Dataset Information

Cloning, expression and characterization of a glycoside hydrolase family 51 α-l-arabinofuranosidase from Thermoanaerobacterium thermosaccharolyticum DSM 571.

ABSTRACT: The present study focused on the characterization of a glycoside hydrolase 51 family α-l-arabinofuranosidase named TtAbf51 from thermophile Thermoanaerobacterium thermosaccharolyticum DSM 571. The recombinant TtAbf51 with 497 amino acids was successfully expressed in Escherichia coli BL21(DE3) and purified via nickel affinity chromatography, and native protein was a dimer verified by size exclusion chromatography. The TtAbf51 showed an optimum pH and temperature of 5.5 and 55 °C, and was relatively stable at pH 5.0-8.0 and up to 60 °C for 2 h of incubation. In addition, TtAbf51 was significantly inhibited by Cu²⁺, Zn²⁺ and 1 mM or 10 mM SDS. In the presence of 800 mM arabinose, the residual activity remained over 40% of the initial activity. In addition, the recombinant enzyme possessed a good catalytic effect for both synthesized and natural substrates, and the specific enzyme activity toward CM-linear arabinan reached 426.5 μmol min^-1 mg^-1. In summary, this study provides an α-l-arabinofuranosidase with potential in the synergistic hydrolysis of hemicellulose to fermentable sugars in applications such as liquid biofuels, food and beverages, and related industries.

SUBMITTER: Shi H

PROVIDER: S-EPMC9288575 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature

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Cloning, expression and characterization of a glycoside hydrolase family 51 α-l-arabinofuranosidase from Thermoanaerobacterium thermosaccharolyticum DSM 571.

Shi Hao H Gao Feng F Yan Xing X Li Qingfei Q Nie Xinling X

3 Biotech 20220716 8

The present study focused on the characterization of a glycoside hydrolase 51 family α-l-arabinofuranosidase named TtAbf51 from thermophile Thermoanaerobacterium thermosaccharolyticum DSM 571. The recombinant TtAbf51 with 497 amino acids was successfully expressed in Escherichia coli BL21(DE3) and purified via nickel affinity chromatography, and native protein was a dimer verified by size exclusion chromatography. The TtAbf51 showed an optimum pH and temperature of 5.5 and 55 °C, a ...[more]

PMID: 35855476

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Project description:A GH (glycoside hydrolase) family 54 alpha-L-arabinofuranosidase from Trichoderma koningii G-39 (termed Abf) was successfully expressed in Pichia pastoris and purified to near homogeneity by cation-exchange chromatography. To determine the amino acid residues essential for the catalytic activity of Abf, extensive mutagenesis of 24 conserved glutamate and aspartate residues was performed. Among the mutants, D221N, E223Q and D299N were found to decrease catalytic activity significantly. The kcat values of the D221N and D299N mutants were 7000- and 1300-fold lower respectively, than that of the wild-type Abf. E223Q was nearly inactive. These results are consistent with observations obtained from the Aspergillus kawachii alpha-L-arabinofuranosidase three-dimensional structure. This structure indicates that Asp221 of T. koningii Abf is significant for substrate binding and that Glu223 as well as Asp299 function as a nucleophile and a general acid/base catalyst for the enzymatic reaction respectively. The catalytic mechanism of wild-type Abf was further investigated by NMR spectroscopy and kinetic analysis. The results showed that Abf is a retaining enzyme. It catalyses the hydrolysis of various substrates via the formation of a common intermediate that is probably an arabinosyl-enzyme intermediate. A two-step, double-displacement mechanism involving first the formation, and then the breakdown, of an arabinosyl-enzyme intermediate was proposed. Based on the kcat values of a series of aryl-alpha-L-arabinofuranosides catalytically hydrolysed by wild-type Abf, a relatively small Brønsted constant, beta(lg)=-0.18, was obtained, suggesting that the rate-limiting step of the enzymatic reaction is the dearabinosylation step. Further kinetic studies with the D299G mutant revealed that the catalytic activity of this mutant depended largely on the pK(a) values (>6) of leaving phenols, with beta(lg)=-1.3, indicating that the rate-limiting step of the reaction becomes the arabinosylation step. This kinetic outcome supports the idea that Asp299 is the general acid/base residue. The pH activity profile of D299N provided further evidence strengthening this suggestion.

Dataset Information

Cloning, expression and characterization of a glycoside hydrolase family 51 α-l-arabinofuranosidase from Thermoanaerobacterium thermosaccharolyticum DSM 571.

Publications

Cloning, expression and characterization of a glycoside hydrolase family 51 α-l-arabinofuranosidase from <i>Thermoanaerobacterium thermosaccharolyticum</i> DSM 571.

Similar Datasets

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