Cloning,Expression And Molecular Modification Of ?-glucosidase BGL3 Gene From Chaetomium Thermophilus

Cellulose is a natural polymer polysaccharide that is widely distributed on the earth.Its efficient use can solve major problems such as resource depletion and environmental pollution.Cellulase is a multi-component enzyme in which?-glucosidase is the key rate-limiting enzyme for the degradation of cellulose.This study uses Chaetomium thermophilum as experimental material,non-conserved amino acids of?-glucosidase active channel and conserved amino acids that may interact with the substrate at the catalytic center of enzyme from GH3 to have site-directed mutagenesis,enzymatic properties of mutanted enzymes were studied,and key amino acid sites for enzyme-substrate binding and the thermostable mutanted enzymes were screened out.The main findings are as follows:Gene bgl3 derived of GH3?-glucosidase from Chaetium thermophilum was cloned and introduced into Pichia pastoris,the expressed protein was isolated and purified.The protein purified product was detected by SDS-PAGE electrophoresis,and protein size was 120 kDa,which was larger than the predicted theoretical molecular weight,indicating that the glycosylation modification occurred during protein translation,and the wild-type enzyme BGL3 was obtained.The specific activity of BGL3 was 36.1U/mg,and the optimum temperature and pH of the enzymatic reaction were 60?and 5.0.The K_m,k_cat,and k_cat/K_m values were 4.27±0.032 mM,20.16±0.13 s^-11 and 4.72±0.061 s^-1×mM^-1.The wild enzyme was incubated at 60?and 70?for 10 minutes,maintaining enzyme activities of 65.6%and 7.0%,respectively.In order to obtain well-characterized mutanted enzymes,site-directed mutagenesis was performed on eight non-conserved amino acids G148,N187,A236,M267,Q273,M288,W438 and R508 in the active channel of the enzyme,resulting in mutanted enzymes G148A,N187Y,A236S,M267,Q273H,M288A,W438G and R508M.4mM to salicin as a substrate,measuring the enzymatic properties of the mutanted enzymes.Compared with the wild-type enzyme BGL3,the K_m value of N187Y decreased,and the K_m value of other mutanted enzymes increased;the k_catat value of R508M increased,and the k_catat value of other mutanted enzymes decreased;the optimal temperature of the eight mutanted enzymes was 60?,the optimal pH for reaction of A236S was 4.0,and the optimal pH of other mutants was 5.0.The relative enzymatic activities of mutanted enzymes W438G and M288A retain 30.9%and62.2%after incubation for 10 minutes at 70?.In order to screen the key amino acid sites for binding of the enzyme to the substrate,homologous modeling was performed on BGL3,and 11 amino acid sites that may bind to the substrate were mutated to alanine A,which was then introduced into Pichia pastoris for expression,to get V67A,D85A,R91A,L134A,R149A,K182A,H183A,R193A,M234A,Y237A and W270A mutanted enzymes.4mM to salicin as a substrate,the enzymatic properties of the mutanted enzyme were determined.Compared with the wild-type enzyme BGL3,the enzyme activities of the mutanted enzymes R149A,K182A and M234A were completely lost,and the enzyme activities of other mutanted enzymes also decreased to varying degrees;the optimal temperature for reaction and pH of 11 mutanted enzymes were60?and 5.0;The k_catat of the mutanted enzymes V67A and Y237A increased and the other mutanted k_catat decreased.The K_m value of V67A,D85A and Y237A increased.This experiment screened the key amino acid sites for enzyme and substrate binding,providing reference for the further molecular modification of GH3?-glucosidase.