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Expression and characterization of a GH43 endo-arabinanase from Thermotoga thermarum.

ABSTRACT: BACKGROUND: Arabinan is an important plant polysaccharide degraded mainly by two hydrolytic enzymes, endo-arabinanase and ?-L-arabinofuranosidase. In this study, the characterization and application in arabinan degradation of an endo-arabinanase from Thermotoga thermarum were investigated. RESULTS: The recombinant endo-arabinanase was expressed in Escherichia coli BL21 (DE3) and purified by heat treatment followed by purification on a nickel affinity column chromatography. The purified endo-arabinanase exhibited optimal activity at pH 6.5 and 75°C and its residual activity retained more than 80% of its initial activity after being incubated at 80°C for 2 h. The results showed that the endo-arabinanase was very effective for arabinan degradation at higher temperature. When linear arabinan was used as the substrate, the apparent K(m) and V(max) values were determined to be 12.3?±?0.15 mg ml?¹ and 1,052.1?±?12.7 ?mol ml?¹ min?¹, respectively (at pH 6.5, 75°C), and the calculated kcat value was 349.3?±?4.2 s?¹. CONCLUSIONS: This work provides a useful endo-arabinanase with high thermostability andcatalytic efficiency, and these characteristics exhibit a great potential for enzymatic conversion of arabinan.

SUBMITTER: Shi H

PROVIDER: S-EPMC4021227 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Expression and characterization of a GH43 endo-arabinanase from Thermotoga thermarum.

Shi Hao H Ding Huaihai H Huang Yingjuan Y Wang Liangliang L Zhang Yu Y Li Xun X Wang Fei F

BMC biotechnology 20140430

<h4>Background</h4>Arabinan is an important plant polysaccharide degraded mainly by two hydrolytic enzymes, endo-arabinanase and α-L-arabinofuranosidase. In this study, the characterization and application in arabinan degradation of an endo-arabinanase from Thermotoga thermarum were investigated.<h4>Results</h4>The recombinant endo-arabinanase was expressed in Escherichia coli BL21 (DE3) and purified by heat treatment followed by purification on a nickel affinity column chromatography. The purif ...[more]

PMID: 24886412

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Project description:?-Xylosidase is an important constituent of the hemicellulase system and it plays an important role in hydrolyzing xylooligosaccharides to xylose. Xylose, a useful monose, has been utilized in a wide range of applications such as food, light, chemical as well as energy industry. Therefore, the xylose-tolerant ?-xylosidase with high specific activity for bioconversion of xylooligosaccharides has a great potential in the fields as above.A ?-xylosidase gene (Tth xynB3) of 2,322 bp was cloned from the extremely thermophilic bacterium Thermotoga thermarum DSM 5069 that encodes a protein containing 774 amino acid residues, and was expressed in Escherichia coli BL21 (DE3). The phylogenetic trees of ?-xylosidases were constructed using Neighbor-Joining (NJ) and Maximum-Parsimony (MP) methods. The phylogeny and amino acid analysis indicated that the Tth xynB3 ?-xylosidase was a novel ?-xylosidase of GH3. The optimal activity of the Tth xynB3 ?-xylosidase was obtained at pH 6.0 and 95°C and was stable over a pH range of 5.0-7.5 and exhibited 2 h half-life at 85°C. The kinetic parameters Km and Vmax values for p-nitrophenyl-?-D-xylopyranoside and p-nitrophenyl-?-L-arabinofuranoside were 0.27 mM and 223.3 U/mg, 0.21 mM and 75 U/mg, respectively. The kcat/Km values for p-nitrophenyl-?-D-xylopyranoside and p-nitrophenyl-?-L-arabinofuranoside were 1,173.4 mM-1 s-1 and 505.9 mM-1 s-1, respectively. It displayed high tolerance to xylose, with Ki value approximately 1000 mM. It was stimulated by xylose at higher concentration up to 500 mM, above which the enzyme activity of Tth xynB3 ?-xylosidase was gradually decreased. However, it still remained approximately 50% of its original activity even if the concentration of xylose was as high as 1000 mM. It was also discovered that the Tth xynB3 ?-xylosidase exhibited a high hydrolytic activity on xylooligosaccharides. When 5% substrate was incubated with 0.3 U Tth xynB3 ?-xylosidase in 200 ?L reaction system for 3 h, almost all the substrate was biodegraded into xylose.The article provides a useful and novel ?-xylosidase displaying extraordinary and desirable properties: high xylose tolerance and catalytic activity at temperatures above 75°C, thermally stable and excellent hydrolytic activity on xylooligosaccharides.

Dataset Information

Expression and characterization of a GH43 endo-arabinanase from Thermotoga thermarum.

Publications

Expression and characterization of a GH43 endo-arabinanase from Thermotoga thermarum.

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