粪便宏基因组来源低分子量碱性β-半乳糖苷酶的异源表达及酶学性质

Heterologous expression and enzyme properties of low-molecular-weight and basic β-galactosidase derived from the metagenome of faeces

摘要:

【背景】β-半乳糖苷酶在食品加工、临床医疗及基因工程等领域有重要的应用价值，开发酶活性高、热稳定性强的β-半乳糖苷酶已成为研究热点。【目的】从西黑冠长臂猿(Nomascusconcolor)粪便微生物宏基因组中挖掘新型β-半乳糖苷酶并进行酶学性质研究。【方法】以西黑冠长臂猿粪便微生物宏基因组DNA为模板扩增β-半乳糖苷酶基因GalNC1-8，构建重组表达质粒pEASY-E2/GalNC1-8，转化至大肠杆菌(Escherichia coli)BL21(DE3)异源表达，研究其酶学性质。【结果】获得GH35家族碱性β-半乳糖苷酶GalNC1-8，其分子量为28.18 kDa，最适温度为50℃，最适pH为8.0。将该酶在30−50℃下处理1 h，剩余酶活仍保持在80%以上；pH 7.0−9.0下处理1 h，剩余酶活大于54%。在含乙醇的反应体系中，其酶活性几乎不受影响；β-巯基乙醇、丙三醇、甲醇、Na⁺、K⁺和Li⁺对其酶活性有促进作用。在0.5−3.5 mol/L NaCl下处理1 h后，仍保留50%以上的酶活性。【结论】从粪便微生物宏基因组中克隆得到β-半乳糖苷酶基因GalNC1-8，并在E.coli BL21(DE3)中成功表达。GalNC1-8是已知宏基因组来源蛋白质分子量最小的β-半乳糖苷酶，其pH作用范围较广、温度稳定性和NaCl耐受性较好，具有较好的应用前景。

Abstract:

[Background] β-galactosidase has important application value in many fields such as food processing, clinical medicine, and genetic engineering. Thus, the development of β-galactosidase with high activity and strong thermal stability has become a research focus. [Objective] This paper aims to screen novel β-galactosidases from the fecal microbial metagenome of Nomascus concolor and study its enzymatic properties. [Methods] β-galactosidase gene GalNC1-8 was cloned by PCR with the fecal microbial metagenomic DNA of N. concolor as template. Then, the recombinant plasmid pEASY-E2/GalNC1-8 was constructed and transformed into Escherichia coli BL21(DE3) for expression, and the enzymatic properties of recombinase GalNC1-8 was tested. [Results] The basic β-galactosidase GalNC1-8, belonging to GH35 family, had the molecular weight of 28.18 kDa. The optimal conditions for GalNC1-8 were pH 8.0 and 50 °C. After incubation at 30, 37, 40, and 50 °C for 1 h, the relative enzyme activity was still above 80%. After 1 h treatment at pH 7.0−9.0, the enzyme kept more than 54% of the activity. The activity of GalNC1-8 was hardly influenced by the reaction system containing ethanol. β-mercaptoethanol, glycerol, methanol, Na⁺, K⁺, and Li⁺ enhanced the enzyme activity. It retained above 50% activity after being treated with 0.5−3.5 mol/L NaCl at 50 °C for 1 h. [Conclusion] a novel β-galactosidase gene GalNC1-8 was screened from the N. concolor fecal microbial metagenome, which expressed in E. coli BL21(DE3). GalNC1-8 has the lowest molecular weight among the known metagenome-derived β-galactosidases. With adaptability to a wide pH range, thermal stability, and salt tolerance, it has good application prospects.