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Puchart V, Biely P Ref: Essays Biochem, 67:479, 2023 : PubMed ESTHER: Puchart_2023_Essays.Biochem_67_479 PubMedSearch: Puchart 2023 Essays.Biochem 67 479 PubMedID: 36468678 Abstract This article reviews microbial esterases participating in the degradation of the major plant hemicellulose, xylan. The main chain of this polysaccharide built of beta-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. Title: Structural characterization of hemicellulose released from corn cob in continuous flow type hydrothermal reactor Arai T, Biely P, Uhliarikova I, Sato N, Makishima S, Mizuno M, Nozaki K, Kaneko S, Amano Y Ref: J Biosci Bioeng, 127:222, 2019 : PubMed ESTHER: Arai_2019_J.Biosci.Bioeng_127_222 PubMedSearch: Arai 2019 J.Biosci.Bioeng 127 222 PubMedID: 30143337 Abstract 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 (1)H-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 beta-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. Title: Glucuronoyl esterases: diversity, properties and biotechnological potential. A review Monrad RN, Eklof J, Krogh K, Biely P Ref: Critical Reviews in Biotechnology, 38:1121, 2018 : PubMed ESTHER: Monrad_2018_Crit.Rev.Biotechnol_38_1121 PubMedSearch: Monrad 2018 Crit.Rev.Biotechnol 38 1121 PubMedID: 29739247 Abstract 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. Title: Phylogeny, classification and metagenomic bioprospecting of microbial acetyl xylan esterases Adesioye FA, Makhalanyane TP, Biely P, Cowan DA Ref: Enzyme Microb Technol, 93-94:79, 2016 : PubMed ESTHER: Adesioye_2016_Enzyme.Microb.Technol_93-94_79 PubMedSearch: Adesioye 2016 Enzyme.Microb.Technol 93-94 79 PubMedID: 27702488 Abstract 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. Title: Towards enzymatic breakdown of complex plant xylan structures: State of the art Biely P, Singh S, Puchart V Ref: Biotechnol Adv, 34:1260, 2016 : PubMed ESTHER: Biely_2016_Biotechnol.Adv_34_1260 PubMedSearch: Biely 2016 Biotechnol.Adv 34 1260 PubMedID: 27620948 Abstract 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 beta-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. Title: Microbial Glucuronoyl Esterases: 10 Years after Discovery Biely P Ref: Applied Environmental Microbiology, 82:7014, 2016 : PubMed ESTHER: Biely_2016_Appl.Environ.Microbiol_82_7014 PubMedSearch: Biely 2016 Appl.Environ.Microbiol 82 7014 PubMedID: 27694239 Abstract 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. Title: beta-Glucuronidase-coupled assays of glucuronoyl esterases Franova L, Puchart V, Biely P Ref: Analytical Biochemistry, 510:114, 2016 : PubMed ESTHER: Franova_2016_Anal.Biochem_510_114 PubMedSearch: Franova 2016 Anal.Biochem 510 114 PubMedID: 27452816 Abstract 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 beta-D-glucuronides for qualitative and quantitative GE assay coupled with beta-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. Title: Glucuronoyl esterases are active on the polymeric substrate methyl esterified glucuronoxylan Biely P, Malovikova A, Uhliarikova I, Li XL, Wong DWS Ref: FEBS Letters, 589:2334, 2015 : PubMed ESTHER: Biely_2015_FEBS.Lett_589_2334 PubMedSearch: Biely 2015 FEBS.Lett 589 2334 PubMedID: 26216754 Gene_locus related to this paper: rumfl-CESA Abstract Alkali extracted beechwood glucuronoxylan methyl ester prepared by esterification of 4-O-methyl-d-glucuronic acid side residues by methanol was found to serve as substrate of microbial glucuronoyl esterases from Ruminococcus flavefaciens, Schizophyllum commune and Trichoderma reesei. The enzymatic deesterification was monitored by (1)H NMR spectroscopy and evaluated on the basis of the decrease of the signal of the ester methyl group and increase of the signal of methanol. The results show for the first time the action of enzymes on polymeric substrate, which imitates more closely the natural substrate in plant cell walls than the low molecular mass artificial substrates used up to present. Title: Microbial carbohydrate esterases deacetylating plant polysaccharides Biely P Ref: Biotechnol Adv, 30:1575, 2012 : PubMed ESTHER: Biely_2012_Biotechnol.Adv_30_1575 PubMedSearch: Biely 2012 Biotechnol.Adv 30 1575 PubMedID: 22580218 Abstract Several plant polysaccharides are partially esterified with acetic acid One of the roles of this modification is protection of plant cell walls against invading microorganisms. Acetylation of glycosyl residues of polysaccharides prevents hydrolysis of their glycosidic linkages by the corresponding glycoside hydrolases. In this way the acetylation also represents an obstacle of enzymatic saccharification of plant hemicelluloses to fermentable sugars which appears to be a hot topic of current research. We can eliminate this obstacle by alkaline extraction or pretreatment leading to saponification of ester linkages. However this task has been accomplished in a different way in the nature. The acetyl groups became targets of microbial carbohydrate esterases that evolved to overcome the complexity of the plant cell walls and that cooperate with glycoside hydrolases in plant polysaccharide degradation. This article concentrates on enzymes deacetylating plant hemicelluloses excluding pectin. They are currently grouped in at least 8 families specifically in CE families 1-7 and 16 originally assigned as acetylxylan esterases the enzymes acting on hardwood acetyl glucuronoxylan and its fragments generated by endo-beta-1,4-xylanases. There are esterases deacetylating softwood galactoglucomannan but they have not been classified yet. The enzymes present in CE families 1-7 differ in structure and substrate and positional specificity. There are families behaving as endo-type and exo-type deacetylates i.e esterases deacetylating internal sugar residues of partially acetylated polysaccharides and also esterases deacetylating non-reducing end sugar residues in oligosaccharides. With one exception the enzymes of all mentioned CE families belong to serine type esterases CE family 4 harbors enzymes that are metal-dependent aspartic esterases. Three-dimensional structures have been solved for members of the first seven CE families however there is still insufficient knowledge about their substrate specificity and real physiological role. Current knowledge on catalytic properties of the selected families of CEs is summarized in this review. Some of the families are emerging also as new biocatalysts for regioselective acylation and deacylation of carbohydrates. Title: Functional and structural characterization of a thermostable acetyl esterase from Thermotoga maritima Levisson M, Han GW, Deller MC, Xu Q, Biely P, Hendriks S, Ten Eyck LF, Flensburg C, Roversi P and Wilson IA <9 more author(s)> Levisson M, Han GW, Deller MC, Xu Q, Biely P, Hendriks S, Ten Eyck LF, Flensburg C, Roversi P, Miller MD, McMullan D, von Delft F, Kreusch A, Deacon AM, Van der Oost J, Lesley SA, Elsliger MA, Kengen SW, Wilson IA (- 9) Ref: Proteins, 80:1545, 2012 : PubMed ESTHER: Levisson_2012_Proteins_80_1545 PubMedSearch: Levisson 2012 Proteins 80 1545 PubMedID: 22411095 Gene_locus related to this paper: thema-TM0077 Inhibitor(s) related to this paper: Paraoxon Abstract TM0077 from Thermotoga maritima is a member of the carbohydrate esterase family 7 and is active on a variety of acetylated compounds, including cephalosporin C. TM0077 esterase activity is confined to short-chain acyl esters (C2-C3), and is optimal around 100degC and pH 7.5. The positional specificity of TM0077 was investigated using 4-nitrophenyl--D-xylopyranoside monoacetates as substrates in a -xylosidase-coupled assay. TM0077 hydrolyzes acetate at positions 2, 3, and 4 with equal efficiency. No activity was detected on xylan or acetylated xylan, which implies that TM0077 is an acetyl esterase and not an acetyl xylan esterase as currently annotated. Selenomethionine-substituted and native structures of TM0077 were determined at 2.1 and 2.5 resolution, respectively, revealing a classicalpha/beta-hydrolase fold. TM0077 assembles into a doughnut-shaped hexamer with small tunnels on either side leading to an inner cavity, which contains the six catalytic centers. Structures of TM0077 with covalently bound phenylmethylsulfonyl fluoride and paraoxon were determined to 2.4 and 2.1 , respectively, and confirmed that both inhibitors bind covalently to the catalytic serine (Ser188). Upon binding of inhibitor, the catalytic serine adopts an altered conformation, as observed in other esterase and lipases, and supports a previously proposed catalytic mechanism in which Ser hydroxyl rotation prevents reversal of the reaction and allows access of a water molecule for completion of the reaction. Title: Functional Cloning and Expression of the Schizophyllum commune Glucuronoyl Esterase Gene and Characterization of the Recombinant Enzyme Wong DW, Chan VJ, McCormack AA, Hirsch J, Biely P Ref: Biotechnol Res Int, 2012:951267, 2012 : PubMed ESTHER: Wong_2012_Biotechnol.Res.Int_2012_951267 PubMedSearch: Wong 2012 Biotechnol.Res.Int 2012 951267 PubMedID: 22844600 Gene_locus related to this paper: hypjq-cip2 Abstract The gene encoding Schizophyllum commune glucuronoyl esterase was identified in the scaffold 17 of the genome, containing two introns of 50 bp and 48 bp, with a transcript sequence of 1179 bp. The gene was synthesized and cloned into Pichia pastoris expression vector pGAPZalpha to achieve constitutive expression and secretion of the recombinant enzyme in soluble active form. The purified protein was 53 kD with glycosylation and had an acidic pI of 3.7. Activity analysis on several uronic acids and their derivatives suggests that the enzyme recognized only esters of 4-O-methyl-D-glucuronic acid derivatives, even with a 4-nitrophenyl aglycon but did not hydrolyze the ester of D-galacturonic acid. The kinetic values were K(m) 0.25 mM, V(max) 16.3 microM.min(-1), and k(cat) 9.27 s(-1) with 4-nitrophenyl 2-O-(methyl 4-O-methyl-alpha-D-glucopyranosyluronate)-beta-D-xylopyranoside as the substrate. Title: Structure of the catalytic domain of glucuronoyl esterase Cip2 from Hypocrea jecorina Pokkuluri PR, Duke NE, Wood SJ, Cotta MA, Li XL, Biely P, Schiffer M Ref: Proteins, 79:2588, 2011 : PubMed ESTHER: Pokkuluri_2011_Proteins_79_2588 PubMedSearch: Pokkuluri 2011 Proteins 79 2588 PubMedID: 21661060 Gene_locus related to this paper: hypjq-cip2 Abstract The structure of the catalytic domain of glucuronoyl esterase Cip2 from the fungus H. jecorina was determined at a resolution of 1.9 . This is the first structure of the newly established carbohydrate esterase family 15. The structure has revealed the residues Ser278-His411-Glu301 present in a triad arrangement as the active site. Ser278 is present in the novel consensus sequence GCSRXG reported earlier in the members of CE-15 family. The active site is exposed on the surface of the protein which has implications for the ability of the enzyme to hydrolyze ester bonds of large substrates. Efforts are underway to obtain crystals of Cip2_GE complexed with inhibitor and synthetic substrates. The activity of the glucuronoyl esterase could play a significant role in plant biomass degradation as its expected role is to separate the lignin from hemicelluloses by hydrolysis of the ester bond between 4-O-methyl-D-glucuronic acid moieties of glucuronoxylans and aromatic alcohols of lignin. Title: Fungal glucuronoyl esterases and substrate uronic acid recognition Duranova M, Hirsch J, Kolenova K, Biely P Ref: Biosci Biotechnol Biochem, 73:2483, 2009 : PubMed ESTHER: Duranova_2009_Biosci.Biotechnol.Biochem_73_2483 PubMedSearch: Duranova 2009 Biosci.Biotechnol.Biochem 73 2483 PubMedID: 19897892 Gene_locus related to this paper: wolco-gce1, phacr-gce1, phacr-gce2 Abstract Glucuronoyl esterases are enzymes involved in microbial plant cell-wall degradation. In this study we purified and characterized two recombinant Phanerochaete chrysosporium glucuronoyl esterases, PcGE1 and PcGE2. The catalytic activity of these and previously described glucuronoyl esterases was investigated on new synthetic substrates, methyl esters of uronic acids and their glycosides, prepared by esterification with ethereal diazomethane.The data obtained indicate that the enzymes hydrolyzed efficiently not only esters of 4-O-methyl-D-glucuronic acid, but also methyl esters of D-glucuronic acid carrying a 4-nitrophenyl aglycon. Moreover, the fact that they did not recognize the 4-epimers of these compounds, the D-galacturonic acid derivatives, supports the hypothesis that these carbohydrate esterases attack ester linkages between 4-O-methyl-D-glucuronic acid of glucuronoxylan and lignin alcohols. Title: Two glucuronoyl esterases of Phanerochaete chrysosporium Duranova M, Spanikova S, Wosten HA, Biely P, de Vries RP Ref: Arch Microbiol, 191:133, 2009 : PubMed ESTHER: Duranova_2009_Arch.Microbiol_191_133 PubMedSearch: Duranova 2009 Arch.Microbiol 191 133 PubMedID: 18854978 Gene_locus related to this paper: wolco-gce1, phacr-gce1 Abstract The white-rot fungus Phanerochaete chrysosporium produces glucuronoyl esterase, a recently discovered carbohydrate esterase, during growth on sugar beet pulp. Two putative genes encoding this enzyme, ge1 and ge2, were isolated and cloned. Heterologous expression in Aspergillus vadensis, Pycnoporus cinnabarinus and Schizophyllum commune resulted in extracellular glucuronoyl esterase activity, demonstrating that these genes encode this enzymatic function. The amino acid sequence of GE1 was used to identify homologous genes in the genomes of twenty-four fungi. Approximately half of the genomes, both from ascomycetes and basidiomycetes, contained putative orthologues, but their presence could not be assigned to any of fungal class or subclass. Comparison of the amino acid sequences of identified and putative glucuronoyl esterases to other types of carbohydrate esterases (CE) confirmed that they form a separate family of CEs. These enzymes are interesting candidates for biotechnological applications such as the separation of lignin and hemicellulose. Title: Purification, characterization and mass spectrometric sequencing of a thermophilic glucuronoyl esterase from Sporotrichum thermophile. Vafiadi C, Topakas E, Biely P, Christakopoulos P Ref: FEMS Microbiology Letters, 296:178, 2009 : PubMed ESTHER: Vafiadi_2009_FEMS.Microbiol.Lett_296_178 PubMedSearch: Vafiadi 2009 FEMS.Microbiol.Lett 296 178 PubMedID: 19459957 Gene_locus related to this paper: chagb-Q2GMN0, thiha-cip2 Abstract The cellulolytic system of the thermophilic fungus Sporotrichum thermophile contains a recently discovered esterase that may hydrolyze the ester linkage between the 4-O-methyl-D-glucuronic acid of glucuronoxylan and lignin alcohols. The glucuronoyl esterase named StGE1 was purified to homogeneity with a molecular mass of M(r) 58 kDa and pI 6.7. The enzyme activity was optimal at pH 6.0 and 60 degrees C. The esterase displayed a narrow pH range stability at pH 8.0 and retained 50% of its activity after 430 and 286 min at 50 and 55 degrees C, respectively. The enzyme was active on substrates containing glucuronic acid methyl ester, showing a lower catalytic efficiency on 4-nitrophenyl 2-O-(methyl-4-O-methyl-alpha-d-glucopyranosyluronate)-beta-D-xylopyranoside than its mesophilic counterparts reported in the literature, which is typical of thermophilic enzymes. StGE1 was proved to be a modular enzyme containing a noncatalytic carbohydrate-binding module. LC-MS/MS analysis provided peptide mass and sequence information that facilitated the identification and classification of StGE1 as a family 15 glucuronoyl esterase that showed the highest homology with the hypothetical glucuronoyl esterase CHGG_10774 of Chaetomium globosum CBS 148.51. This work represents the first example of the purification and identification of a thermophilic glucuronoyl esterase from S. thermophile. Title: Novel family of carbohydrate esterases, based on identification of the Hypocrea jecorina acetyl esterase gene Li XL, Skory CD, Cotta MA, Puchart V, Biely P Ref: Applied Environmental Microbiology, 74:7482, 2008 : PubMed ESTHER: Li_2008_Appl.Environ.Microbiol_74_7482 PubMedSearch: Li 2008 Appl.Environ.Microbiol 74 7482 PubMedID: 18978092 Abstract Plant cell walls have been shown to contain acetyl groups in hemicelluloses and pectin. The gene aes1, encoding the acetyl esterase (Aes1) of Hypocrea jecorina, was identified by amino-terminal sequencing, peptide mass spectrometry, and genomic sequence analyses. The coded polypeptide had 348 amino acid residues with the first 19 serving as a secretion signal peptide. The calculated molecular mass and isoelectric point of the secreted enzyme were 37,088 Da and pH 5.89, respectively. No significant homology was found between the predicated Aes1 and carbohydrate esterases of known families, but putative aes1 orthologs were found in genomes of many fungi and bacteria that produce cell wall-degrading enzymes. The aes1 transcript levels were high when the fungal cells were induced with sophorose, cellulose, oat spelt xylan, lactose, and arabinose. The recombinant Aes1 produced by H. jecorina transformed with aes1 under the cellobiohydrolase I promoter displayed properties similar to those reported for the native enzyme. The enzyme hydrolyzed acetate ester bond specifically. Using 4-nitrophenyl acetate as substrate, the activity of the recombinant enzyme was enhanced by D-xylose, D-glucose, cellobiose, D-galactose, and xylooligosaccharides but not by arabinose, mannose, or lactose. With the use of 4-nitrophenyl-beta-D-xylopyranoside monoacetate as substrate in a beta-xylosidase-coupled assay, Aes1 hydrolyzed positions 3 and 4 with the same efficiency while the H. jecorina acetylxylan esterase 1 exclusively deacetylated the position 2 acetyl group. Aes1 was capable of transacetylating methylxyloside in aqueous solution. The data presented demonstrate that Aes1 and other homologous microbial proteins may represent a new family of esterases for lignocellulose biodegradation. Title: Identification of genes encoding microbial glucuronoyl esterases Li XL, Spanikova S, de Vries RP, Biely P Ref: FEBS Letters, 581:4029, 2007 : PubMed ESTHER: Li_2007_FEBS.Lett_581_4029 PubMedSearch: Li 2007 FEBS.Lett 581 4029 PubMedID: 17678650 Gene_locus related to this paper: hypjq-cip2 Abstract One type of covalent linkages connecting lignin and hemicellulose in plant cell walls is the ester linkage between 4-O-methyl-D-glucuronic acid of glucuronoxylan and lignin alcohols. An enzyme that could hydrolyze such linkages, named glucuronoyl esterase, occurs in the cellulolytic system of the wood-rotting fungus Schizophyllum commune. Here we report partial amino acid sequences of the enzyme and the results of subsequent search for homologous genes in sequenced genomes. The homologous genes of unknown functions were found in genomes of several filamentous fungi and one bacterium. The gene corresponding to the cip2 gene of Hypocrea jecorina (Trichoderma reesei), known to be up-regulated under conditions of induction of cellulolytic and hemicellulolytic enzymes, was over-expressed in H. jecorina. The product of the cip2 gene was purified to homogeneity and shown to exhibit glucuronoyl esterase activity. Title: Substrate and positional specificity of feruloyl esterases for monoferuloylated and monoacetylated 4-nitrophenyl glycosides Puchart V, Vrsanska M, Mastihubova M, Topakas E, Vafiadi C, Faulds CB, Tenkanen M, Christakopoulos P, Biely P Ref: J Biotechnol, 127:235, 2007 : PubMed ESTHER: Puchart_2007_J.Biotechnol_127_235 PubMedSearch: Puchart 2007 J.Biotechnol 127 235 PubMedID: 16901567 Abstract 4-Nitrophenyl glycosides of 2-, 3-, and 5-O-(E)-feruloyl- and 2- and 5-O-acetyl-alpha-L-arabinofuranosides and of 2-, 3-, and 4-O-(E)-feruloyl- and 2-, 3- and 4-O-acetyl-beta-D-xylopyranosides, compounds mimicking natural substrates, were used to investigate substrate and positional specificity of type-A, -B, and -C feruloyl esterases. All the feruloyl esterases behave as true feruloyl esterases showing negligible activity on sugar acetates. Type-A enzymes, represented by AnFaeA from Aspergillus niger and FoFaeII from Fusarium oxysporum, are specialized for deferuloylation of primary hydroxyl groups, with a very strong preference for hydrolyzing 5-O-feruloyl-alpha-L-arabinofuranoside. On the contrary, type-B and -C feruloyl esterases, represented by FoFaeI from F. oxysporum and TsFaeC from Talaromyces stipitatus, acted on almost all ferulates with exception of 4- and 3-O-feruloyl-beta-D-xylopyranoside. 5-O-Feruloyl-alpha-L-arabinofuranoside was the best substrate for both TsFaeC and FoFaeI, although catalytic efficiency of the latter enzyme toward 2-O-feruloyl-alpha-L-arabinofuranoside was comparable. In comparison with acetates, the corresponding ferulates served as poor substrates for the carbohydrate esterase family 1 feruloyl esterase from Aspergillus oryzae. The enzyme hydrolyzed all alpha-L-arabinofuranoside and beta-D-xylopyranoside acetates. It behaved as a non-specific acetyl esterase rather than a feruloyl esterase, with a preference for 2-O-acetyl-beta-D-xylopyranoside. Title: Synthetic esters recognized by glucuronoyl esterase from Schizophyllum commune Spanikova S, Polakova M, Joniak D, Hirsch J, Biely P Ref: Arch Microbiol, 188:185, 2007 : PubMed ESTHER: Spanikova_2007_Arch.Microbiol_188_185 PubMedSearch: Spanikova 2007 Arch.Microbiol 188 185 PubMedID: 17440709 Abstract Glucuronoyl esterase is a novel carbohydrate esterase recently discovered in the cellulolytic system of the wood-rotting fungus Schizophyllum commune on the basis of its ability to hydrolyze methyl ester of 4-O-methyl-D-glucuronic acid. This substrate was not fully corresponding to the anticipated function of the enzyme to hydrolyze esters between xylan-bound 4-O-methyl-D-glucuronic acid and lignin alcohols occurring in plant cell walls. In this work we showed that the enzyme was capable of hydrolyzing two synthetic compounds that mimic the ester linkages described in lignin-carbohydrate complexes, esters of 4-O-methyl-D-glucuronic and D-glucuronic acid with 3-(4-methoxyphenyl)propyl alcohol. A comparison of kinetics of hydrolysis of methyl and 3-(4-methoxyphenyl)propyl esters indicated that the glucuronoyl esterase recognizes the uronic acid part of the substrates better than the alcohol type. The catalytic efficiency of the enzyme was much higher with the ester of 4-O-methyl-D-glucuronic acid than with that of D-glucuronic acid. Examination of the action of glucuronoyl esterase on a series of methyl esters of 4-O-methyl-D-glucopyranuronosyl residues alpha-1,2-linked to xylose and several xylooligosaccharides suggested that the rate of deesterification is independent of the character of the carbohydrate part glycosylated by the 4-O-methyl-D-glucuronic acid. Title: The acetates of p-nitrophenyl alpha-L-arabinofuranoside--regioselective preparation by action of lipases Mastihubova M, Szemesova J, Biely P Ref: Bioorganic & Medicinal Chemistry, 14:1805, 2006 : PubMed ESTHER: Mastihubova_2006_Bioorg.Med.Chem_14_1805 PubMedSearch: Mastihubova 2006 Bioorg.Med.Chem 14 1805 PubMedID: 16288881 Abstract All possible di-O-acetates and mono-O-acetates of p-nitrophenyl alpha-L-arabinofuranoside were prepared by chemoenzymatic way using lipases. The 2,3-di-O-acetate was obtained in 90% yield by deacetylation of the primary acetyl group of per-O-acetylated p-nitrophenyl alpha-L-arabinofuranoside by Candida cylindracea lipase (CCL) or Candida rugosa lipase (LAY). The 2,5- and 3,5-di-O-acetates were obtained by acetylation of p-nitrophenyl alpha-L-arabinofuranoside by Pseudomonas cepacia lipase (LPS-30) in organic solvents. The 5-O-acetate was regioselectively synthesised in 95% yield by acetylation of p-nitrophenyl alpha-L-arabinofuranoside catalysed by porcine pancreas lipase. Finally, the 2- and 3-O-acetates of p-nitrophenyl alpha-L-arabinofuranoside were obtained in two steps. The enzymatic di-O-acetylation of p-nitrophenyl alpha-L-arabinofuranoside by LPS-30 was followed by enzymatic hydrolysis of the primary acetyl group by CCL or LAY. Title: Glucuronoyl esterase--novel carbohydrate esterase produced by Schizophyllum commune Spanikova S, Biely P Ref: FEBS Letters, 580:4597, 2006 : PubMed ESTHER: Spanikova_2006_FEBS.Lett_580_4597 PubMedSearch: Spanikova 2006 FEBS.Lett 580 4597 PubMedID: 16876163 Abstract 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. Title: Lipase-catalysed preparation of acetates of 4-nitrophenyl beta-D-xylopyranoside and their use in kinetic studies of acetyl migration Mastihubova M, Biely P Ref: Carbohydr Res, 339:1353, 2004 : PubMed ESTHER: Mastihubova_2004_Carbohydr.Res_339_1353 PubMedSearch: Mastihubova 2004 Carbohydr.Res 339 1353 PubMedID: 15113674 Abstract Di-O-acetates and mono-O-acetates of 4-nitrophenyl beta-D-xylopyranoside were prepared by use of lipase PS-30. Polarity of organic solvents and reaction time affected the regioselectivity of the di-O-acetylation as well as the yields of monoacetates. The kinetics of acetyl groups migration in these derivatives was studied in aqueous media using HPLC. Migration of the acetyl group strongly depended on pH. The highest rate of acetyl migration was observed from O-2 to O-3 in both 2,4-di-O-acetate and 2-O-acetate. On the contrary, acetyl exchange between O-3 and O-4 in both directions was slower than between O-2 and O-3. The 2,3-di-O-acetate and 4-O-acetate showed to be the most stable towards acetyl migration. The 3,4-di-O-acetate and 4-O-acetate were dominant in the corresponding equilibration mixtures. Title: Purification and characterization of a type B feruloyl esterase (StFAE-A) from the thermophilic fungus Sporotrichum thermophile Topakas E, Stamatis H, Biely P, Christakopoulos P Ref: Applied Microbiology & Biotechnology, 63:686, 2004 : PubMed ESTHER: Topakas_2004_Appl.Microbiol.Biotechnol_63_686 PubMedSearch: Topakas 2004 Appl.Microbiol.Biotechnol 63 686 PubMedID: 14615854 Gene_locus related to this paper: myctt-faeb Abstract A feruloyl esterase (StFAE-A) produced by Sporotrichum thermophile was purified to homogeneity. The purified homogeneous preparation of native StFAE-A exhibited a molecular mass of 57.0+/-1.5 kDa, with a mass of 33+/-1 kDa on SDS-PAGE. The pI of the enzyme was estimated by cation-exchange chromatofocusing to be at pH 3.1. The enzyme activity was optimal at pH 6.0 and 55-60 degrees C. The purified esterase was stable at the pH range 5.0-7.0. The enzyme retained 70% of activity after 7 h at 50 degrees C and lost 50% of its activity after 45 min at 55 degrees C and after 12 min at 60 degrees C. Determination of k(cat)/ K(m) revealed that the enzyme hydrolyzed methyl p-coumarate 2.5- and 12-fold more efficiently than methyl caffeate and methyl ferulate, respectively. No activity on methyl sinapinate was detected. The enzyme was active on substrates containing ferulic acid ester linked to the C-5 and C-2 linkages of arabinofuranose and it hydrolyzed 4-nitrophenyl 5- O- trans-feruloyl-alpha- l-arabinofuranoside (NPh-5-Fe-Ara f) 2-fold more efficiently than NPh-2-Fe-Ara f. Ferulic acid (FA) was efficiently released from destarched wheat bran when the esterase was incubated together with xylanase from S. thermophile (a maximum of 34% total ferulic acid released after 1 h incubation). StFAE-A by itself could release FA, but at a level almost 47-fold lower than that obtained in the presence of xylanase. The potential of StFAE-A for the synthesis of various phenolic acid esters was tested using a ternary water-organic mixture consisting of n-hexane, 1-butanol and water as a reaction system. Title: Regioselective deacetylation of cellulose acetates by acetyl xylan esterases of different CE-families Altaner C, Saake B, Tenkanen M, Eyzaguirre J, Faulds CB, Biely P, Viikari L, Siika-aho M, Puls J Ref: J Biotechnol, 105:95, 2003 : PubMed ESTHER: Altaner_2003_J.Biotechnol_105_95 PubMedSearch: Altaner 2003 J.Biotechnol 105 95 PubMedID: 14511913 Abstract Cellulose acetate (CA) was found to be a substrate of several acetyl xylan esterases (AXE). Eight AXE from different carbohydrate esterase (CE) families were tested on their activity against CA with a degree of substitution of 0.7 and 1.4. The classification of the AXEs into CE families according to their structure by hydrophobic cluster analysis followed clearly their activity against CA. Within the same CE family similar, and between the CE families different deacetylation behaviours could be observed. Furthermore, each esterase family showed a distinct regioselective mode of action. The CE 1 family enzymes regioselectively cleaved the substituents in C2- and C3-position, while CE 5 family enzymes only cleaved the acetyl groups in C2-position. CE 4 family enzymes seemed to interact only with the substituents in C3-position. Evidence was found that the deacetylation reaction of the CE 1 family enzymes proceeded faster in C2- than in C3-position of CA. The enzymes were able to cleave acetyl groups from fully substituted anhydroglucose units. Title: Purification and properties of a feruloyl esterase involved in lignocellulose degradation by Aureobasidium pullulans Rumbold K, Biely P, Mastihubova M, Gudelj M, Gubitz G, Robra KH, Prior BA Ref: Applied Environmental Microbiology, 69:5622, 2003 : PubMed ESTHER: Rumbold_2003_Appl.Environ.Microbiol_69_5622 PubMedSearch: Rumbold 2003 Appl.Environ.Microbiol 69 5622 PubMedID: 12957952 Abstract The lignocellulolytic fungus Aureobasidium pullulans NRRL Y 2311-1 produces feruloyl esterase activity when grown on birchwood xylan. Feruloyl esterase was purified from culture supernatant by ultrafiltration and anion-exchange, hydrophobic interaction, and gel filtration chromatography. The pure enzyme is a monomer with an estimated molecular mass of 210 kDa in both native and denatured forms and has an apparent degree of glycosylation of 48%. The enzyme has a pI of 6.5, and maximum activity is observed at pH 6.7 and 60 degrees C. Specific activities for methyl ferulate, methyl p-coumarate, methyl sinapate, and methyl caffeate are 21.6, 35.3, 12.9, and 30.4 micro mol/min/mg, respectively. The pure feruloyl esterase transforms both 2-O and 5-O arabinofuranosidase-linked ferulate equally well and also shows high activity on the substrates 4-O-trans-feruloyl-xylopyranoside, O-[5-O-[(E)-feruloyl]-alpha-L-arabinofuranosyl]-(1,3)-O-beta-D-xylopyranosyl-(1,4)-D-xylopyranose, and p-nitrophenyl-acetate but reveals only low activity on p-nitrophenyl-butyrate. The catalytic efficiency (k(cat)/K(m)) of the enzyme was highest on methyl p-coumarate of all the substrates tested. Sequencing revealed the following eight N-terminal amino acids: AVYTLDGD. Title: Purification and characterization of a feruloyl esterase from Fusarium oxysporum catalyzing esterification of phenolic acids in ternary water-organic solvent mixtures Topakas E, Stamatis H, Biely P, Kekos D, Macris BJ, Christakopoulos P Ref: J Biotechnol, 102:33, 2003 : PubMed ESTHER: Topakas_2003_J.Biotechnol_102_33 PubMedSearch: Topakas 2003 J.Biotechnol 102 33 PubMedID: 12668312 Gene_locus related to this paper: fusox-a0a1d3s5h0 Abstract An extracellular feruloyl esterase (FAE-II) from the culture filtrates of Fusarium oxysporum F3 was purified to homogeneity by SP-Sepharose, t-butyl-HIC and Sephacryl S-200 column chromatography. The protein corresponded to molecular mass and pI values of 27 kDa and 9.9, respectively. The enzyme was optimally active at pH 7 and 45 degrees C. The purified esterase was fully stable at pH 7.0-9.0 and temperature up to 45 degrees C after 1 h incubation. Determination of k(cat)/K(m) revealed that the enzyme hydrolysed methyl sinapinate 6, 21 and 40 times more efficiently than methyl ferulate, methyl coumarate and methyl caffeate, respectively. The enzyme was active on substrates containing ferulic acid ester linked to the C-5 but inactive to the C-2 positions of arabinofuranose such as 4-nitrophenyl 5-O-trans-feruloyl-alpha-L-arabinofuranoside and 4-nitrophenyl 2-O-trans-feruloyl-alpha-L-arabinofuranoside. In the presence of Sporotrichum thermophile xylanase, there was a significant release of ferulic acid from destarched wheat bran by FAE-II, indicating a synergistic interaction between FAE-II and S. thermophile xylanase. FAE-II by itself could release only little ferulic acid from destarched wheat bran. The potential of FAE-II for the synthesis of various phenolic acid esters was tested using as a reaction system a surfactantless microemulsion formed in ternary mixture consisting of n-hexane, 1-propanol and water. Title: Purification and characterization of a Fusarium oxysporum feruloyl esterase (FoFAE-I) catalysing transesterification of phenolic acid esters Topakas E, Stamatis H, Mastihubova M, Biely P, Kekos D, Macris BJ, Christakopoulos P Ref: Enzyme Microb Technol, 33:729, 2003 : PubMed ESTHER: Topakas_2003_Enzyme.Microb.Technol_33_729 PubMedSearch: Topakas 2003 Enzyme.Microb.Technol 33 729 Abstract An extracellular feruloyl esterase (FoFAE-I) from the culture filtrates of Fusarium oxysporum F3 was purified to homogeneity by ion-exchange, hydrophobic interaction and gel filtration chromatographies. The protein corresponded to molecular mass and pI values of 31kDa and 9.5, respectively. The enzyme was optimally active at pH 7.0 and 55C. The purified esterase was fully stable at pH 7.0-9.0 and temperature up to 30C. Determination of kcat/Km revealed that the enzyme hydrolysed methyl p-coumarate (MpCA) 4.5, 9, and 239 times more efficiently than methyl caffeate (MCA), methyl ferulate (MFA) and methyl sinapinate (MSA), respectively. The enzyme was active on substrates containing ferulic acid ester linked to the C-5 and C-2 linkages of arabinofuranose but showed preference for the ester at position 2. 4-Nitrophenyl-2-O-trans-feruloyl--l-arabinofuranoside (NPh-5-Fe-Araf) was hydrolysed 100 times more efficiently than 4-nitrophenyl-5-O-trans-feruloyl--l-arabinofuranoside (NPh-2-Fe-Araf). Ferulic acid (FA) was efficiently released from destarched wheat bran (DSWB) when the esterase was incubated together with xylanase from Sporotrichum thermophile (a maximum of 92% total ferulic acid released after 4h incubation). FoFAE-I by itself could release FA but at a level almost five-fold lower than that obtained in the presence of xylanase. The potential of FAE-I for the synthesis of various phenolic acid esters was tested using as a reaction system a surfactantless microemulsions formed in ternary mixture consisting of n-hexane, 1-butanol and water. Title: Differentiation of feruloyl esterases on synthetic substrates in alpha-arabinofuranosidase-coupled and ultraviolet-spectrophotometric assays Biely P, Mastihubova M, van Zyl WH, Prior BA Ref: Analytical Biochemistry, 311:68, 2002 : PubMed ESTHER: Biely_2002_Anal.Biochem_311_68 PubMedSearch: Biely 2002 Anal.Biochem 311 68 PubMedID: 12441154 Abstract 4-Nitrophenyl 5-O-trans-feruloyl-alpha-L-arabinofuranoside and 4-nitrophenyl 2-O-trans-feruloyl-alpha-L-arabinofuranoside, synthesized by our group (M. Mastihubov, J. Szemesov, and P. Biely), were found to be suitable substrates for determination of activity of feruloyl esterases (FeEs) exhibiting affinity for 5-O- and 2-O-feruloylated alpha-L-arabinofuranosyl residues. One assay is based on coupling the FeE-catalyzed formation of 4-nitrophenyl alpha-L-arabinofuranoside with its efficient hydrolysis by alpha-L-arabinofuranosidase to release 4-nitrophenol. An alternative assay explores the difference in the molar absorbances at 340 nm of the substrate (ferulic acid esters) and the reaction products, which are (1) free ferulic acid and 4-nitrophenyl alpha-L-arabinofuranoside in samples free of alpha-L-arabinofuranosidase and (2) ferulic acid, 4-nitrophenyl alpha-L-arabinofuranoside, and/or 4-nitrophenol in samples containing alpha-L-arabinofuranosidase. The new substrates represent convenient tools to differentiate FeEs on the basis of substrate specificity. Title: Inverting character of alpha-glucuronidase A from Aspergillus tubingensis Biely P, de Vries RP, Vrsanska M, Visser J Ref: Biochimica & Biophysica Acta, 1474:360, 2000 : PubMed ESTHER: Biely_2000_Biochim.Biophys.Acta_1474_360 PubMedSearch: Biely 2000 Biochim.Biophys.Acta 1474 360 PubMedID: 10779688 Abstract Alpha-glucuronidase A from Aspergillus tubingensis was found to be capable of liberating 4-O-methyl-D-glucuronic acid (MeGlcA) only from those beechwood glucuronoxylan fragments in which the acid is attached to the non-reducing terminal xylopyranosyl residue. Reduced aldotetrauronic acid, 4-O-methyl-D-glucuronosyl-alpha-1,2-D-xylopyranosyl-beta-1,4-xylopyranosyl-beta-1 ,4-xylitol, was found to be a suitable substrate to follow the stereochemical course of the hydrolytic reaction catalyzed by the purified enzyme. The configuration of the liberated MeGlcA was followed in a D(2)O reaction mixture by (1)H-NMR spectroscopy. It was unambiguously established that MeGlcA was released from the substrate as its beta-anomer from which the alpha-anomer was formed on mutarotation. This result represents the first experimental evidence for the inverting character of a microbial alpha-glucuronidase, a member of glycosyl hydrolase family 67 (EC 3.1.1.139). Title: A chromogenic substrate for a beta-xylosidase-coupled assay of alpha-glucuronidase Biely P, Hirsch J, la Grange DC, van Zyl WH, Prior BA Ref: Analytical Biochemistry, 286:289, 2000 : PubMed ESTHER: Biely_2000_Anal.Biochem_286_289 PubMedSearch: Biely 2000 Anal.Biochem 286 289 PubMedID: 11067752 Abstract 4-Nitrophenyl 2-(4-O-methyl-alpha-d-glucopyranuronosyl)-beta-d-xylopyranoside obtained on deesterification of 4-nitrophenyl 2-O-(methyl 4-O-methyl-alpha-d-glucopyranosyluronate)-beta-d-xylopyranoside (Hirsch et al., Carbohydr. Res. 310, 145-149, 1998) was found to be an excellent substrate for the measurement of hemicellulolytic alpha-glucuronidase activity. A new precise alpha-glucuronidase assay was developed by coupling the alpha-glucuronidase-catalyzed formation of 4-nitrophenyl beta-d-xylopyranoside with its efficient hydrolysis by beta-xylosidase. A recombinant strain of Saccharomyces cerevisiae, harboring and expressing the beta-xylosidase gene xlnD of Aspergillus niger under control of the alcohol dehydrogenase II promoter on a multicopy plasmid, was used as a source of beta-xylosidase. The activity values of beta-xylosidase in the assay required to achieve a steady-state rate of 4-nitrophenol formation shortly after starting the alpha-glucuronidase reaction were obtained both experimentally and by calculation using the kinetics of coupled enzyme reactions. Title: Effects of purified endo-beta-1,4-xylanases of family 10 and 11 and acetyl xylan esterases on eucalypt sulfite dissolving pulp Christov L, Biely P, Kalogeris E, Christakopoulos P, Prior BA, Bhat MK Ref: J Biotechnol, 83:231, 2000 : PubMed ESTHER: Christov_2000_J.Biotechnol_83_231 PubMedSearch: Christov 2000 J.Biotechnol 83 231 PubMedID: 11051420 Abstract Sulfite dissolving pulp from Eucalyptus grandis contained approximately 3.8% O-acetyl-4-O-methylglucuronoxylan with a molar ratio of xylose:4-O-methylglucuronic acid:acetyl group close to 13.6:1:6.2. The effects produced by purified endo-xylanases from two different glycosyl hydrolase families (family 10 and 11) as well as acetyl xylan esterases were examined and assessed on pulp in relation to their bleaching abilities. The purified endo-xylanases hydrolyzed only a limited portion (less than 30%) of the acetylglucuronoxylan present in the pulp. The enzymes of family 10 produced acetylated xylobiose and xylotriose whereas acetylated xylobiose was not observed among the products released from the pulp by the family 11 xylanases. The esterases however were not capable of deacetylating the acetylated aldouronic acids generated by the xylanases. Regardless of the different mode of action of the endo-xylanases on dissolving pulp, their effect on pulp bleaching was not related to the amount and nature of sugars generated or the glycosyl hydrolase family. No additional brightness gain was obtained when endo-xylanases were used in conjunction with acetyl xylan esterases, suggesting that the latter do not play an important role in biobleaching of eucalypt sulfite dissolving pulps. Title: Action of acetylxylan esterase from Trichoderma reesei on acetylated methyl glycosides Biely P, Cote GL, Kremnicky L, Greene RV, Tenkanen M Ref: FEBS Letters, 420:121, 1997 : PubMed ESTHER: Biely_1997_FEBS.Lett_420_121 PubMedSearch: Biely 1997 FEBS.Lett 420 121 PubMedID: 9459293 Abstract Substrate specificity of purified acetylxylan esterase (AcXE) from Trichoderma reesei was investigated on partially and fully acetylated methyl glycopyranosides. Methyl 2,3,4-tri-O-acetyl-beta-D-xylopyranoside was deacetylated at positions 2 and 3, yielding methyl 4-O-acetyl-beta-D-xylopyranoside in almost 90% yield. Methyl 2,3-di-O-acetyl beta-D-xylopyranoside was deacetylated at a rate similar to the fully acetylated derivative. The other two diacetates (2,4- and 3,4-), which have a free hydroxyl group at either position 3 or 2, were deacetylated one order of magnitude more rapidly. Thus the second acetyl group is rapidly released from position 3 or 2 after the first acetyl group is removed from position 2 or 3. The results strongly imply that in degradation of partially acetylated beta-1,4-linked xylans, the enzyme deacetylates monoacetylated xylopyranosyl residues more readily than di-O-acetylated residues. The T. reesei AcXE attacked acetylated methyl beta-D-glucopyranosides and beta-D-mannopyranosides in a manner similar to the xylopyranosides. Title: Substrate specificity and mode of action of acetylxylan esterase from Streptomyces lividans Biely P, Cote GL, Kremnicky L, Greene RV, Dupont C, Kluepfel D Ref: FEBS Letters, 396:257, 1996 : PubMed ESTHER: Biely_1996_FEBS.Lett_396_257 PubMedSearch: Biely 1996 FEBS.Lett 396 257 PubMedID: 8914998 Abstract The substrate specificity of purified acetylxylan esterase (AcXE) from Streptomyces lividans was investigated on partially and fully acetylated methyl glycopyranosides. The enzyme exhibited deacetylation regioselectivity on model compounds which provided insights pertaining to its function in acetylxylan degradation. The enzyme catalyzed double deacetylation of methyl 2,3,4-tri-O-acetyl-beta-D-xylopyranoside and methyl 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside at positions 2 and 3. Two methyl xylopyranoside diacetates, which had a free hydroxyl group at position 2 or 3, i.e. the derivatives that most closely mimic monoacetylated xylopyranosyl residues in acetylxylan, were deacetylated 1 to 2 orders of magnitude faster than methyl 2,3,4-tri-O-acetyl-beta-D-xylopyranoside and methyl 2,3-di-O-acetyl-beta-D-xylopyranoside. These observations explain the double deacetylation. The second acetyl group is released immediately after the first one is removed from the fully acetylated methyl beta-D-xylo- and -glucopyranoside. The results suggest that in acetylxylan degradation the enzyme rapidly deacetylates monoacetylated xylopyranosyl residues, but attacks doubly acetylated residues much more slowly. Evidence is also presented that the St. lividans enzyme could be the first real substrate-specific AcXE. Title: Substrate specificity of acetylxylan esterase from Schizophyllum commune: mode of action on acetylated carbohydrates Biely P, Cote GL, Kremnicky L, Weisleder D, Greene RV Ref: Biochimica & Biophysica Acta, 1298:209, 1996 : PubMed ESTHER: Biely_1996_Biochim.Biophys.Acta_1298_209 PubMedSearch: Biely 1996 Biochim.Biophys.Acta 1298 209 PubMedID: 8980647 Abstract Substrate specificity of a purified acetylxylan esterase from Schizophyllum commune was investigated on a variety of methyl per-O-acetyl glycopyranosides, methyl di-O-acetyl-beta-D-xylopyranosides and acetylated polysaccharides. The enzyme preferentially deacetylated the 3-position of methyl 2,3,4-tri-O-acetyl-beta-D-xylopyranoside and 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside. Removal of the 3-acetyl group from the xylopyranoside was accompanied by a slower deacetylation at positions 2 and 4. A similarly slower, accompanying deacetylation occurred primarily at position 2 with the glucopyranoside. Such specificity corresponds well to the expected function of the esterase in acetylxylan degradation. Of the three possible diacetates of methyl beta-D-xylopyranoside, the 3,4-diacetate was found to be the most rapidly deacetylated. Unexpectedly, products of its deacetylation were a mixture of 2- and 4-monoacetate. The formation of the methyl 2-O-acetyl-beta-D-xylopyranoside involved an enzyme-mediated acetyl group transfer because the rate of the enzyme-catalyzed reaction exceeded the rate of spontaneous migration of acetyl groups. This is the likely mechanism for acetyl removal from position 2 in the native substrate. The enzyme exhibited the highest regioselectivity with methyl 2,3,4,6-tetra-O-acetyl-beta-D-mannopyranoside. An 80% conversion of this substrate to methyl 4,6-di-O-acetyl-beta-D-mannopyranoside, a new mannose derivative, was achieved. In contrast to the majority of lipases and esterases exploited for regioselective deacetylation, the S. commune acetylxylan esterase did not attack the C-6 acetyl linkages in methyl hexopyranosides when other acetyl groups were available. | |||||||
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