Co-directed Evolution Of Optimum Temperature,Activity And Product Tolerance Of ?-glucosidase

Cellulose is widely distributed on the earth and is an available natural high-molecular substance.?-glucosidase?BGL?is a rate-limiting enzyme in the process of cellulose hydrolysis.At present,BGL is still unable to improve product tolerance and activity simultaneously through rational design.In addition,there is a large gap between the optimal temperature of ethanol fermentation of Saccharomyces cerevisiae and the optimal temperature of BGL.In this study,BGL in Penicillium oxalicum 16 screened by the lab was looked on as research object.Total RNA was extracted from it,and BGL cDNA was obtained by reverse transcription PCR.Then PCR amplification was performed to obtain the16BGL gene without its signal peptide.Using the 16BGL gene sequence as a template,a round of error-prone PCR was carried out,heptagoside and ferric ammonium citrate were used as substrates of BGL,which was combined with high-throughput screening method of 96-well plate for second screening,and then shaking flask was used for third screening.Finally,the target mutant Y-1-B1 was obtained.The optimal temperature of Y-1-B1 is 50?,and it is 20?lower than 16BGL.The specific activity of Y-1-B1 at 40,45,50and 80?reached 213.3,405.6,505.3and 151.7 IU/mg,respectively,and it was 70%,120%,80%and 190%higher than16BGL.The catalytic efficiency constant k_cat/K_m of Y-1-B1 reached 1354.94 mM^-1S^-1,and its product inhibition strength K_appm/K_I value was 1.13?when 20 mmol/L glucose?,which was 1.68 times and 0.89 times of 16BGL?Y-1-B1 glucose tolerance increased by 12%?,reflecting higher product tolerance and activity.In addition,when Y-1-B1 was added in the simultaneous saccharification and fermentation of cellulose,the bioethanol yield of Y-1-B1 was 6.85 g/L,and 22%higher than that of 16BGL.The results of model construction and molecular docking showed that the mutation sites of Y-1-B1 were S414,V421 and S441,which were far away from the active site pocket.Therefore,it is speculated that these improved enzymatic properties are not related to the active site pocket.This reminds us not to ignore other sites far away from the catalytic center.In a conclusion,through a round of co-directed evolution,the mutants with simultaneous improvement of product tolerance,activity and optimum temperature were obtained in this study,which have potential application value in bioethanol production.