土壤宏基因组文库中3个新β-葡萄糖苷酶基因的克隆及其酶学特性

Cloning and Enzymatic Characteristics of Three New β-glucosidases in Soil Metagenomic Library

为从喀斯特土壤宏基因组DNA中筛选获得新型β-葡萄糖苷酶基因,采用β-葡萄糖苷酶活性筛选策略,从宏基因组文库中筛选β-葡萄糖苷酶基因,并对其进行初步酶学性质分析。结果表明:构建宏基因组文库中外源DNA总量约为5.4×107bp,平均插入片段约为2.7kb。筛选获得3个编号为pGLU-001、pGLU-002和pGLU-003的表达β-葡萄糖苷酶活性的克隆。其外源片段分别包含有1 383bp、1 437bp和1 227bp的ORF,分别编码460、478和408氨基酸组成的蛋白质,PI分别为5.12、4.78和8.78,蛋白质分子量分别为52.3KD、52.6KD和47.7KD。BLAST软件分析结果,pGLU-001和pGLU-002分别与来自Di-ctyoglomus thermophilum和Bacillus subtilis TU-B-10的β-葡萄糖苷酶的氨基酸组成上有78.3%和66.7%的相似性,而pGLU-003经BLAST比对后未发现与目前报道的β-葡萄糖苷酶基因的核酸及氨基酸水平上有相似性,从而推测pGLU-003蛋白是来源于未培养微生物的新型β-葡萄糖苷酶。进一步酶学性质研究表明,3个β-葡萄糖苷酶最适温度分别约为30℃、40℃及50℃,最适pH分别约为6.0、9.0及10.0。Mg2+对3个酶有较强的促进作用,同时Zn2+对pGLU-001也有较强的促进作用,而EDTA和Fe3+对于3个酶都有不同程度的抑制作用。

Three β-glucosidases were successfully slected from metagenomic library by determining β- glucosidase activity and analyzing their enzymatic property. The results showed that the exogenous DNA amount in the established metagenomic library was about 5.4 ×107 bp and the average inserted fragment was 2.7 kb. Three β-glucosidase clones respectively named as pGLU-001, pGLU-002 and pGLU-Q03 were obtained and their exogenous fragments encoded as 460, 478 and 408 amino acids contained 1 383 bp, 1 437 bp and 1 227 bp ORFs respectively. Their PI were 5.12, 4.78 and 8.78, and the size of protein molecular weight were 52.3 KD, 52.6 KD and 47.7 KD respectively. The BLAST software analysis results showed that pGLU-001 and pGLU-002 had 78. 3% and 66.7% similarity with animo acids of β-glueosidase from Dietyoglomus thermophilum and Bacillus subtilis TU-B-10 separately but pGLU-003 had no similarity with the known β-glucosidase genes in the Genbank, which indicates that pGLU-003 may be a new β-glucosidase from uncultured microorganisms. The optimal temepature and pH for pGLU-001, pGLU-002 and pGLU- 003 were 30℃, 40℃ and 50℃, and 6.0, 9.0 and 10.0 respectively. Mg^2＋ could improve the enzymatic activity of three β-glucosidases significantly but Zn^2＋ could enhance the enzymatic activity of pGLU-001. EDTA and Fe^3＋ had different inhibition effect on enzymatic activity of three β-glucosidases.