Biochemical and kinetic analysis of the GH3 family β-xylosidase from Aspergillus awamori X-100

Eneyskaya, Elena V.; Ivanen, Dina R.; Bobrov, Kirill S.; Isaeva-Ivanova, Lyudmila S.; Shabalin, Konstantin A.; Savel'ev, Andrew N.; Golubev, A. M.; Kulminskaya, Anna A.

doi:10.1016/j.abb.2006.10.024

Cited by 39 publications

(28 citation statements)

References 47 publications

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“…When we cloned the putative ␤-D-glucosidase gene (ORF02829) and characterized the recombinant protein, we found that it releases pNP from pNPX and pNP-␣-L-arabinofuranoside but not pNP-␤-D-glucopyranoside. The hydrolysis of both pNPX and pNP-␣-L-arabinofuranoside (Xyl/ Ara) has been reported for some members of GH family 3 (20,46,51,74). With one exception (68), all of the characterized family 3 ␤-D-xylosidases possess substrate specificity for Xyl/ Ara and do not possess ␤-D-glucosidase activity.…”

Section: Discussionmentioning

confidence: 99%

Biochemical Analysis of a β- d -Xylosidase and a Bifunctional Xylanase-Ferulic Acid Esterase from a Xylanolytic Gene Cluster in Prevotella ruminicola 23

Dodd

Kocherginskaya²,

Spies

et al. 2009

J Bacteriol

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Prevotella ruminicola 23 is an obligate anaerobic bacterium in the phylum Bacteroidetes that contributes to hemicellulose utilization within the bovine rumen. To gain insight into the cellular machinery that this organism elaborates to degrade the hemicellulosic polymer xylan, we identified and cloned a gene predicted to encode a bifunctional xylanase-ferulic acid esterase (xyn10D-fae1A) and expressed the recombinant protein in Escherichia coli. Biochemical analysis of purified Xyn10D-Fae1A revealed that this protein possesses both endo-␤-1,4-xylanase and ferulic acid esterase activities. A putative glycoside hydrolase (GH) family 3 ␤-Dglucosidase gene, with a novel PA14-like insertion sequence, was identified two genes downstream of xyn10D-fae1A. Biochemical analyses of the purified recombinant protein revealed that the putative ␤-D-glucosidase has activity for pNP-␤-D-xylopyranoside, pNP-␣-L-arabinofuranoside, and xylo-oligosaccharides; thus, the gene was designated xyl3A. When incubated in combination with Xyn10D-Fae1A, Xyl3A improved the release of xylose monomers from a hemicellulosic xylan substrate, suggesting that these two enzymes function synergistically to depolymerize xylan. Directed mutagenesis studies of Xyn10D-Fae1A mapped the catalytic sites for the two enzymatic functionalities to distinct regions within the polypeptide sequence. When a mutation was introduced into the putative catalytic site for the xylanase domain (E280S), the ferulic acid esterase activity increased threefold, which suggests that the two catalytic domains for Xyn10D-Fae1A are functionally coupled. Directed mutagenesis of conserved residues for Xyl3A resulted in attenuation of activity, which supports the assignment of Xyl3A as a GH family 3 ␤-D-xylosidase.␤-1,4-linked xylopyranose is the principal component of plant cell wall hemicellulose, which represents the second largest reservoir of fixed carbon in the biosphere (11,30,34,38,42,72). The catabolic breakdown of hemicellulose thus represents a critical step in the recycling of carbon in nature and has been targeted as a subject of intense research with respect to renewable energy resources. Two enzymes of principal importance for recycling hemicellulosic material are endo-1,4-␤-xylanases (EC 3.2.1.8), which cleave the xylan backbone, and ␤-D-xylosidases (EC 3.2.1.37), which cleave xylose monomers from the nonreducing end of xylo-oligosaccharides (17).Prevotella ruminicola 23 is an important member of the anaerobic rumen microbiota (60) that contributes to the utilization of noncellulosic polysaccharides, such as starch and xylan (15,25,35,55). Despite its documented importance in rumen physiology, relatively little is known about the cellular machinery that P. ruminicola 23 employs to harvest energy from hemicellulosic substrates. Several studies have explored the carbohydrate active enzymes from the related taxon Prevotella bryantii B 1 4 (previously classified as P. ruminicola B 1 4) (25,27,28,50,52,66,73); however, less is known about the xylanolytic system that P. rumi...

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Section: Discussionmentioning

confidence: 99%

Biochemical Analysis of a β- d -Xylosidase and a Bifunctional Xylanase-Ferulic Acid Esterase from a Xylanolytic Gene Cluster in Prevotella ruminicola 23

Dodd

Kocherginskaya²,

Spies

et al. 2009

J Bacteriol

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“…Further studies reported a great number of fungal b-xylosidases with transxylosylating ability (Shinoyama et al 1988;Iizuka et al 1992;Sulistyo et al 1995;Herrmann et al 1997;Matsuo et al 1998;Ito et al 2003;Eneyskaya et al 2007). Transglycosylation activity was also reported in filamentous fungal xylanases (Christakopoulos et al 1996;Jiang et al 2004).…”

Section: B-xylosidase Localizationmentioning

confidence: 90%

“…Sulistyo et al (1995) reported that b-xylosidases from Aspergillus pulverulentus have broad acceptor specificity in transferring the xylosyl residue of xylooligosaccharides to various acceptors, such as alcohols and phenolic compounds. The b-xylosidase from Aspergillus awamori X-100 revealed a remarkable transglycosylating ability to produce xylooligosaccharides with degree of polymerization higher than seven (Eneyskaya et al 2007). …”

Section: B-xylosidase Localizationmentioning

confidence: 99%

β-Xylosidases from filamentous fungi: an overview

Knob

Terrasan

Carmona

2009

World J Microbiol Biotechnol

159

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b-Xylosidases are hydrolytic enzymes which play an important role in xylan degradation, hydrolyzing xylobiose and xylooligosaccharides to xylose from the nonreducing end. Filamentous fungi are particularly interesting producers of this enzyme from an industrial point of view, due to the fact that they secrete b-xylosidases into the medium. Besides, fungal b-xylosidases are highly advantageous for their elevated activity levels and specificity. Interest in xylanolytic enzymes has been increasing, for their possible application in many biotechnological processes. This fact has driven the isolation, purification and characterization of several b-xylosidases. In this review, the mechanisms of action, substrate specificities, physicochemical characteristics, regulation at molecular level, molecular cloning and classification of filamentous fungal b-xylosidases are described. The potential industrial applications of fungal b-xylosidases will also be presented.

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“…Talaromyces emersonii (␤-XTE) and T. reesei (␤-XTR) hydrolyze xylobiose, producing ␤-D-xylose (160). In Aspergillus awamori, the enzymatic hydrolysis of p-nitrophenyl ␤-Dxylopyranoside occurs with overall retention of the substrate anomeric configuration, suggesting cleavage of xylosidic bonds through a double-displacement mechanism (161,162). The catalytic amino acid residues of the extracellular ␤-D-xylosidase, which hydrolyzes p-nitrophenyl ␤-D-xyloside as a substrate, involve a carboxylate and a protonated group part for binding of the substrate; however, only a carboxylate group is needed for cleavage (163).…”

Section: Hemicellulasesmentioning

confidence: 99%

Genomics Review of Holocellulose Deconstruction by Aspergilli

Segato

Damásio

Lucas

et al. 2014

Microbiol Mol Biol Rev

105

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SUMMARY Biomass is constructed of dense recalcitrant polymeric materials: proteins, lignin, and holocellulose, a fraction constituting fibrous cellulose wrapped in hemicellulose-pectin. Bacteria and fungi are abundant in soil and forest floors, actively recycling biomass mainly by extracting sugars from holocellulose degradation. Here we review the genome-wide contents of seven Aspergillus species and unravel hundreds of gene models encoding holocellulose-degrading enzymes. Numerous apparent gene duplications followed functional evolution, grouping similar genes into smaller coherent functional families according to specialized structural features, domain organization, biochemical activity, and genus genome distribution. Aspergilli contain about 37 cellulase gene models, clustered in two mechanistic categories: 27 hydrolyze and 10 oxidize glycosidic bonds. Within the oxidative enzymes, we found two cellobiose dehydrogenases that produce oxygen radicals utilized by eight lytic polysaccharide monooxygenases that oxidize glycosidic linkages, breaking crystalline cellulose chains and making them accessible to hydrolytic enzymes. Among the hydrolases, six cellobiohydrolases with a tunnel-like structural fold embrace single crystalline cellulose chains and cooperate at nonreducing or reducing end termini, splitting off cellobiose. Five endoglucanases group into four structural families and interact randomly and internally with cellulose through an open cleft catalytic domain, and finally, seven extracellular β-glucosidases cleave cellobiose and related oligomers into glucose. Aspergilli contain, on average, 30 hemicellulase and 7 accessory gene models, distributed among 9 distinct functional categories: the backbone-attacking enzymes xylanase, mannosidase, arabinase, and xyloglucanase, the short-side-chain-removing enzymes xylan α-1,2-glucuronidase, arabinofuranosidase, and xylosidase, and the accessory enzymes acetyl xylan and feruloyl esterases.

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Biochemical and kinetic analysis of the GH3 family β-xylosidase from Aspergillus awamori X-100

Cited by 39 publications

References 47 publications

Biochemical Analysis of a β- d -Xylosidase and a Bifunctional Xylanase-Ferulic Acid Esterase from a Xylanolytic Gene Cluster in Prevotella ruminicola 23

Biochemical Analysis of a β- d -Xylosidase and a Bifunctional Xylanase-Ferulic Acid Esterase from a Xylanolytic Gene Cluster in Prevotella ruminicola 23

β-Xylosidases from filamentous fungi: an overview

Genomics Review of Holocellulose Deconstruction by Aspergilli

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