Dataset Information

Novel Trifunctional Xylanolytic Enzyme Axy43A from Paenibacillus curdlanolyticus Strain B-6 Exhibiting Endo-Xylanase, ?-d-Xylosidase, and Arabinoxylan Arabinofuranohydrolase Activities.

ABSTRACT: The axy43A gene encoding the intracellular trifunctional xylanolytic enzyme from Paenibacillus curdlanolyticus B-6 was cloned and expressed in Escherichia coli Recombinant PcAxy43A consisting of a glycoside hydrolase family 43 and a family 6 carbohydrate-binding module exhibited endo-xylanase, ?-xylosidase, and arabinoxylan arabinofuranohydrolase activities. PcAxy43A hydrolyzed xylohexaose and birch wood xylan to release a series of xylooligosaccharides, indicating that PcAxy43A contained endo-xylanase activity. PcAxy43A exhibited ?-xylosidase activity toward a chromogenic substrate, p-nitrophenyl-?-d-xylopyranoside, and xylobiose, while it preferred to hydrolyze long-chain xylooligosaccharides rather than xylobiose. In addition, surprisingly, PcAxy43A showed arabinoxylan arabinofuranohydrolase activity; that is, it released arabinose from both singly and doubly arabinosylated xylose, ?-l-Araf-(1?2)-d-Xylp or ?-l-Araf-(1?3)-d-Xylp and ?-l-Araf-(1?2)-[?-l-Araf-(1?3)]-?-d-Xylp Moreover, the combination of PcAxy43A and P. curdlanolyticus B-6 endo-xylanase Xyn10C greatly improved the efficiency of xylose and arabinose production from the highly substituted rye arabinoxylan, suggesting that these two enzymes function synergistically to depolymerize arabinoxylan. Therefore, PcAxy43A has the potential for the saccharification of arabinoxylan into simple sugars for many applications. IMPORTANCE In this study, the glycoside hydrolase 43 (GH43) intracellular multifunctional endo-xylanase, ?-xylosidase, and arabinoxylan arabinofuranohydrolase (AXH) from P. curdlanolyticus B-6 were characterized. Interestingly, PcAxy43A AXH showed a new property that acted on both the C(O)-2 and C(O)-3 positions of xylose residues doubly substituted with arabinosyl, which usually obstruct the action of xylanolytic enzymes. Furthermore, the studies here show interesting properties for the processing of xylans from cereal grains, particularly rye arabinoxylan, and show a novel relationship between PcAxy43A and endo-xylanase Xyn10C from strain B-6, providing novel metabolic potential for processing arabinoxylans into xylose and arabinose.

SUBMITTER: Teeravivattanakit T

PROVIDER: S-EPMC5103093 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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Novel Trifunctional Xylanolytic Enzyme Axy43A from Paenibacillus curdlanolyticus Strain B-6 Exhibiting Endo-Xylanase, β-d-Xylosidase, and Arabinoxylan Arabinofuranohydrolase Activities.

Teeravivattanakit Thitiporn T Baramee Sirilak S Phitsuwan Paripok P Waeonukul Rattiya R Pason Patthra P Tachaapaikoon Chakrit C Sakka Kazuo K Ratanakhanokchai Khanok K

Applied and environmental microbiology 20160923 23

The axy43A gene encoding the intracellular trifunctional xylanolytic enzyme from Paenibacillus curdlanolyticus B-6 was cloned and expressed in Escherichia coli Recombinant PcAxy43A consisting of a glycoside hydrolase family 43 and a family 6 carbohydrate-binding module exhibited endo-xylanase, β-xylosidase, and arabinoxylan arabinofuranohydrolase activities. PcAxy43A hydrolyzed xylohexaose and birch wood xylan to release a series of xylooligosaccharides, indicating that PcAx ...[more]

PMID: 27663030

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Project description:Xylanolytic enzymes, with both endo-xylanase and arabinoxylan arabinofuranohydrolase (AXH) activities, are attractive for the economically feasible conversion of recalcitrant arabinoxylan. However, their characterization and utilization of these enzymes in biotechnological applications have been limited. Here, we characterize a novel bifunctional enzyme, rAbf43A, cloned from a bacterial consortium that exhibits AXH and endo-xylanase activities. Hydrolytic pattern analyses revealed that the AXH activity belongs to AXHd3 because it attacked only the C(O)-3-linked arabinofuranosyl residues of double-substituted xylopyranosyl units of arabinoxylan and arabinoxylan-derived oligosaccharides, which are usually resistant to hydrolysis. The enzyme rAbf43A also liberated a series of xylo-oligosaccharides (XOSs) from beechwood xylan, xylohexaose and xylopentaose, indicating that rAbf43A exhibited endo-xylanase activity. Homology modelling based on AlphaFold2 and site-directed mutagenesis identified three non-catalytic residues (H161, A270 and L505) located in the substrate-binding pocket essential for its dual-functionality, while the mutation of A117 located in the -1 subsite to the proline residue only affected its endo-xylanase activity. Additionally, rAbf43A showed significant synergistic action with the bifunctional xylanase/feruloyl esterase rXyn10A/Fae1A from the same bacterial consortium on insoluble wheat arabinoxylan and de-starched wheat bran degradation. When rXyn10A/Fae1A was added to the rAbf43A pre-hydrolyzed reactions, the amount of released reducing sugars, xylose and ferulic acid increased by 9.43% and 25.16%, 189.37% and 93.54%, 31.39% and 32.30%, respectively, in comparison with the sum of hydrolysis products released by each enzyme alone. The unique characteristics of rAbf43A position it as a promising candidate not only for designing high-performance enzyme cocktails but also for investigating the structure-function relationship of GH43 multifunctional enzymes.

Project description:BackgroundCellulose and hemicellulose are the two largest components in lignocellulosic biomass. Enzymes with activities towards cellulose and xylan have attracted great interest in the bioconversion of lignocellulosic biomass, since they have potential in improving the hydrolytic performance and reducing the enzyme costs. Exploring glycoside hydrolases (GHs) with good thermostability and activities on xylan and cellulose would be beneficial to the industrial production of biofuels and bio-based chemicals.ResultsA novel GH10 enzyme (XynA) identified from a xylanolytic strain Bacillus sp. KW1 was cloned and expressed. Its optimal pH and temperature were determined to be pH 6.0 and 65 °C. Stability analyses revealed that XynA was stable over a broad pH range (pH 6.0-11.0) after being incubated at 25 °C for 24 h. Moreover, XynA retained over 95% activity after heat treatment at 60 °C for 60 h, and its half-lives at 65 °C and 70 °C were about 12 h and 1.5 h, respectively. More importantly, in terms of substrate specificity, XynA exhibits hydrolytic activities towards xylans, microcrystalline cellulose (filter paper and Avicel), carboxymethyl cellulose (CMC), cellobiose, p-nitrophenyl-β-d-cellobioside (pNPC), and p-nitrophenyl-β-d-glucopyranoside (pNPG). Furthermore, the addition of XynA into commercial cellulase in the hydrolysis of pretreated corn stover resulted in remarkable increases (the relative increases may up to 90%) in the release of reducing sugars. Finally, it is worth mentioning that XynA only shows high amino acid sequence identity (88%) with rXynAHJ14, a GH10 xylanase with no activity on CMC. The similarities with other characterized GH10 enzymes, including xylanases and bifunctional xylanase/cellulase enzymes, are no more than 30%.ConclusionsXynA is a novel thermostable GH10 xylanase with a wide substrate spectrum. It displays good stability in a broad range of pH and high temperatures, and exhibits activities towards xylans and a wide variety of cellulosic substrates, which are not found in other GH10 enzymes. The enzyme also has high capacity in saccharification of pretreated corn stover. These characteristics make XynA a good candidate not only for assisting cellulase in lignocellulosic biomass hydrolysis, but also for the research on structure-function relationship of bifunctional xylanase/cellulase.

Dataset Information

Novel Trifunctional Xylanolytic Enzyme Axy43A from Paenibacillus curdlanolyticus Strain B-6 Exhibiting Endo-Xylanase, ?-d-Xylosidase, and Arabinoxylan Arabinofuranohydrolase Activities.

Publications

Novel Trifunctional Xylanolytic Enzyme Axy43A from Paenibacillus curdlanolyticus Strain B-6 Exhibiting Endo-Xylanase, β-d-Xylosidase, and Arabinoxylan Arabinofuranohydrolase Activities.

Similar Datasets

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