Microbial xylanolytic carbohydrate esterases.

This article reviews microbial esterases participating in the degradation of the major plant hemicellulose, xylan. The main chain of this polysaccharide built of ß-1,4-glycosidically linked xylopyranosyl residues is substituted by other sugars and also partially acetylated. Besides esters of acetic acid, there are two other types of ester linkages in plant xylans. L-Arabinofuranosyl side chains form esters with phenolic acids, predominantly with ferulic acid. The dimerization of ferulic acid residues leads to cross-links connecting the hemicellulose molecules. Ferulic acid cross-links were shown to serve as covalent linkage between lignin and hemicellulose. Another cross-linking between lignin and hemicellulose is provided by esters between the xylan side residues of glucuronic or 4-O-methyl-D-glucurononic acid and lignin alcohols. Regardless of the cross-linking, the side residues prevent xylan main chains from association that leads to crystallization similar to that of cellulose. Simultaneously, xylan decorations hamper the action of enzymes acting on the main chain. The enzymatic breakdown of plant xylan, therefore, requires a concerted action of glycanases attacking the main chain and enzymes catalyzing debranching, called accessory xylanolytic enzymes including xylanolytic esterases. While acetylxylan esterases and feruloyl esterases participate directly in xylan degradation, glucuronoyl esterases catalyze its separation from lignin. The current state of knowledge of diversity, classification and structure-function relationship of these three types of xylanolytic carbohydrate esterases is discussed with emphasis on important aspects of their future research relevant to their industrial applications.

Cellulose- and xylan-degrading yeasts: Enzymes, applications and biotechnological potential.

Microbes and their carbohydrate-active enzymes are central for depolymerization of complex lignocellulosic polysaccharides in the global carbon cycle. Their unique abilities to degrade and ferment carbohydrates are also utilized in many industrial processes such as baking, brewing and production of biofuels and drugs. Effective degradation and utilization of cellulose and hemicelluloses is important for the shift towards green bioeconomy, and requires microbes equipped with proper sets of carbohydrate-active enzymes (CAZymes). Knowledge of cellulolytic and xylanolytic CAZymes has mainly been generated from bacteria and filamentous fungi, while yeasts have been largely overlooked and may represent an untapped resource in natural CAZymes with industrial relevance. Cellulose and xylan-degrading yeasts with the ability to ferment saccharides are also promising candidates for consolidated bioprocesses (CBPs), as they can degrade lignocellulose and utilize its constituents to produce desired products at the same time. Cellulolytic yeasts able to utilize insoluble crystalline cellulose are rare while xylanolytic yeasts are rather widespread in nature. The lack of particular enzymes in yeasts can be remediated by introducing the missing enzymes into strains having outstanding product-forming attributes. In this review, we provide a comprehensive overview of the cellulose- and xylan-degrading ascomycetous and basidiomycetous yeasts known to date. We describe how these yeasts can be identified through bioprospecting and bioinformatic approaches and summarize available growth and enzymatic assays for strain characterization. Known and predicted CAZymes are extensively analyzed, both in individual species and in a phylogenetic perspective. We also describe the strategies used for construction of recombinant cellulolytic and xylanolytic strains as well as current applications for polysaccharide-degrading yeasts. Finally, we discuss the great potential of these yeasts as industrial cell factories, identify open research questions and provide suggestions for future investigations.

Catalytic Diversity of GH30 Xylanases.

Catalytic properties of GH30 xylanases belonging to subfamilies 7 and 8 were compared on glucuronoxylan, modified glucuronoxylans, arabinoxylan, rhodymenan, and xylotetraose. Most of the tested bacterial GH30-8 enzymes are specific glucuronoxylanases (EC 3.2.1.136) requiring for action the presence of free carboxyl group of MeGlcA side residues. These enzymes were not active on arabinoxylan, rhodymenan and xylotetraose, and conversion of MeGlcA to its methyl ester or its reduction to MeGlc led to a remarkable drop in their specific activity. However, some GH30-8 members are nonspecific xylanases effectively hydrolyzing all tested substrates. In terms of catalytic activities, the GH30-7 subfamily is much more diverse. In addition to specific glucuronoxylanases, the GH30-7 subfamily contains nonspecific endoxylanases and predominantly exo-acting enzymes. The activity of GH30-7 specific glucuronoxylanases also depend on the presence of the MeGlcA carboxyl, but not so strictly as in bacterial enzymes. The modification of the carboxyl group of glucuronoxylan had only weak effect on the action of predominantly exo-acting enzymes, as well as nonspecific xylanases. Rhodymenan and xylotetraose were the best substrates for exo-acting enzymes, while arabinoxylan represented hardly degradable substrate for almost all tested GH30-7 enzymes. The results expand current knowledge on the catalytic properties of this relatively novel group of xylanases.

Non-Specific GH30_7 Endo-ß-1,4-xylanase from Talaromyces leycettanus.

This study describes the catalytic properties of a GH30_7 xylanase produced by the fungus Talaromyces leycettanus. The enzyme is an ando-ß-1,4-xylanase, showing similar specific activity towards glucuronoxylan, arabinoxylan, and rhodymenan (linear ß-1,3-ß-1,4-xylan). The heteroxylans are hydrolyzed to a mixture of linear as well as branched ß-1,4-xylooligosaccharides that are shorter than the products generated by GH10 and GH11 xylanases. In the rhodymenan hydrolyzate, the linear ß-1,4-xylooligosaccharides are accompanied with a series of mixed linkage homologues. Initial hydrolysis of glucuronoxylan resembles the action of other GH30_7 and GH30_8 glucuronoxylanases, resulting in a series of aldouronic acids of a general formula MeGlcA2Xyln. Due to the significant non-specific endoxylanase activity of the enzyme, these acidic products are further attacked in the unbranched regions, finally yielding MeGlcA2Xyl2-3. The accommodation of a substituted xylosyl residue in the -2 subsite also applies in arabinoxylan depolymerization. Moreover, the xylose residue may be arabinosylated at both positions 2 and 3, without negatively affecting the main chain cleavage. The catalytic properties of the enzyme, particularly the great tolerance of the side-chain substituents, make the enzyme attractive for biotechnological applications. The enzyme is also another example of extraordinarily great catalytic diversity among eukaryotic GH30_7 xylanases.

Xylanases of glycoside hydrolase family 30 - An overview.

Xylan is the most abundant hemicellulose in nature and as such it is a huge source of renewable carbon. Its bioconversion requires a battery of xylanolytic enzymes. Of them the most important are the endo-ß-1,4-xylanases which depolymerize the polysaccharide into smaller fragments. Most of the xylanases are members of glycoside hydrolase (GH) families 10 and 11, although they are classified in some other GH families. The relatively new xylanases of GH30 are of special interest. Initially, they appeared to be specific glucuronoxylanases, however, other specificities were found later among prokaryotic and in particular eukaryotic enzymes. This review gives an overview of the substrate and product specificities observed for the GH30 xylanases characterized to date. An emphasis is given to the structure-activity relationship in order to explain how minor differences in catalytic centre and its vicinity can alter catalytic properties from the endoxylanase into the reducing end xylose releasing exoxylanase or into the non-reducing end xylobiohydrolase. Biotechnological potential of the GH30 xylanases is also considered.

A novel bacterial GH30 xylobiohydrolase from Hungateiclostridium clariflavum.

Typical bacterial GH30 xylanases are glucuronoxylanases requiring 4-O-methylglucuronic acid (MeGlcA) substitution of a xylan main chain for their action. They do not exhibit a significant activity on neutral xylooligosaccharides, arabinoxylan (AraX), or rhodymenan (Rho). In this work, the biochemical characterization of the bacterial Clocl_1795 xylanase from Hungateiclostridium (Clostridium) clariflavum DSM 19732 (HcXyn30A) is presented. Amino acid sequence analysis of HcXyn30A revealed that the enzyme does not contain amino acids known to be responsible for MeGlcA coordination in the -2b subsite of glucuronoxylanases. This suggested that the catalytic properties of HcXyn30A may differ from those of glucuronoxylanases. HcXyn30A shows similar specific activity on glucuronoxylan (GX) and Rho, while the specific activity on AraX is about 1000 times lower. HcXyn30A releases Xyl2 as the main product from the non-reducing end of different polymeric and oligomeric substrates. Catalytic properties of HcXyn30A resemble the properties of the fungal GH30 xylobiohydrolase from Acremonium alcalophilum, AaXyn30A. HcXyn30A is the first representative of a prokaryotic xylobiohydrolase. Its unique specificity broadens the catalytic diversity of bacterial GH30 xylanases. KEY POINTS: • Bacterial GH30 xylobiohydrolase from H. clariflavum (HcXyn30A) has been characterized. • HcXyn30A releases xylobiose from the non-reducing end of different substrates. • HcXyn30A is the first representative of bacterial xylobiohydrolase.

A novel GH30 xylobiohydrolase from Acremonium alcalophilum releasing xylobiose from the non-reducing end.

Xylanases of the GH30 family are grouped to subfamilies GH30-7 and GH30-8. The GH30-8 members are of bacterial origin and well characterized, while the GH30-7 members are from fungal sources and their properties are quite diverse. Here, a heterologous expression and characterization of the GH30-7 xylanase AaXyn30A from a cellulolytic fungus Acremonium alcalophilum is reported. From various polymeric and oligomeric substrates AaXyn30A generates xylobiose as the main product. It was proven that xylobiose is released from the non-reducing end of all tested substrates, thus the enzyme behaves as a typical non-reducing-end acting xylobiohydrolase. AaXyn30A is active on different types of xylan, exhibiting the highest activity on rhodymenan (linear ß-1,3-ß-1,4-xylan) from which also an isomeric xylotriose Xyl-ß-1,3-Xyl-ß-1,4-Xyl is formed. Production of xylobiose from glucuronoxylan is at later stage accompanied by a release of aldouronic acids differing from those liberated by the bacterial GH30-8 glucuronoxylanases.

Positional specificity of Flavobacterium johnsoniae acetylxylan esterase and acetyl group migration on xylan main chain.

A new Flavovacterium johnsoniae isolate encodes an enzyme that is essentially identical with a recently discovered novel acetylxylan esterase, capable of liberating 3-O-acetyl group from 4-O-methyl-d-glucuronic acid-substituted xylopyranosyl (Xylp) residues (Razeq et al., 2018). In addition to deesterification of the 2-O-MeGlcA-substituted Xylp residues in acetylglucuronoxylan, the enzyme acts equally well on doubly acetylated Xylp residues from which it liberates only the 3-O-acetyl groups, leaving the 2-O-acetyl groups untouched. 3-O-Monoacetylated Xylp residues are attacked with a significantly reduced affinity. The resulting 2-O-acetylated xylan was used to investigate for the first time the migration of the 2-O-acetyl group to position 3 within the polysaccharide. In contrast to easy acetyl group migration along the monomeric xylopyranosides or non-reducing-end terminal Xylp residues of xylooligosaccharides, such a migration in the polymer required much longer heating at 100 °C. The specificity of the xylan 3-O-deacetylase was, however, no so strict on acetylated methyl and 4-nitrophenyl xylopyranosides.

Xylan from bambara and cowpea biomass and their structural elucidation.

This work is the first report on the isolation and structural elucidation of xylan from bambara and cowpea biomass. The xylans, isolated using acidic delignification followed by NaOH extraction method gave 12.3% and 13.6% yield, respectively. 1H NMR analyses revealed that both the xylans were glucuronoxylan. The presence of xylose and glucuronic acid was confirmed by monosaccharide analysis and uronic acid assay. Further, xylooligosaccharide production from bambara and cowpea xylans was carried out using xylanase from three different glycoside hydrolase families, and the products were analyzed by TLC and MALDI-ToF MS. The hydrolysis products of both xylans resembled hardwood glucuronoxylan fragments, generated under similar conditions. The most common oligosaccharides observed in the hydrolysates were Xyl2, Xyl3, MeGlcA3Xyl3, MeGlcAXyl4 and MeGlcAXyl5. A series of computational approaches were also used to study the interactions of the three different xylanases with xylan. Thus, untapped biomass such as bambara and cowpea could serve as a potential source for xylan which could further be converted to xylooligosaccharides and many other value-added chemicals.

Characterization of Acetylxylan Esterase from White-Rot Fungus Irpex lacteus.

The carbohydrate esterase family 1 (CE1) in CAZy contains acetylxylan esterases (AXEs) and feruloyl esterases (FAEs). Here we cloned a gene coding for an AXE belonging to CE1 from Irpex lacteus (IlAXE1). IlAXE1 was heterologously expressed in Pichia pastoris, and the recombinant enzyme was purified and characterized. IlAXE1 hydrolyzed p-nitrophenyl acetate, α-naphthyl acetate and 4-methylumbelliferyl acetate, however, it did not show any activity on ethyl ferulate and methyl p-coumarate. We also examined the activity on partially acetylated and feruloylated xylan extracted from corncob by hydrothermal reaction. Similarly, ferulic and p-coumaric acids were not liberated, and acetic acid was only detected in the reaction mixture. The results indicated that IlAXE1 is an acetylxylan esterase actually reacted to acetyl xylan. However, since IlAXE1 was unable to completely release acetic acid esterifying xylopyranosyl residues, it is assumed that acetyl groups exhibiting resistance to deacetylation by IlAXE1 are present in corn cob xylan.

Structural characterization of hemicellulose released from corn cob in continuous flow type hydrothermal reactor.

Hydrothermal reaction is known to be one of the most efficient procedures to extract hemicelluloses from lignocellulosic biomass. We investigated the molecular structure of xylooligosaccharides released from corn cob in a continuous flow type hydrothermal reactor designed in our group. The fraction precipitable from the extract with four volumes of ethanol was examined by 1H-NMR spectroscopy and MALDI-TOF MS before and after enzymatic treatment with different purified enzymes. The released water-soluble hemicellulose was found to correspond to a mixture of wide degree of polymerization range of acetylarabinoglucuronoxylan fragments (further as corn cob xylan abbreviated CX). Analysis of enzymatic hydrolyzates of CX with an acetylxylan esterase, GH3 ß-xylosidase, GH10 and GH11 xylanases revealed that the main chain contains unsubstituted regions mixed with regions of xylopyranosyl residues partially acetylated and occasionally substituted by 4-O-methyl-d-glucuronic acid and arabinofuranose esterified with ferulic or coumaric acid. Single 2- and 3-O-acetylation was accompanied by 2,3-di-O-acetylation and 3-O-acetylation of Xylp residues substituted with MeGlcA. Most of the non-esterified arabinofuranose side residues were lost during the hydrodynamic process. Despite reduced branching, the acetylation and ferulic acid modification of pentose residues contribute to high yields and high solubility of the extracted CX. It is also shown that different enzyme treatments of CX may lead to various types of xylooligosaccharides of different biomedical potential.

Glucuronoxylan 3-O-acetylated on uronic acid-substituted xylopyranosyl residues and its hydrolysis by GH10, GH11 and GH30 endoxylanases.

Glucuronoxylan selectively 3-O-acetylated on uronic acid-substituted xylopyranosyl residues was prepared by deacetylation of steam explosion-extracted aspenwood acetylglucuronoxylan by the CE6 acetylxylan esterase from Orpinomyces sp. The 3-O-acetylation of MeGlcA-substituted xylopyranosyl residues did not influence the mode of action of GH10, 11 and 30 xylanases, resulting in similar aldouronic acids as are found in alkali-extracted glucuronoxylan hydrolysates. In all three hydrolysates of the selectively acetylated glucuronoxylan, however, 3-O-acetylated aldouronic acids predominated over non-acetylated ones, suggesting that in native aspenwood xylan almost all MeGlcA-substituted Xylp residues are 3-O-acetylated. The results contribute to current knowledge of the mode of action of xylanases and also point to a possibility to produce novel types of xylooligosaccharides. The 3-O-acetylated aldouronic acids, along with the specifically 3-O-acetylated glucuronoxylan, may serve as model substrates for searching for a novel type of esterase able to liberate this MeGlcA-shielded acetyl group. Such esterases are important to improve significantly saccharification yields.

Glucuronoyl esterases: diversity, properties and biotechnological potential. A review.

Glucuronoyl esterases (GEs) belonging to the carbohydrate esterase family 15 (CE15) are involved in microbial degradation of lignocellulosic plant materials. GEs are capable of degrading complex polymers of lignin and hemicellulose cleaving ester bonds between glucuronic acid residues in xylan and lignin alcohols. GEs promote separation of lignin, hemicellulose and cellulose which is crucial for efficient utilization of biomass as an energy source and feedstock for further processing into products or chemicals. Genes encoding GEs are found in both fungi and bacteria, but, so far, bacterial GEs are essentially unexplored, and despite being discovered >10 years ago, only a limited number of GEs have been characterized. The first laboratory scale example of improved xylose and glucuronic acid release by the synergistic action of GE with cellulolytic enzymes was only reported recently (improved C5 sugar and glucuronic acid yields) and, until now, not much is known about their biotechnology potential. In this review, we discuss the diversity, structure and properties of microbial GEs and consider the status of their action on natural substrates and in biological systems in relation to their future industrial use.

Glucuronoxylan recognition by GH 30 xylanases: A study with enzyme and substrate variants.

XynA from Erwinia chrysanthemi (EcXyn30A), belonging to glycoside hydrolase family 30 subfamily 8, is specialized for hydrolysis of 4-O-methylglucuronoxylan (GX). Carboxyl group of 4-O-methylglucuronic acid serves as a substrate recognition element interacting ionically with positively charged Arg293 of the enzyme. We determined kinetic parameters of EcXyn30A on GX, its methyl ester (GXE) and 4-O-methylglucoxylan (GXR) and compared them with behavior of the enzyme variant in which Arg293 was replaced by Ala. The modifications of the substrate carboxyl groups resulted in several thousand-fold decrease in catalytic efficiency of EcXyn30A. In contrast, the R293A replacement reduced catalytic efficiency on GX only 18-times. The main difference was in catalytic rate (kcat) which was much lower for EcXyn30A acting on the modified substrates than for the variant which exhibited similar kcat values on all three polymers. The R293A variant cleaved GX, GXE and GXR on the second glycosidic bond from branch towards the reducing end, similarly to EcXyn30A. The R293A replacement caused 15-times decrease in specific activity on MeGlcA3Xyl4, but it did not influence low activity on linear xylooligosaccharides. Docking experiments showed that MeGlcA3Xyl4 and its esterified and reduced forms were bound to both enzymes in analogous way but with different binding energies.

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.

Microbial Glucuronoyl Esterases: 10 Years after Discovery.

A carbohydrate esterase called glucuronoyl esterase (GE) was discovered 10 years ago in a cellulolytic system of the wood-rotting fungus Schizophyllum commune Genes coding for GEs were subsequently found in a number of microbial genomes, and a new family of carbohydrate esterases (CE15) has been established. The multidomain structures of GEs, together with their catalytic properties on artificial substrates and positive effect on enzymatic saccharification of plant biomass, led to the view that the esterases evolved for hydrolysis of the ester linkages between 4-O-methyl-d-glucuronic acid of plant glucuronoxylans and lignin alcohols, one of the crosslinks in the plant cell walls. This idea of the function of GEs is further supported by the effects of cloning of fungal GEs in plants and by very recently reported evidence for changes in the size of isolated lignin-carbohydrate complexes due to uronic acid de-esterification. These facts make GEs interesting candidates for biotechnological applications in plant biomass processing and genetic modification of plants. This article is a brief summary of current knowledge of these relatively recent and unexplored esterases.

Phylogeny, classification and metagenomic bioprospecting of microbial acetyl xylan esterases.

Acetyl xylan esterases (AcXEs), also termed xylan deacetylases, are broad specificity Carbohydrate-Active Enzymes (CAZymes) that hydrolyse ester bonds to liberate acetic acid from acetylated hemicellulose (typically polymeric xylan and xylooligosaccharides). They belong to eight families within the Carbohydrate Esterase (CE) class of the CAZy database. AcXE classification is largely based on sequence-dependent phylogenetic relationships, supported in some instances with substrate specificity data. However, some sequence-based predictions of AcXE-encoding gene identity have proved to be functionally incorrect. Such ambiguities can lead to mis-assignment of genes and enzymes during sequence data-mining, reinforcing the necessity for the experimental confirmation of the functional properties of putative AcXE-encoding gene products. Although one-third of all characterized CEs within CAZy families 1-7 and 16 are AcXEs, there is a need to expand the sequence database in order to strengthen the link between AcXE gene sequence and specificity. Currently, most AcXEs are derived from a limited range of (mostly microbial) sources and have been identified via culture-based bioprospecting methods, restricting current knowledge of AcXEs to data from relatively few microbial species. More recently, the successful identification of AcXEs via genome and metagenome mining has emphasised the huge potential of culture-independent bioprospecting strategies. We note, however, that the functional metagenomics approach is still hampered by screening bottlenecks. The most relevant recent reviews of AcXEs have focused primarily on the biochemical and functional properties of these enzymes. In this review, we focus on AcXE phylogeny, classification and the future of metagenomic bioprospecting for novel AcXEs.

Towards enzymatic breakdown of complex plant xylan structures: State of the art.

Significant progress over the past few years has been achieved in the enzymology of microbial degradation and saccharification of plant xylan, after cellulose being the most abundant natural renewable polysaccharide. Several new types of xylan depolymerizing and debranching enzymes have been described in microorganisms. Despite the increasing variety of known glycoside hydrolases and carbohydrate esterases, some xylan structures still appear quite recalcitrant. This review focuses on the mode of action of different types of depolymerizing endoxylanases and their cooperation with ß-xylosidase and accessory enzymes in breakdown of complex highly branched xylan structures. Emphasis is placed on the enzymatic hydrolysis of alkali-extracted deesterified polysaccharide as well as acetylated xylan isolated from plant cell walls under non-alkaline conditions. It is also shown how the combination of selected endoxylanases and debranching enzymes can determine the nature of prebiotic xylooligosaccharides or lead to complete hydrolysis of the polysaccharide. The article also highlights the possibility for discovery of novel xylanolytic enzymes, construction of multifunctional chimeric enzymes and xylanosomes in parallel with increasing knowledge on the fine structure of the polysaccharide.

Comparison of fungal carbohydrate esterases of family CE16 on artificial and natural substrates.

The enzymatic conversion of acetylated hardwood glucuronoxylan to functional food oligomers, biochemicals or fermentable monomers requires besides glycoside hydrolases enzymes liberating acetic acid esterifying position 2 and/or 3 in xylopyranosyl (Xylp) residues. The 3-O-acetyl group at internal Xylp residues substituted by MeGlcA is the only acetyl group of hardwood acetylglucuronoxylan and its fragments not attacked by acetylxylan esterases of carbohydrate esterase (CE) families 1, 4, 5 and 6 and by hemicellulolytic acetyl esterases classified in CE family 16. Monoacetylated aldotetraouronic acid 3″-Ac(3)MeGlcA(3)Xyl3, generated from the polysaccharide by GH10 endoxylanases, appears to be one of the most resistant fragments. The presence of the two substituents on the non-reducing-end Xylp residue prevents liberation of MeGlcA by α-glucuronidase of family GH67 and blocks the action of acetylxylan esterases. The Ac(3)MeGlcA(3)Xyl3 was isolated from an enzymatic hydrolysate of birchwood acetylglucuronoxylan and characterized by (1)H NMR spectroscopy as a mixture of two positional isomers, 3″-Ac(3)MeGlcA(3)Xyl3 and 4″-Ac(3)MeGlcA(3)Xyl3, the latter being the result of acetyl group migration. The mixture was used as a substrate for three members of CE16 family of fungal origin. Trichoderma reesei CE16 esterase, inactive on polymeric substrate, deacetylated both isomers. Podospora anserina and Aspergillus niger esterases, active on acetylglucuronoxylan, deesterified effectively only the 4″-isomer. The results indicate catalytic diversity among CE16 enzymes, but also their common and unifying catalytic ability to exo-deacetylate positions 3 and 4 on non-reducing-end Xylp residues, which is an important step in plant hemicellulose saccharification.

ß-Glucuronidase-coupled assays of glucuronoyl esterases.

Glucuronoyl esterases (GEs) are microbial enzymes with potential to cleave the ester bonds between lignin alcohols and xylan-bound 4-O-methyl-d-glucuronic acid in plant cell walls. This activity renders GEs attractive research targets for biotechnological applications. One of the factors impeding the progress in GE research is the lack of suitable substrates. In this work, we report a facile preparation of methyl esters of chromogenic 4-nitrophenyl and 5-bromo-4-chloro-3-indolyl ß-D-glucuronides for qualitative and quantitative GE assay coupled with ß-glucuronidase as the auxiliary enzyme. The indolyl derivative affording a blue indigo-type product is suitable for rapid and sensitive assay of GE in commercial preparations as well as for high throughput screening of microorganisms and genomic and metagenomic libraries.