. Master

49
2. Product stock solution: 50 mM p-nitrophenol (pNP) in DMSO
(see Note 2). Weigh 0.07 g of pNP (139.11 g/mol) using a
digital balance (0.1 mg readability), transfer to a 10 mL volumetric flask, and add DMSO up to the mark.
3. Stock solution of the test enzyme in water or reaction buffer
(see Note 3).
1. Substrate stock solution: 100 mM α -naphthyl acetate in DMSO
(see Note 4). Weigh 0.19 g of α -naphthyl acetate (186.21 g/mol)
using a digital balance (0.1 mg readability), transfer to a 10 mL
volumetric flask, and add DMSO up to the mark.
2. Product stock solution: 100 mM α-naphthol in DMSO. Weigh
0.14 g of α-naphthol (144.17 g/mol) using a digital balance
(0.1 mg readability), transfer to a 10 mL volumetric flask, and add DMSO up to the mark.
3. Fast Corinth Salt stopping and dye solution (see Note 5):
0.01% Fast Corinth V Salt (zinc chloride double salt) in 1 M sodium acetate buffer (pH 4.3) containing 10% Tween 20. To
prepare 1 M sodium acetate buffer, prepare separately 1 M
sodium acetate and 1 M acetic acid solution and adjust pH of sodium acetate with acetic acid.
4. Stock solution of the test enzyme in water or reaction buffer
(see Note 3).
1. Substrate stock solution: 100 mM 4 MUA in dimethyl sulfoxide
(DMSO) (see Note 6). Weigh 0.22 g of 4MUA (218.2 g/mol)
using a digital balance (0.1 mg readability), transfer to a 10 mL volumetric flask, and add DMSO up to the mark.
2. Product stock solution: 100 mM 4MU in DMSO (see Note 7 ).
Weigh 0.18 g of 4MU (176.17 g/mol) using a digital balance
(0.1 mg readability), transfer to a 10 mL volumetric flask, and
add DMSO up to the mark.
3. Stopping solution: 50 mM citric acid (pH 2.2). Weigh 4.8 g of
citric acid salt (192.12 g/mol), transfer to a graduated cylinder,
and make up to 500 mL with milliQ water. Filter through
0.45 μM filter and transfer into a glass bottle.
4. Stock solution of the test enzyme in water or reaction buffer (see Note 3).
1. Substrate stock solution: 3% of acetylated oligosaccharides or polysaccharides prepared in milliQ water. Weigh 0.3 g of substrate, transfer to a 10 mL volumetric flask, and add water
up to the mark.
2. Stopping solution: 0.33 M H
2SO 4 prepared in milliQ water.
Transfer 50 mL of milliQ water to a graduated cylinder. Weight 2.2 Assay Using
α-Naphthyl Acetate
2.3 Assay Using
4MUA
2.4 Assay Using
Acetylated Natural
Substrates
Acetyl (Xylan) Esterase Detection

50
3.23 g of concentrated H 2SO 4 (98.08 g/mol), transfer into
the cylinder, and fill up to 100 mL with milliQ water.
3. Detection method: commercially available acetic acid kits (e.g.,
Acetic Acid Assay Kit (K-ACET; Megazyme) or Acetate
Colorimetric Assay Kit (MAK086; Sigma) or HPLC [5, 48].
4. Stock solution of the test enzyme in water or reaction buffer
(see Note 3).
5. Water bath with shaker.
1. Substrate stock solutions (200 mM) in DMSO: 2-O -, 3-O -, or
4-O-acetyl p-nitrophenyl β-d-xylopyranoside, and p – nitrophenyl
β-d-xylopyranoside. Weigh 0.60 g of mono-O-acetyl p-
nitrophenyl β-d-xylopyranoside (C 12H15NO 8; calculated molec-
ular weight: 301.25 g/mol) or 0.54 g of p -nitrophenyl
β-d-xylopyranoside (271.22 g/mol), using a digital balance
(0.1 mg readability), transfer to a 10 mL volumetric flask, and add DMSO up to the mark.
2. Product stock solution: 50 mM p-nitrophenol (pNP) in
DMSO. Weigh 0.07 g of pNP (139.11 g/mol) using a digital balance (0.1 mg readability), transfer to a 10 mL volumetric
flask, and add DMSO up to the mark.
3. Enzyme mixture: comprising the test enzyme (acetyl xylan ester –
ase) and 1.5 nkat (equates to ~0.09 U or 1.5 nmol/s) of a
β-xylosidase determined using p -nitrophenyl β-
d-xylopyranoside
(e.g., recombinant XlnD from Aspergillus niger (AnGH3) pro-duced in Saccharomyces cerevisiae Y293 [49– 51] or β -xylosidase
XloA (locus tag: TM0076) from T. maritima introduced into pET24d vector and expressed in E. coli DL41 [36]. Commercial
β-
d-xylosidases are also available (e.g., E-BXSR from Megazyme;
X3504 from Sigma) (see Note 8).
4. Stopping solution: saturated Na 2B4O7 (~0.12 M). Dissolve
15 g of Na 2B4O7∙10 H 2O in 350 mL milliQ water, stir for sev-
eral hours, and then decant the solution from the residual
solid.
3 Methods
1. To prepare a final reaction volume of 1 mL containing 50 mM buffer pH 6.5 (or lower pH) and 1 mM p NP-acetate (see Note 9 ),
transfer 100 μL of a 500 mM stock-buffer (e.g., sodium citrate
buffer) solution to 780 μL of milliQ water and add 20 μL of
the 50 mM pNP-acetate stock solution.
2. Control samples are prepared as above without adding enzyme.
3. Mix and incubate the solution in a preheated water bath for 5 min.2.5 The
β-Xylosidase- Coupled
Assay with 4-Nitrophenyl β-
d-Xylopyranosides
3.1 Assay Using
pNP-Acetate
Galina Mai-Gisondi and Emma R. Master

51
4. To initiate the reaction, add 100 μL of enzyme solution at an
appropriate dilution for product detection. Negative control
samples will substitute the enzyme solution for 100 μL of
water or storage buffer for the enzyme. If measuring reaction
rates, the enzyme dose must also ensure a linear relationship
between reaction time and product release (see Note 10).
5. Directly measure p-nitrophenol formation at A405 nm using a 1.5 mL cuvette; use milliQ water as the blank (see Note 11).
6. The absorbance of p -nitrophenol is pH dependent (Fig. 1 ).
Therefore, a standard curve is required for each tested pH condi-tion. The following p-nitrophenol concentrations are recom-
mended for 1 mL cuvette (i.e., 1 cm path length) at the specified pH value: pH 3: 0.75–9 mM; pH 4: 0.3–9 mM; pH 5: 0.03–0.6 mM; pH 6: 0.05–0.5 mM; pH 7: 0.01–0.1 mM; pH 8:
0.01–0.1 mM; pH 9: 0.01–0.05 mM; pH 10: 0.005–0.5 mM.
1. To prepare a final reaction volume of 2 mL containing 1 mM α-naphtyl acetate in final 50 mM buffer, pre-equilibrate 20 μL
of the 100 mM substrate stock solution with 200 μL of
500 mM buffer and 1.58 mL milliQ water at the reaction
temperature.
2. Control samples are prepared as above without adding enzyme.
3. Initiate the reaction by adding 200 μL of enzyme solution at
an appropriate dilution for product detection (see Note 10).
Negative control samples will substitute the enzyme solution for 200 μL of water or storage buffer for the enzyme.3.2 Assay Using
α-Naphthyl Acetate
pH5pH6pH7pH8
pH9pH10
pH3pH402468 10
00.40.81.2
00.61.21.8
0 0.2 0.4 0.6 0.81A405(nm)
pNP (mM)
Fig. 1 Impact of pH on standard curves generated using p-nitrophenol (pNP)
Acetyl (Xylan) Esterase Detection

52
4. Incubate the reaction mix for 10 min at a specified temperature.
5. Add 1 mL of 0.01 Fast Corinth Salt stopping and dye
solution.
6. Read absorbance at A535 nm exactly 10 min after addition of
the dye; milliQ water containing dye is used as the blank.
7. To prepare a standard curve, dilute 100 mM α-naphthol 1:10
with milliQ water and use 10 mM solution for dilution series
containing 0.01–0.1 mM α-naphtol in cuvette with 1 cm path
length after addition of stopping reagent.
1. To prepare a final reaction volume of 400 μL containing 100 mM buffer and 1 mM 4MUA, transfer 80 μL of a 500 mM
stock buffer solution to 306 μL milliQ water and add 4 μL of
the 100 mM 4MUA stock solution (see Note 12).
2. Control samples are prepared as above without adding
enzyme.
3. Mix and incubate the solution in a preheated water bath for
5 min.
4. To initiate the reaction, add 10 μL of enzyme solution at an
appropriate dilution for product detection. If measuring reac-tion rates, the enzyme dose must also ensure a linear relation
between reaction time and product release. Negative control
samples will substitute the enzyme solution for 10 μL of water
or storage buffer for the enzyme.
5. Incubate the reaction mix for 10 min at a specified temperature (see Note 10).
6. After 10 min, add 600 μL of 50 mM citric acid (pH 2.2) and vortex rigorously to stop the reaction.
7. Filter the samples through a 0.2 μm GHP Acrodiscs 13 filter
(PALL) to remove insoluble particles prior to measurement
(see Note 13).
8. Transfer filtered samples into disposable 1.5 mL polystyrene
cuvettes and read the absorbance at 354 nm; milliQ water is
used as the blank.
9. To prepare a standard curve, dilute 100 mM 4 MU stock solu-tion to 10 mM in DMSO. Use 0.01–1 mM of 4 MU for 1 cm
path length after addition of stopping solution (for 96-well plate use 0.05–2.5 mM of 4 MU without adding of stopping
solution).
1. To prepare a final reaction volume of 200 μL containing
50 mM buffer and 1.5% (w/v) oligosaccharide, mix 20 μL of 500 mM buffer, 30 μL water, and 100 μL of a 3% (w/v) oligo-
saccharide to stock solution (see Note 14).
2. Control samples are prepared as above without adding enzyme.3.3 Assay Using
4MUA
3.4 Assay Using
Acetylated Natural
Substrates
Galina Mai-Gisondi and Emma R. Master

53
3. Preheat the tubes containing substrate–buffer–water mix to
the reaction temperature for 5 min.
4. Initiate reaction by adding 50 μL enzyme dilution. Negative
control samples will substitute the enzyme solution for 50 μL
of water or storage buffer for the enzyme.
5. Incubate with shaking (80–200 rpm) at a temperature and
duration suitable for measuring reaction rates (see Note 15).
6. Detect acetic acids released using a commercial acetic acid kit
or by HPLC after stopping the reaction with 40 μL of 0.33 M H
2SO 4 or by heating at 100 °C for 10–20 min (see Note 16).
1. To prepare a final reaction volume of 250 μL containing 100 mM buffer (pH 5–6) (see Note 17) and 4 mM substrate,
pre-warm 50 μL of 500 mM buffer together with 145 μL mil-liQ water and add 5 μL of 200 mM mono-acetylated substrate
stock solution.
2. Control samples are prepared as above without adding enzyme.
3. Initiate the reaction by adding 50 μL of enzyme mixture directly after the substrate addition. Negative control samples will substitute the enzyme solution for 50 μL of water or stor –
age buffer for the enzyme. Approximately 0.16 nkat of AcXE is reported to correspond to an A
405 of 1.75 in 1 cm light path
cuvette; absorbance values over 0.1 are recommended [35].
4. Incubate the reaction mix for 10 min at 40 °C or less (see
Note 18).
5. Add 800 μL of saturated Na 2B4O7 solution (see Note 19) and
measure absorbance at 405 nm.
6. To prepare a standard curve use 0.003–0.3 mM 4-nitrophenol
for both 1 cm light path cuvette or 96 well plate after addition
of stopping solution.
4 Notes
1. Acetonitrile was alternatively used to prepare 80 mM pNP- acetate stock solution [28]. In the product sheet of Sigma-
Aldrich, methanol is recommended. Methanol (52%) was
confirmed experimentally in our group to prepare a 10 mM
pNP-acetate solution. Higher pNP-acetate concentrations or less than 52% of methanol led to precipitation. In methanol,
stock solutions can be stored for about one week at 2–8 °C
with only a small increase in free nitrophenol. By contrast, aqueous solutions should be freshly prepared each day (prod-
uct sheet of Sigma-Aldrich).
2. 10 mM pNP can also be dissolved in 40% methanol (deter –
mined experimentally by our group).3.5 The
β-Xylosidase- Coupled
Assay with 4-Nitrophenyl β-
d-Xylopyranosides
Acetyl (Xylan) Esterase Detection

54
3. The concentration of an enzyme stock solution is dependent
on the enzyme preparation and dilution required in the assay.
4. A 100 mM stock solution of α-naphthyl acetate in DMSO is
insoluble if diluted to 10 mM with water. Alternative methods
describe using 1 mM substrate in buffer (buffer is dependent
on enzyme used) [25] or 5 mM stock solution in methanol [24]. The maximal concentration of α-naphthyl acetate that is
soluble in aqueous solution is reported to be 2 mM [14].
5. Fast Corinth Salt stopping solution contains insoluble parti-
cles. However, filtration can reduce product detection. This
developing procedure is also pH sensitive.
6. Alternatively, 4MUA is reported to solubilize as a 5 mM ace-tone solution [28].
7. Use of DMSO to dissolve 4MU is recommended in the prod-
uct information sheet provided by Sigma-Aldrich. 4MU is
soluble at 1 mM in solutions containing 1% DMSO.
8. β-Xylosidase should not be capable of hydrolyzing the mono-
acetates of 4-nitrophenyl β-
d-xylopyranosides (NPh-Xyl),
should be free of deacetylating esterases, and activity on 4-nitrophenyl β-
d-xylopyranosides should not be inhibited by
acetic acid released by AcXEs.
9. Concentrations of 1–2 mM of pNP-acetate are described for this assay.
10. When determining rates of reaction, the reaction time is typically
between 5–20 min, to minimize the chance of enzyme dena-
turation and conversion of more than 10% substrate.
11. If absorbance of substrate is high, solutions containing sub-
strate but no enzyme should also be prepared as a negative reference.
12. The 4MUA assay reported by Shao et al. [7] begins by mixing
the enzyme sample with the reaction buffer prior to substrate
addition. This may be because the substrate can precipitate
once suspended in aqueous solutions, depending on substrate
concentration and reaction temperature.
13. Depending on the reaction temperature, 1 mM of substrate in
aqueous solvent may lead to precipitation, which can interfere
with product detection. In this case, residual substrate can be
removed by filtration prior to absorbance readings.
14. Acetate buffer should not be used in this assay. The final substrate concentration will depend on the sensitivity of the detection method used.
15. 200 rpm is indicated in Juturu et al. [23]. The time needed for
the reaction depends on the substrate concentration, enzyme
performance, and sensitivity of the acetic acid detection method.
Galina Mai-Gisondi and Emma R. Master

55
16. Heating to stop the reaction is preferred in the cases where pH
changes caused by addition of H 2SO 4 are not desirable. If the
reaction products will also be analyzed by NMR to determine
the regioselectivity of the enzyme, then the precautions against
acetyl group migration that are described in Notes 17 and 18
should also be applied.
17. Acetyl group migration is avoided at pH 4, and is very low
below pH 6 [34].
18. Acetyl group migration is also mitigated by running reactions below 40 °C and for short incubation times (i.e., less than 30 min) [34].
19. Saturated Na
2B4O7 solution intensifies the yellow color of
product but can also increase background detection of remain-
ing substrate.
Acknowledgments
This work was supported by a grant to G.M. from Ella and Georg
Ehrnrooth foundation, Finland and an ERC Consolidator Grant to
E.M. (BHIVE—648925). We thank Professor M. Tenkanen for her
critical review of the manuscript.
References
1. Biely P (2012) Microbial carbohydrate ester –
ases deacetylating plant polysaccharides.
Biotechnol Adv 30:1575–1588. doi:10.1016/j.biotechadv.2012.04.010
2. Alalouf O, Balazs Y, Volkinshtein M et al (2011) A new family of carbohydrate esterases is repre-sented by a GDSL hydrolase/acetylxylan ester –
ase from Geobacillus stearothermophilus. J Biol Chem 286:41993–42001. doi:10.1074/jbc.M111.301051
3. Lombard V, Golaconda Ramulu H, Drula E et al (2014) The carbohydrate-active enzymes
database (CAZy) in 2013. Nucleic Acids Res 42:D490–D495
4. Taylor EJ, Gloster TM, Turkenburg JP et al (2006) Structure and activity of two metal ion- dependent acetylxylan esterases involved in plant cell wall degradation reveals a close simi-larity to peptidoglycan deacetylases. J Biol
Chem 281:10968–10975. doi:10.1074/jbc.M513066200
5. Biely P, Puls J, Schneider H (1985) Acetyl xylan esterases in fungal cellulolytic systems. FEBS Lett 186:80–84. doi:10.1016/ 0014-5793(85)81343-0 6. Johnson KG, Fontana JD, MacKenzie CR (1988) Measurement of acetylxylan esterase in Streptomyces. Methods Enzymol 160:551–560. doi:10.1016/0076-6879(88)60168-6
7. Shao W, Wiegel J (1995) Purification and characterization of two thermostable acetyl xylan esterases from Thermoanaerobacterium sp. strain JW/SL-YS485. Appl Environ Microbiol 61:729–733
8. Christakopoulos P, Mamma D, Kekos D et al (1999) Enhanced acetyl esterase production by Fusarium oxysporum. World J Microbiol
Biotechnol 15:443–446.
doi:10.1023/A:1008936204368
9. Biely P, Côté G, Kremnický L et al (1996) Substrate specificity of acetylxylan esterase from
Schizophyllum commune: mode of action on acet-
ylated carbohydrates. Biochim Biophys Acta 1298:209–222. doi:10.1016/S0167-4838(96)
00132-X
10. Lee H, To RJ, Latta RK et al (1987) Some
properties of extracellular acetylxylan ester –
ase produced by the yeast Rhodotorula muci-
laginosa. Appl Environ Microbiol
53:2831–2834
Acetyl (Xylan) Esterase Detection

56
11. Degrassi G, Okeke BC, Bruschi CV et al (1998)
Purification and characterization of an acetyl xylan esterase from Bacillus pumilus. Appl Environ Microbiol 64:789–792
12. Chung HJ, Park SM, Kim HR et al (2002) Cloning the gene encoding acetyl xylan ester –
ase from Aspergillus ficuum and its expression
in Pichia pastoris. Enzyme Microb Technol
31:384–391. doi:10.1016/S0141-0229(02) 00122-9
13. Halgasová N, Kutejová E, Timko J (1994)
Purification and some characteristics of the acetylxylan esterase from Schizophyllum com-
mune. Biochem J 298(Pt 3):751–755
14. McDermid KP, Forsberg CW, Mackenzie CR
(1990) Purification and properties of an acetylxylan esterase from Fibrobacter succino-genes S85. Appl Environ Microbiol 56:3805–3810. doi:10.1016/j.enzmictec.2007.09.007
15. Merino-Trigo A, Sampedro L, Rodrguez- Berrocal FJ et al (1999) Activity and partial characterisation of xylanolytic enzymes in the earthworm Eisenia andrei fed on organic
wastes. Soil Biol Biochem 31:1735–1740. doi:10.1016/S0038-0717(99)00092-9
16. Bauer S, Vasu P, Persson S et al (2006) Development and application of a suite of polysaccharide- degrading enzymes for analyz-
ing plant cell walls. Proc Natl Acad Sci USA 103:11417–11422. doi:10.1073/pnas. 0604632103
17. Chungool W, Thongkam W, Raweesri P et al
(2008) Production, purification, and charac-
terization of acetyl esterase from Streptomyces sp. PC22 and its action in cooperation with xyl-anolytic enzymes on xylan degradation. World J Microbiol Biotechnol 24:549–556. doi:10.1007/s11274-007-9509-1
18. Hespell RB, O’Bryan-Shah PJ (1988) Esterase
activities in Butyrivibrio fibrisolvens strains.
Appl Environ Microbiol 54:1917–1922
19. Blum DL, Li XL, Chen H, Ljungdahl LG (1999) Characterization of an acetyl xylan ester –
ase from the anaerobic fungus Orpinomyces sp. strain PC-2. Appl Environ Microbiol 65:3990–3995
20. Westlake K, Mackie RI, Dutton MF (1987) T-2 toxin metabolism by ruminal bacteria and its effect on their growth. Appl Environ Microbiol 53:587–592
21. Navarro-Fernández J, Martínez-Martínez I,
Montoro-García S et al (2008) Characterization
of a new rhamnogalacturonan acetyl esterase from Bacillus halodurans C-125 with a new putative carbohydrate binding domain. J Bacteriol 190:1375–1382. doi:10.1128/JB.01104-07
22. Neumueller KG, Streekstra H, Gruppen H et al (2014) Trichoderma longibrachiatum acetyl xylan esterase 1 enhances hemicellulo-lytic preparations to degrade corn silage poly-saccharides. Bioresour Technol 163:64–73. doi:10.1016/j.biortech.2014.04.001
23. Juturu V, Aust C, Wu J (2013) Heterologous expression and biochemical characterization of
acetyl xylan esterase from Coprinopsis cinerea.
World J Microbiol Biotechnol 29:597–605.
doi:10.1007/s11274-012-1215-y
24. Koseki T, Furuse S, Iwano K et al (1997) An Aspergillus awamori acetylesterase: purification of the enzyme, and cloning and sequencing of the gene. Biochem J 326:485–490
25. Poutanen K, Sundberg M (1988) An acetyl esterase of Trichoderma reesei and its role in the hydrolysis of acetyl xylans. Appl Microbiol Biotechnol 28:419–424
26. Poutanen K, Sundberg M, Korte H et al
(1990) Deacetylation of xylans by acetyl ester –
ases of Trichoderma reesei. Appl Microbiol Biotechnol 33:506–510
27. He X (2003) A continuous spectrophotomet-ric assay for the determination of diamondback moth esterase activity. Arch Insect Biochem Physiol 54:68–76. doi:10.1002/arch.10103
28. Garcia-Sastre A, Villar E, Manuguerra JC et al (1991) Activity of influenza C virus O-acetylesterase with O-acetyl-containing compounds. Biochem J 273(Pt2):435–441
29. Biely P, Côté G, Kremnický L et al (1996)
Substrate specificity and mode of action of
acetylxylan esterase from Streptomyces lividans. FEBS Lett 396:257–260. doi:10.1016/0014-5793(96)01080-0
30. Biely P, Mastihubová M, Côté GL et al (2003) Mode of action of acetylxylan esterase from Streptomyces lividans: a study with deoxy and
deoxy-fluoro analogues of acetylated methyl β -d-
xylopyranoside. Biochim Biophys Acta 1622:82–
88. doi:10.1016/S0304-4165(03)00130-2
31. Horton D, Lauterback JH (1969) Relative reactivities of hydroxyl groups in carbohydrate derivatives. Specific NMR spectral assignments of acetyl groups in methyl tetra-O-acetyl-
alpha-D-glucopyranoside and related deriva-
tives. J Org Chem 34:86–92. doi:10.1021/jo00838a021
32. Mastihubová M, Biely P (2001) A common access to 2- and 3-substituted methyl β-D- xylopyranosides. Tetrahedron Lett 42:9065–9067. doi:10.1016/S0040-4039(01)01957-8
33. Mastihubová M, Biely P (2004) Deoxy and deoxyfluoro analogues of acetylated methyl beta-D-xylopyranoside-substrates for acetylx-ylan esterases. Carbohydr Res 339:2101–2110. doi:10.1016/j.carres.2004.06.001
34. Mastihubová M, Biely P (2004) Lipase- catalysed preparation of acetates of
Galina Mai-Gisondi and Emma R. Master

57
4- nitrophenyl β-D-xylopyranoside and their
use in kinetic studies of acetyl migration.
Carbohydr Res 339:1353–1360. doi:10.1016/j.carres.2004.02.016
35. Biely P, Mastihubová M, la Grange DC et al (2004) Enzyme-coupled assay of acetylxylan
esterases on monoacetylated 4-nitrophenyl
beta-D-xylopyranosides. Anal Biochem 332:109–115. doi:10.1016/j.ab.2004.04.022
36. Levisson M, Han GW, Deller MC et al (2012) Functional and structural characterization of a thermostable acetyl esterase from Thermotoga maritima. Proteins 80:1545–1559.
doi:10.1002/prot.24041
37. Biely P, Cziszárová M, Agger JW et al (2014)
Trichoderma reesei CE16 acetyl esterase and its role in enzymatic degradation of acetylated hemi-cellulose. Biochim Biophys Acta 1840:516–525. doi:10.1016/j.bbagen.2013.10.008
38. Biely P, Cziszárová M, Uhliariková I et al (2013) Mode of action of acetylxylan esterases on acetyl glucuronoxylan and acetylated oligosaccharides generated by a GH10 endox-
ylanase. Biochim Biophys Acta 1830:5075–5086. doi:10.1016/j.bbagen.2013.07.018
39. Rantanen H, Virkki L, Tuomainen P et al (2007) Preparation of arabinoxylobiose from rye xylan using family 10 Aspergillus aculeatus endo-1,4-β-d-xylanase. Carbohydr Polym 68:350–359. doi:10.1016/j.carbpol.2006.11.022
40. Pastell H, Tuomainen P, Virkki L et al (2008)
Step-wise enzymatic preparation and structural
characterization of singly and doubly substi-tuted arabinoxylo-oligosaccharides with non- reducing end terminal branches. Carbohydr Res 343:3049–3057. doi:10.1016/j.carres.2008.09.013
41. Dalrymple BP, Cybinski DH, Layton I et al
(1997) Three Neocallimastix patriciarum
esterases associated with the degradation of complex polysaccharides are members of a new family of hydrolases. Microbiology 143:2605–2614. doi:10.1099/00221287-143-8-2605
42. Pouvreau L, Jonathan MC, Kabel MA et al (2011) Characterization and mode of action of
two acetyl xylan esterases from Chrysosporium
lucknowense C1 active towards acetylated xylans. Enzyme Microb Technol 49:312–320. doi:10.1016/j.enzmictec.2011.05.010 43. Martínez-Martínez I, Montoro-García S, Lozada-Ramírez JD et al (2007) A colorimetric assay for the determination of acetyl xylan ester –
ase or cephalosporin C acetyl esterase activities using 7-amino cephalosporanic acid, cephalo-sporin C, or acetylated xylan as substrate. Anal Biochem 369:210–217. doi:10.1016/j.ab.2007.06.030
44. Cybinski DH, Layton I, Lowry JB et al (1999) An acetylxylan esterase and a xylanase expressed from genes cloned from the ruminal fungus Neocallimastix patriciarum act synergistically to degrade acetylated xylans. Appl Microbiol Biotechnol 52:221–225
45. Chong SL, Virkki L, Maaheimo H et al (2014) O-Acetylation of glucuronoxylan in Arabidopsis thaliana wild type and its change in xylan bio-synthesis mutants. Glycobiology 24:494–506. doi:10.1093/glycob/cwu017
46. Uhliariková I, Vršanská M, McCleary BV et al
(2013) Positional specifity of acetylxylan ester –
ases on natural polysaccharide: an NMR study.
Biochim Biophys Acta 1830:3365–3372. doi:10.1016/j.bbagen.2013.01.011
47. Neumüller KG, de Souza AC, van Rijn JH et al (2015) Positional preferences of acetyl ester –
ases from different CE families towards acety-
lated 4-O-methyl glucuronic acid-substituted
xylo-oligosaccharides. Biotechnol Biofuels 8:1–11. doi:10.1186/s13068-014-0187-6
48. Neumüller KG, de Souza AC, Van Rijn J et al (2013) Fast and robust method to determine phenoyl and acetyl esters of polysaccharides by quantitative 1H NMR. J Agric Food Chem
61:6282–6287. doi:10.1021/jf401393c
49. Biely P, Mastihubová M, Tenkanen M et al
(2011) Action of xylan deacetylating enzymes on monoacetyl derivatives of 4-nitrophenyl glyco-sides of β -D-xylopyranose and α -L-
arabinofuranose. J Biotechnol 151:137–142.
doi:10.1016/j.jbiotec.2010.10.074
50. Biely P, Hirsch J, la Grange DC et al (2000)
A chromogenic substrate for a beta-xylosidase- coupled assay of alpha-glucuronidase. Anal Biochem 286:289–294. doi:10.1006/abio.2000.4810
51. Topakas E, Kyriakopoulos S, Biely P et al (2010) Carbohydrate esterases of family 2 are 6-O-deacetylases. FEBS Lett 584:543–548. doi:10.1016/j.febslet.2009.11.095
Acetyl (Xylan) Esterase Detection