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

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.

Key words:β-galactosidase;fecal microorganism;metagenome;enzymatic properties