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Cloning and identification of GH11 family thermostable xylanase gene from biomass degradation strains
Pages: 12-17
Year: Issue:  11
Journal: Food and Fermentation Industries

Keyword:  biomassmetagenomemTAIL-PCRthermostable xylanasegene expressionenzymic properties;
Abstract: The biomass degradation strains in cow dung were enrichment cultured using corncob powder as substrates. And then the metagenome DNA of the biomass degradation strains were extracted and purified. Various DNA segments encoding partial of xylanases,which were about 200bp,were obtained through degenerate primer and the metagenome DNA model. In the PCR segments,the most abundant DNA sequence with the highest similarity with thermostable xylanases was used to amplify the whole gene sequence by mTAIL-PCR. The new gene( xynHAD4) was1 059bp encoding 352 amino acids. The new xylanase( XynHAD4) was belonged to Family GH11 of xylanases and showed the highest similarity by 93% with the thermostable xylanase 229B from Dictyoglomus thermophilum. The new gene was cloned into the expression vector pET-22b and expressed in E. coli BL21( DE3). The recombinant xylanase was purified by Ni2 +-NTA affinity chromatography. The molecular weight of the purified XynHAD4 was estimated to be 36kDa by SDS-PAGE. It revealed optimal activity at 80° C and pH 6. 0 and remained high activity over a large range of pH( 4. 0 ~ 10. 0). It remained at least 82% residual activity at pH 4. 0 for 1 h. Therefore,the thermostable xylanase( XynHAD4) with high stability at acidic condition is a promising candidate for food and feed industry.
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