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

利用宏基因组文库方法,通过功能筛选法从文库中筛选到一个新型β-葡萄糖苷酶基因bgl2238(Gen Bank:KU320675.1),对新基因进行生物信息学分析和原核表达,并研究其酶学性质。结果表明,该基因全长2 238 bp,可编码745个氨基酸,通过氨基酸序列分析,预测bgl2238蛋白分子质量为80.67 k Da,p I值为4.95,是一种结构较稳定、疏水性高且无信号肽序列的酸性蛋白质;经同源性分析,发现其与来源于Bacteroides thetaiotaomicron的β-葡萄糖苷酶具有58%的相似性,属于GH3超家族和GH3C超家族酶。将重组质粒p ET-32a(+)-bgl2238转化入Escherichia coli BL2l(DE3)细胞进行异源表达,酶学性质测定结果显示,以对硝基苯基-β-D-吡喃葡萄糖苷(p-nitrophenyl-β-D-glucopyranoside,p NPG)为底物,β-葡萄糖苷酶bgl2238最适反应p H值和温度分别为6.10、44℃,此时该酶最大比活力达到29.1 U/mg;且在4~50℃范围内,热处理β-葡萄糖苷酶bgl2238 4 h后仍保留70%以上的酶活力,热稳定性较好;其动力学参数Vmax为576?μmo L/(L·min),Km为0.296 mmol/L;不同浓度的K+、Na~+、Fe~(2+)、Mg~(2+)、Mn~(2+)、Ca~(2+)、Co~(2+)和Ni~(2+)对酶活力均有较大促进作用,其中Fe~(2+)对bgl2238酶活力的促进作用最大,10 mmol/L Fe~(2+)使相对酶活力高达260%,而重金属离子Ag~+、Hg~(2+)及Cu~(2+)对酶活力有抑制作用。bgl2238具有良好的酶学性质,可以尝试应用于工业上纤维素降解的生产。

Screening,Cloning and Characterization of a Novel β-Glucosidase from Soil Metagenomic Library

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 Km and Vmax values 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.