Abstract:[Background] β-glucosidase (EC 3.2.1.21) is an important component of cellulase system. At present, most β-glucosidases used in industry come from plants and fungi, but few come from bacteria, and there are some problems such as low enzyme activity, poor thermal stability, narrow reaction conditions, products inhibition, which increase the economic cost. Thermophilic microorganisms have special genetic information resources, so it is possible to excavate novel β-glucosidases with good enzymatic characterization from the genome to solve the industrial problems. [Objective] A novel β-glucosidase gene was extracted from the genome of Bacillus thermoamylovorans, and purified protein was obtained by gene recombination, heterologous expression and protein purification. The enzymatic characterization was studied systematically. It can lay the foundation for the application of β-glucosidase in the fields of cellulose hydrolysis. [Methods] The recombinant plasmid pET22b-bgl52 was constructed and transformed into Escherichia coli BL21(DE3) by electric pulse method. The recombinant protein was expressed in soluble form and purified by Ni-NTA affinity chromatography. [Results] The recombinant plasmid pET22b-bgl52 was expressed in E. coli BL21(DE3) and purified β-glucosidase Bgl52 protein was obtained. The molecular weight of the Bgl52 was 52 kD and it showed the best activity at 70 °C and pH 6.5. When p-nitrophenyl-β-D-glucopyranoside (pNPG) as substrate, the specific enzyme activity was 223.7±5.3 U/mg, Km was 9.3±1.2 mmol/L, Vmax was 270.3±4.3 μmol/(min·mg). Bgl52 preferred substrate for hydrolysis of β-1,4 glycosidic bond. Fe2+ and Mg2+ activated the enzyme activity obviously, Co2+, Cu2+ and SDS inhibited the activity of enzyme. Bgl52 is one of the few glucose and xylose-activated glucosidases. A maximal 2.84-fold stimulation by glucose was observed at 0.2 mol/L, and a maximal 3.24-fold stimulation by xylose was found at 0.4 mol/L. At the same time, under physiological conditions, Bgl52 was not substantially inhibited by the feedback of the product glucose. [Conclusion] Using the genetic information resources contained in the genomes of thermophilic microorganisms, and through modern biotechnological methods such as gene synthesis, we can excavate the β-glucosidase with excellent enzymatic characterization, it lays a foundation for its application in cellulose degradation and other industrial fields.
