Overexpression of celB gene coding for beta-glucosidase from Pyrococcus furiosus using a baculovirus expression vector system in silkworm, Bombyx mori.
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 Pyrococcus 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 haemolymph 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.
Lin
Xu'Ai
X
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, China.
Zhang
Wei
W
Chen
Yin
Y
Yao
Bin
B
Zhang
Zhi Fang
ZF
D000818:Animals; D019843:Archaeal Proteins / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics*; D012831:Bombyx / Q000201:enzymology; D017931:DNA Primers; D019848:Gene Expression Regulation, Archaeal; D015971:Gene Expression Regulation, Enzymologic; D020745:Genome, Archaeal; D007700:Kinetics; D007814:Larva; D020027:Pyrococcus furiosus / Q000201:enzymology / Q000235:genetics*; D011994:Recombinant Proteins / Q000378:metabolism; D001617:beta-Glucosidase / Q000235:genetics*
D019843:Archaeal Proteins; D001426:Bacterial Proteins; D017931:DNA Primers; D011994:Recombinant Proteins; C406923:CelB protein, Pyrococcus furiosus; D001617:beta-Glucosidase