Purification and characterization of GH1 β-glucosidase from fecal microbes metagenome

Abstract:

[Background] β-glucosidase is an important class of cellulolytic enzymes. At present, β-glucosidase (Bgl) derived from cultivable microorganisms has the problems of poor thermal stability and acid-base stability and a narrow range of action. [Objective] Discover new β-glucoside genes from the fecal microbial metagenomics of Rhinopithecus bieti, express heterologously and study its enzymatic properties, and provide new enzyme resources for food and other fields. [Methods] Starting from the fecal microbial metagenome, the β-glucosidase genes BglRBS_26 and BglRBS_9 were amplified and heterologously expressed, and the enzymatic properties were studied. [Results] The GH1 family recombinant β-glucosidase BglRBS_26 and BglRBS_9 were obtained, with molecular weights of 60 kD and 50 kD, respectively. The optimal conditions for BglRBS_26 are pH 6.0 and 45 ℃; the optimal conditions for BglRBS_9 are pH 5.0 and 40 ℃. The K_m of BglRBS_26 and BglRBS_9 are (0.681 6±0.164 2) μmol/L and (3.317 0±0.871 4) μmol/L, respectively. BglRBS_26 has good acid-base tolerance. After treatment at pH 5.0–6.0 for 1 h, the remaining enzyme activity is greater than 110%; in the range of pH 7.0–8.0, the remaining enzyme activity remains above 100%. In addition, sucrose can activate BglRBS_26 and BglRBS_9 to varying degrees. When 20% (M/V) sucrose is added to the reaction system, the enzyme activity of BglRBS_26 can be increased to 140%; 10% (M/V) sucrose can increase the enzyme activity of BglRBS_9 to 180%. In addition, BglRBS_26 has better NaCl tolerance and stability. It retains 80% enzyme activity after being treated at 37 ℃ and 2.5 mol/L NaCl for 1 h. [Conclusion] In this study, two novel β-glucosidase genes BglRBS_26 and BglRBS_9 were obtained from the R. bieti fecal microbial metagenome, and they were successfully expressed in E. coli BL21(DE3). BglRBS_26 and BglRBS_9 have good pH stability and sucrose tolerance, making them have potential applications in food, fermentation and other industries.