Detection and production condition of acetylxylan esterase from a wood-rotting fungus,Coriolus versicolor

Acetyl esterase production was detected in a wood-rotting fungus,Coriolus versicolor, by the formation of a clear zone on a double layer agar plate containing glucose β-d-pentaacetate. Two polysaccharide acetates, carboxymethyl cellulose acetate and xylan acetate, also served as detectable substrates in place of glucose acetate to form clear zone. In an esterase assay, this fungal esterase showed a higher specificity to acetylxylan than did porcine liver esterase, indicating that it is an acetylxylan esterase.     

Action of xylan deacetylating enzymes on monoacetyl derivatives of 4-nitrophenyl glycosides of β-D-xylopyranose and α-L-arabinofuranose

Measurements of esterase activity by enzyme-coupled assays on monoacetates of 4-nitrophenyl β-d-xylopyranoside and 4-nitrophenyl α-l-arabinofuranoside showed that acetylxylan esterases of families 1, 4 and 5 produced by Trichoderma reesei and Penicillium purpurogenum have a strong preference for deacetylation of position 2 in xylopyranosides. The acetylxylan esterases exhibit only weak activity on acetylated arabinofuranosides, with 2-acetate as the best substrate. Acetyl esterases of family 16 produced by the same two fungi deacetylate in xylopyranosides preferentially positions 3 and 4. Their specific activity on arabinofuranosides is also much lower than on xylopyranosides, however, substantially greater than that in the case of typical acetylxylan esterases.     

Transacetylations to carbohydrates catalyzed by acetylxylan esterase in the presence of organic solvent

Various conditions were applied to test the ability of acetylxylan esterase (AcXE) from Schizophyllum commune to catalyze acetyl group transfer to methyl beta-D-xylopyranoside (Me-beta-Xylp) and other carbohydrates. The best performance of the enzyme was observed in an n-hexane-vinyl acetate-sodium dioctylsulfosuccinate (DOSS)-water microemulsion at a molar water-detergent ratio (w(0)) of about 4-5. Although the enzyme was found to have a half-life of about 1 h in the system, more than 60% conversion of Me-beta-Xylp to acetylated derivatives was achieved. Under identical reaction conditions, the enzyme acetylated other carbohydrates such as methyl beta-D-cellobioside (Me-beta-Cel), cellotetraose, methyl beta-D-glucopyranoside (Me-beta-Glcp), 2-deoxy-D-glucose, D-mannose, beta-1,4-mannobiose, -mannopentaose, -mannohexaose, beta-1,4-xylobiose and -xylopentaose. This work is the first example of reverse reactions by an acetylxylan esterase and a carbohydrate esterase belonging to family 1.     

Glucuronoyl esterase--novel carbohydrate esterase produced by Schizophyllum commune

The cellulolytic system of the wood-rotting fungus Schizophyllum commune contains an esterase that hydrolyzes methyl ester of 4-O-methyl-d-glucuronic acid. The enzyme, called glucuronoyl esterase, was purified to electrophoretic homogeneity from a cellulose-spent culture fluid. Its substrate specificity was examined on a number of substrates of other carbohydrate esterases such as acetylxylan esterase, feruloyl esterase and pectin methylesterase. The glucuronoyl esterase attacks exclusively the esters of MeGlcA. The methyl ester of free or glycosidically linked MeGlcA was not hydrolysed by other carbohydrate esterases. The results suggest that we have discovered a new type of carbohydrate esterase that might be involved in disruption of ester linkages connecting hemicellulose and lignin in plant cell walls.     

Cloning, overexpression in Escherichia coli, and characterization of a thermostable fungal acetylxylan esterase from Talaromyces emersonii

The gene encoding an acetylxylan esterase (AXE1) from the thermophilic ascomycete Talaromyces emersonii was cloned, expressed in Escherichia coli, and characterized. This form of AXE1, rTeAXE1, exhibits increased thermostability and activity at a higher temperature than other known fungal acetyl esterases, thus having huge potential application in biomass bioconversion to high value chemicals or biofuels.     

Carbohydrate esterases of family 2 are 6-O-deacetylases

Three acetyl esterases (AcEs) from the saprophytic bacteria Cellvibrio japonicus and Clostridium thermocellum, members of the carbohydrate esterase (CE) family 2, were tested for their activity against a series of model substrates including partially acetylated gluco-, manno- and xylopyranosides. All three enzymes showed a strong preference for deacetylation of the 6-position in aldohexoses. This regioselectivity is different from that of typical acetylxylan esterases (AcXEs). In aqueous medium saturated with vinyl acetate, the CE-2 enzymes catalyzed transacetylation to the same position, i.e., to the primary hydroxyl group of mono- and disaccharides. Xylose and xylooligosaccharides did not serve as acetyl group acceptors, therefore the CE-2 enzymes appear to be 6-O-deacetylases.     

Action of different types of endoxylanases on eucalyptus xylan in situ

Most studies of the mode of action of industrially important endoxylanases have been done on alkali extracted-plant xylan. In just few cases, the native form of the polysaccharide, acetylated xylan, was used as a substrate. In this work action of xylanases belonging to three glycoside hydrolase families, GH10, GH11, and GH30 was investigated on acetylglucuronoxylan directly in hardwood cell walls. Powdered eucalyptus wood was used as xylanase substrate. Enzyme-generated fragments were characterized by TLC, MALDI ToF MS, and NMR spectroscopy. All three xylanases generated from eucalyptus wood powder acetylated xylooligosaccharides. Those released by GH10 enzyme were the shortest, and those released by GH30 xylanase were of the largest diversity. For GH30 xylanase the 4-O-methyl-D-glucuronic acid (MeGlcA) side residues function as substrate specificity determinants regardless the acetylation of the neighboring hydroxyl group. Much simpler xylooligosaccharide patterns were observed when xylanases were applied in combination with carbohydrate esterase family 6 acetylxylan esterase. In the presence of the esterase, all aldouronic acids remained 3-O-acetylated on the xylopyranosyl (Xylp) residue substituted with MeGlcA. The 3-O-acetyl group, in contrast to the acetyl groups of otherwise unsubstituted Xylp residues, does not affect the mode of action of endoxylanases, but contributes to recalcitrance of the acidic xylan fragments. The results confirm importance of acetylxylan esterases in microbial degradation of acetylated hardwood glucuronoxylan. They also point to still unresolved question of efficient enzymatic removal of the 3-O-acetyl group on MeGlcA-substituted Xylp residues negatively affecting the saccharification yields.

     

Three-dimensional structure of the catalytic core of acetylxylan esterase from Trichoderma reesei: insights into the deacetylation mechanism

Acetylxylan esterase from Trichoderma reesei removes acetyl side groups from xylan. The crystal structure of the catalytic core of the enzyme was solved at 1.9 A resolution. The core has an alpha/beta/alpha sandwich fold, similar to that of homologous acetylxylan esterase from Penicillium purpurogenum and cutinase from Fusarium solani. All three enzymes belong to family 5 of the carbohydrate esterases and the superfamily of the alpha/beta hydrolase fold. Evidently, the enzymes have diverged from a common ancestor and they share the same catalytic mechanism. The catalytic machinery of acetylxylan esterase from T. reesei was studied by comparison with cutinase, the catalytic site of which is well known. Acetylxylan esterase is a pure serine esterase having a catalytic triad (Ser90, His187, and Asp175) and an oxyanion hole (Thr13 N, and Thr13 O gamma). Although the catalytic triad of acetylxylan esterase has been reported previously, there has been no mention of the oxyanion hole. A model for the binding of substrates is presented on the basis of the docking of xylose. Acetylxylan esterase from T. reesei is able to deacetylate both mono- and double-acetylated residues, but it is not able to remove acetyl groups located close to large side groups such as 4-O-methylglucuronic acid. If the xylopyranoside residue is double-acetylated, both acetyl groups are removed by the catalytic triad: first one acetyl group is removed and then the residue is reorientated so that the nucleophilic oxygen of serine can attack the second acetyl group.     

Purification and properties of an acetylxylan esterase from Fibrobacter succinogenes S85.

An acetylxylan esterase (EC 3.1.1.6) was purified to apparent homogeneity from the nonsedimentable extracellular culture fluid of Fibrobacter succinogenes S85 grown on cellulose. This enzyme had an apparent molecular mass of 55 kDa and an isoelectric point of 4.0. The temperature and pH optima were 45 degrees C and 7.0, respectively. The apparent Km and Vmax were 2.7 mM and 9,100 U/mg, respectively, for the hydrolysis of alpha-naphthyl acetate. The enzyme cleaved acetyl residues from birchwood acetylxylan but did not hydrolyze carboxymethylcellulose, larchwood xylan, ferulic acid-arabinose-xylose polymer, p-nitrophenyl-alpha-L-arab-inofuranoside, or longer-chain naphthyl fatty acid esters. The esterase enzyme may play a role in enhancing hemicellulose degradation by F. succinogenes, thereby allowing it greater access to cellulose present in forage cell walls.     

Purification and properties of an acetylxylan esterase from Thermobifida fusca

An acetylxylan esterase from Thermobifida fusca NTU22 was purified 51-fold as measured by specific activity from crude culture filtrate by ultrafiltration concentration, Sepharose CL-6B and DEAE-Sepharose CL-6B column chromatography. The overall yield of the purified enzyme was 14.4%. The purified enzyme gave an apparent single protein band on an SDS-PAGE. The molecular mass of purified enzyme as estimated by SDS-PAGE and by gel filtration on Sepharose CL-6B was found to be 30 and 28kDa, respectively, indicating that the acetylxylan esterase from T. fusca NTU22 is a monomer. The pI value of the purified enzyme was estimated to be 6.55 by isoelectric focusing gel electrophoresis. The N-terminal amino acid sequence of the purified esterase was ANPYERGP. The optimum pH and temperature for the purified enzyme were 8.0 and 80°C, respectively. The Zn(2+), Hg(2+), PMSF and DIPF inhibited the enzyme activity. The K(m) value for p-nitrophenyl acetate and acetylxylan were 1.86μM and 0.15%, respectively. Co-operative enzymatic degradation of oat-spelt xylan by purified acetylxylan esterase and xylanase significantly increased the acetic acid liberation compared to the acetylxylan esterase action alone.     

Purification and properties of an acetylxylan esterase from Fibrobacter succinogenes S85

An acetylxylan esterase (EC 3.1.1.6) was purified to apparent homogeneity from the nonsedimentable extracellular culture fluid of Fibrobacter succinogenes S85 grown on cellulose. This enzyme had an apparent molecular mass of 55 kDa and an isoelectric point of 4.0. The temperature and pH optima were 45 degrees C and 7.0, respectively. The apparent Km and Vmax were 2.7 mM and 9,100 U/mg, respectively, for the hydrolysis of alpha-naphthyl acetate. The enzyme cleaved acetyl residues from birchwood acetylxylan but did not hydrolyze carboxymethylcellulose, larchwood xylan, ferulic acid-arabinose-xylose polymer, p-nitrophenyl-alpha-L-arab-inofuranoside, or longer-chain naphthyl fatty acid esters. The esterase enzyme may play a role in enhancing hemicellulose degradation by F. succinogenes, thereby allowing it greater access to cellulose present in forage cell walls.     

A simple method of isolation and purification of cultures of wood-rotting fungi

A simple method of isolation and purification of cultures of higher fungi, mainly wood-rotting fungi, without special requirements for the presence of nitrogencontaining nutrients is described. Parts of fruiting bodies, spores or infected wood is inoculated on Petri dishes with an agar medium of the following composition: 1.0 g KH2PO4, 0.2 g MgSO4 - 7 H2O, 0.1 g caSO4, 0.01 g Fe2(SO4)3, 10.0 g glucose, tap water to 1 litre; 20.0 g agar. This medium does not suit most of the contaminants but fungal hyphae overgrow the whole surface of the dish so that a purified culture can be obtained from parts distant from the inoculation site.     

Characteristics of adjacent family 6 acetylxylan esterases from Fibrobacter succinogenes and the interaction with the Xyn10E xylanase in hydrolysis of acetylated xylan

Acetylxylan esterase genes axe6A and axe6B located adjacent to one another on a Fibrobacter succinogenes chromosome have been separately cloned and their properties characterized. The corresponding esterases contained an N-terminal carbohydrate esterase family 6 catalytic domain (CD) and a C-terminal family 6 carbohydrate-binding module (CBM). The amino acid sequences of the CDs and CBMs were found to exhibit 52% and 40% amino acid similarity, respectively. The CDs of the two esterases exhibited the highest similarity to CDs of acetylxylan esterases: AxeA from the ruminal fungi Orpinomyces sp. and BnaA from Neocallimastix patriciarum. Axe6A and Axe6B were optimally active at neutral pH and had low K(m) values of 0.084 and 0.056 mmol x L(-1), respectively. Axe6A and Axe6B were shown to bind to insoluble cellulose and xylan and to soluble arabinoxylan. Axe6A deacetylated acetylated xylan at the same initial rate in the presence and absence of added Xyn10E xylanase from F. succinogenes, but the action of the xylanase on acetylated xylan was dependent upon the initial activity of Axe6A. The capacity of acetylxylan esterases to bind to plant cell wall polymers and to independently deacetylate xylan enabling xylanase to release xylooligo saccharides, documents the central role these enzymes have to improve access of F. succinogenes to cellulose.     

厌氧真菌菌系乙酰酯酶的特性研究

利用厌氧培养技术,采用产酶培养基,培养课题组自行构建的一组厌氧真菌菌系,使之产乙酰酯酶。采用硫酸铵分级沉淀、透析袋透析、DEAE-纤维素离子交换柱层析、Sephadex G-75凝胶过滤柱层析,分离纯化所得到乙酰酯酶,研究其酶学性质。酶活力动态分析表明,乙酰酯酶在产酶培养基上,培养至第3天酶活力达到最高。乙酰酯酶最适温度为41℃,最适pH为9．0,Mg^2＋、K^＋、Ca^2＋对酶有一定的激活作用,Fe^3＋对酶有很强的抑制作用。该厌氧真菌菌系所产的乙酰酯酶,对于发酵木质纤维素类物质具有潜在应用价值。     

Some Properties of Extracellular Acetylxylan Esterase Produced by the Yeast Rhodotorula mucilaginosa

The red yeast Rhodotorula mucilaginosa produced an esterase that accumulated in the culture supernatant on induction with triacetin. The enzyme was specific for substrates bearing an O-acetyl group, but was relatively nonspecific for the rest of the molecule, which could consist of a phenol, a monosaccharide, a polysaccharide, or an aliphatic alcohol. The esterase was more active against acetylxylan and glucose beta-d-pentaacetate than were a number of esterases from plant and animal sources, when activities on 4-nitrophenyl acetate were compared. The enzyme exhibited Michaelis-Menten kinetics and was active over a broad pH range (5.5 to 9.2), with an optimum between pH 8 and 10. In addition, the enzyme retained its activity for 2 h at 55 degrees C. The yeast that produced the enzyme did not produce xylanase and, hence, is of interest for the production of acetylxylan esterase that is free of xylanolytic activity.     

Esterase Activities of Fibrobacter succinogenes subsp. succinogenes S85

Cells of the anaerobic ruminal bacterium Fibrobacter succinogenes subsp. succinogenes S85 (formerly Bacteroides succinogenes) exhibit arylesterase activity. When cells were grown on cellulose, it was found that 69% of the total esterase activity was extracellular while 65% was nonsedimentable upon centrifugation of the culture supernatant at 100,000 x g. Disruption of the cells by various different methods failed to increase the esterase activity, indicating that the substrate was fully accessible to esterase enzymes in intact cells. During growth of cells with either glucose or cellulose as the sole carbon source, the increase in acetylesterase activity corresponded to an increase in cell density, suggesting constitutive production. The enzyme(s) hydrolyzed alpha-naphthyl, p-nitrophenyl, and 4-methylumbelliferyl derivatives of acetic acid; xylose tetraacetate; glucose pentaacetate; acetylxylan; and a polymer composed of ferulic acid, arabinose, and xylose in molar proportions of 1:1.1:2.2 (FAX). These data demonstrate the presence of an acetylxylan esterase and a ferulic acid esterase. The cleavage of FAX also documents the presence of an alpha-l-arabinofuranosidase.     

A new family of carbohydrate esterases is represented by a GDSL hydrolase/acetylxylan esterase from Geobacillus stearothermophilus

Acetylxylan esterases hydrolyze the ester linkages of acetyl groups at positions 2 and/or 3 of the xylose moieties in xylan and play an important role in enhancing the accessibility of xylanases to the xylan backbone. The hemicellulolytic system of the thermophilic bacterium Geobacillus stearothermophilus T-6 comprises a putative acetylxylan esterase gene, axe2. The gene product belongs to the GDSL hydrolase family and does not share sequence homology with any of the carbohydrate esterases in the CAZy Database. The axe2 gene is induced by xylose, and the purified gene product completely deacetylates xylobiose peracetate (fully acetylated) and hydrolyzes the synthetic substrates 2-naphthyl acetate, 4-nitrophenyl acetate, 4-methylumbelliferyl acetate, and phenyl acetate. The pH profiles for k(cat) and k(cat)/K(m) suggest the existence of two ionizable groups affecting the binding of the substrate to the enzyme. Using NMR spectroscopy, the regioselectivity of Axe2 was directly determined with the aid of one-dimensional selective total correlation spectroscopy. Methyl 2,3,4-tri-O-acetyl-β-d-xylopyranoside was rapidly deacetylated at position 2 or at positions 3 and 4 to give either diacetyl or monoacetyl intermediates, respectively; methyl 2,3,4,6-tetra-O-acetyl-β-d-glucopyranoside was initially deacetylated at position 6. In both cases, the complete hydrolysis of the intermediates occurred at a much slower rate, suggesting that the preferred substrate is the peracetate sugar form. Site-directed mutagenesis of Ser-15, His-194, and Asp-191 resulted in complete inactivation of the enzyme, consistent with their role as the catalytic triad. Overall, our results show that Axe2 is a serine acetylxylan esterase representing a new carbohydrate esterase family.     

Trichoderma reesei CE16 acetyl esterase and its role in enzymatic degradation of acetylated hemicellulose

Background

Trichoderma reesei CE16 acetyl esterase (AcE) is a component of the plant cell wall degrading system of the fungus. The enzyme behaves as an exo-acting deacetylase removing acetyl groups from non-reducing end sugar residues.

Methods

In this work we demonstrate this exo-deacetylating activity on natural acetylated xylooligosaccharides using MALDI ToF MS.

Results

The combined action of GH10 xylanase and acetylxylan esterases (AcXEs) leads to formation of neutral and acidic xylooligosaccharides with a few resistant acetyl groups mainly at their non-reducing ends. We show here that these acetyl groups serve as targets for TrCE16 AcE. The most prominent target is the 3-O-acetyl group at the non-reducing terminal Xylp residues of linear neutral xylooligosaccharides or on aldouronic acids carrying MeGlcA at the non-reducing terminus. Deacetylation of the non-reducing end sugar may involve migration of acetyl groups to position 4, which also serves as substrate of the TrCE16 esterase.

Conclusion

Concerted action of CtGH10 xylanase, an AcXE and TrCE16 AcE resulted in close to complete deacetylation of neutral xylooligosaccharides, whereas substitution with MeGlcA prevents removal of acetyl groups from only a small fraction of the aldouronic acids. Experiments with diacetyl derivatives of methyl β-d-xylopyranoside confirmed that the best substrate of TrCE16 AcE is 3-O-acetylated Xylp residue followed by 4-O-acetylated Xylp residue with a free vicinal hydroxyl group.

General significance

This study shows that CE16 acetyl esterases are crucial enzymes to achieve complete deacetylation and, consequently, complete the saccharification of acetylated xylans by xylanases, which is an important task of current biotechnology.     

In vitro saprotrophic basidiomycetes tolerance to pendimethalin

Pendimethalin is a dinitroaniline herbicide classified among the persistent-bioaccumulative toxics. In this paper, the tolerance to this herbicide has been studied in isolates of basidiomycetes (10 species including 9 wood-rotting and 1 litter fungi), collected in different areas of the Campania region (South Italy). The isolates were grown on two different agar media, rich and poor for the presence/absence of dextrose and NH₄NO₃, and amended with 0, 100 and 500 ppm herbicide. The mycelial growth was recorded daily, and statistical analysis of fungal growth rates, determined via linear regression, allowed us to compare the hyphal extension of various macrofungi in presence of herbicide. These data represent the adaptation capacity of the fungal organisms to the peculiar environmental situation. In amended agar, all fungi exhibited a certain tolerance to pendimethalin and, normally, the fungal growth decreased with the increasing of pollutant concentration. Nevertheless, in some cases, the difference of ground reflected the different agar media. In fact, the growth of Agrocybe aegerita, in rich agar decreased with the increase of herbicide dose, while in poor agar it increased with the pollutant. Among the examined fungi, A. aegerita was the species which resulted the most tolerant to herbicide, showing growth to about 70% of the control to the highest concentrations of pollutant and for the two different agar media.Scientific relevance of the paperNo published report is available regarding pendimethalin herbicide biodegradation with basidiomycetes; therefore this work represented the first and preliminary investigation as regards. It's directed to determine the fungal tolerance to the pollutant.     

来源于嗜碱芽孢杆菌N16-5甘露聚糖利用基因簇的乙酰酯酶AesA的克隆及性质分析*

天然来源的多糖底物上常存在乙酰基取代,特异性的乙酰酯酶能够切割这些底物上的乙酰基,从而有利于聚糖底物的进一步降解.对Bacillus sp. N16-5甘露聚糖利用基因簇上编码的乙酰酯酶AesA进行了基因克隆和异源表达,并对其酶学性质进行了研究.aesA基因长957bp,编码318个氨基酸,属于碳水化合物酯酶第7家族.AesA对4-甲基伞形酮乙酸酯(4-methylumbelliferyl-acetate)表现出较好的催化活性,金属离子Fe³⁺,Fe²⁺,Mn²⁺及Cu²⁺对AesA活性均有不同程度的促进作用.AesA与甘露聚糖酶ManA对乙酰化的甘露聚糖底物具有显著的协同作用.此项研究有助于理解嗜碱芽孢杆菌Bacillus sp.N16-5对甘露聚糖的水解机制,并且在甘露聚糖降解中具有潜在的应用前景.