Overexpression of celB gene coding for beta-glucosidase from Pyrococcus furiosus using a baculovirus expression vector system in silkworm, Bombyx mori

文献类型： 外文期刊

第一作者： Lin, Xu'Ai

作者： Lin, Xu'Ai;Zhang, Wei;Chen, Yin;Bin Yao;Zhang, Zhi Fang

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关键词： beta-glucosidase;Pyrococcus furiosus;baculovirus expression vector system;ESCHERICHIA-COLI;ALPHA-AMYLASE;PROTEIN;ENZYME;PURIFICATION;LACTOSE;CLONING;MILK

期刊名称：ZEITSCHRIFT FUR NATURFORSCHUNG C-A JOURNAL OF BIOSCIENCES （ 影响因子：0.72； ）

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收录情况： SCI

摘要： beta-Glucosidase is a member of the glycosyl hydrolases that specifically catalyze the hydrolysis of terminal nonreducing beta-D-glucose residues from the end of various oligosaccharides with the release of beta-D-glucose. CelB gene, encoding the thermostable beta-glucosidase, was amplified from the Pyrococcies furiosus genome and then cloned into the baculoviral transfer vector under the control of the polyhedrin gene promoter. After co-transfection with the genetically modified parental Bombyx mori nucleopolyhedrovirus (BmNPV), the recombinant virus containing celB gene was used to express beta-glucosidase in silkworm. The recombinant beta-glucosidase was purified to about 81% homogeneity in a single heat-treatment step. The optimal activity of the expressed beta-glucosidase was obtained at pH 5.0 and about 105 degrees C; divalent cations and high ionic strength did not affect the activity remarkably. This suggested that the enzymatic characteristics of recombinant beta-glucosidase were similar to the native counterpart. The expressed beta-glucosidase accounted for more than 10% of silkworm total haernolymph proteins according to the protein quantification and densimeter scanning. The expression level reached 10,199.5 U per ml haemolymph and 19,797.4 U per silkworm larva, and the specific activity of the one-step purified crude enzyme was 885 U per mg. It was demonstrated to be an attractive approach for mass production of thermostable beta-glucosidase using this system.

分类号： Q

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