食品科学2018，Vol.39Issue(4)：118-124,7.DOI:10.7506/spkx1002-6630-201804018
宏基因组文库新型β-葡萄糖苷酶的筛选、克隆及酶学性质分析
Screening, Cloning and Characterization of a Novel β-Glucosidase from Soil Metagenomic Library
摘要
Abstract
A novel β-glucosidase gene,bgl2238,was isolated from a metagenomic library by functional screening(GenBank:KU320675.1).The bgl2238 gene was sequenced and expressed in Escherichia coli BL21(DE3).The biochemical properties of the resulting protein were further examined. Amino acid sequence analysis showed that the full length of the gene was 2 238 bp, which encoded a protein of 745 amino acids. The β-glucosidase had a molecular mass of 80.67 kDa with an isoelectric point (pI) of 4.95, and it was identified as an acidic protein with stable structure and high hydrophobicity and without signal peptide sequence.Phylogenetic analysis of the bgl2238 gene indicated 58% similarity to the β-glucosidase from Bacteroides thetaiotaomicron,and it belonged to the GH3 and GH3C superfamily.bgl2238 was ligated into the vector pET-32a(+) and then transformed into Escherichia coli BL21 (DE3) for heterologous expression.The maximal activity (29.1 U/mg) of the enzyme was observed at 44 ℃ and pH 6.10 with 4-nitrophenyl-β-D-glucopyranoside (pNPG) as the substrate; furthermore it retained 70% of its activity within the temperature range of 4–50 ℃ for 4 h. The Kmand Vmaxvalues of bgl2238 were 0.296 mmol/L and 576 μmoL/(L·min), respectively. Its activity was enhanced significantly by K+, Na+, Fe2+, Mg2+, Mn2+, Ca2+, Co2+and Ni2+at various concentrations; among these metal ions, Fe2+was found to have the biggest promoting effect and a relative activity up to 260% was attained at a Fe2+concentration of 10 mmol/L. On the other hand, the presence of heavy metal ions (Ag+, Hg2+and Cu2+) inhibited its activity. To sum up, these characteristics were beneficial for the potential application of bgl2238 in industrial cellulose degradation.关键词
宏基因组文库/β-葡萄糖苷酶/克隆表达/酶学性质Key words
metagenome library/β-glucosidase/cloning and expression/enzymatic properties分类
生物科学引用本文复制引用
唐乐丽,王晓萌,吴秀玲,黄庆,李荷..宏基因组文库新型β-葡萄糖苷酶的筛选、克隆及酶学性质分析[J].食品科学,2018,39(4):118-124,7.基金项目
广东省科技厅项目(2013B010404044 （）
2017A010105011) （）
国家自然科学基金青年科学基金项目(3140068) （3140068）
广东省教育厅项目(2013KJCX0107 （）
2016KTSCX067) （）
广东省攀登计划项目(pdjh2017b0262) （pdjh2017b0262）
