Characterization of a novel beta-glucosidase-like activity from a soil metagenome.
We report the cloning of a novel beta-glucosidase-like gene by function-based screening of a metagenomic library from uncultured soil microorganisms. The gene was named bgllC and has an open reading frame of 1,443 base pairs. It encodes a 481 amino acid polypeptide with a predicted molecular mass of about 57.8 kDa. The deduced amino acid sequence did not show any homology with known beta-glucosidases. The putative beta-glucosidase gene was subcloned into the pETBlue-2 vector and overexpressed in E. coli Tuner (DE3) pLacI; the recombinant protein was purified to homogeneity. Functional characterization with a high performance liquid chromatography method demonstrated that the recombinant BgllC protein hydrolyzed D-glucosyl-beta-(l-4)-D-glucose to glucose. The maximum activity for BgllC protein occurred at pH 8.0 and 42 degrees C using p-nitrophenyl-beta-D-glucoside as the substrate. A CaCl(2) concentration of 1 mM was required for optimal activity. The putative beta-glucosidase had an apparent K(m) value of 0.19 mM, a V(max) value of 4.75 U/mg and a k (cat) value of 316.7/min under the optimal reaction conditions. The biochemical characterization of BgllC has enlarged our understanding of the novel enzymes that can be isolated from the soil metagenome.
College of Life Science and Technology, Guangxi Key Laboratory of Subtropical Bioresources Conservation and Utilization, The Key Laboratory of Ministry of Education for Microbial and Plant Genetic Engineering, Guangxi University, Guangxi, PR China.
Chengjian
C
Jiang
Gefei
G
Ma
Shuangxi
S
Li
Tingting
T
Hu
Zhiqun
Z
Che
Peihong
P
Shen
Bing
B
Yan
Bo
B
Wu
D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D054892:Metagenome; D056186:Metagenomics / Q000379:methods; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012988:Soil Microbiology; D013379:Substrate Specificity; D013696:Temperature; D001617:beta-Glucosidase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
D001617:beta-Glucosidase