High Efficient Expression Of Thermophilic And Ethanol-Resistant ?-glucosidase Gene And It's Application In Lignocellulosic Ethanol Fermenation

Lignocellulose is one of the most abundant renewable resources in the world,including cellulose,hemicellulose,lignin and pectin.However,due to its complex crystal structure and the composition of various components,it is formed by the complex super molecule polymerization,as an anti-degradation natural barrier.Cellulase,as a kind of complex enzyme,can effectively degrade lignocellulose for ethanol fermentation.Among them,beta-glycosidase,a key enzyme of cellulose enzymatic hydrolysis process,can quickly and efficiently produce glucose by hydrolysis of cellobiose.From these years of research,the compositions of cellulase,especially beta-glucosidase activity are generally low,and thermostability is poor,leading to the high cost of enzyme and the biomass conversion with low efficiency.Therefore,how to reduce the cost of beta-glucosidase and improve efficiency of the enzyme is of great significance to the development of bioethanol industry and the sustainable development of society.The present research,with the screening of Hypocrea sp.W63 as the original strain,studies the characteristics of this enzyme.The analysis of the biomass composition,the concentration of protein and the activity of enzymatic hydrolysis were compared with different biomass materials as carbon source,and rice straw was found to be the best inducer of producing beta-glucosidase.Subsequent studies found that the optimal hydrolysis temperature and pH of the enzyme is 65? and 4.8,and the enzyme activity can be improved in the presence of the concentration of 0.1%(v/v)and 1%(v/v)5-Hydroxy Methyl Furfural(5-HMF)by 14.7%and 27.9%,respectively.This enzyme has the catalytic activity of glycosidic bond and transglycosylation,and the enzyme activity is increased by 44%and 83%,respectively,with 10%(v/v)methanol and 15%(v/v)ethanol.On the other hand,other long chain of alcohols could also improve the enzyme activity in the concentration of 1%or 10%(v/v).The characteristics of this enzyme showed that it has great potential in industrial fermentation.including bioethanol fermentation.Recombinant was constructed using cDNA strategy of clone of full length sequence of the BGL,plasmid pPIC9K as vector and pichia pastoris GS115 as host,and the expression of recombinant thermophilic ethanol-resistant beta-glucosidase was extracellular secretion.The recombinant strain was induced for expression by methanol 5 days cultivation;The collected supernatantwas concentrated through ammonium sulfate precipitation,and purified by FPLC protein chromatography system,Bio-Gel P6 desalination and Macro-Prep DEAE anion exchange chromatography,and the specific activity of this enzyme was 194.25 U/mg with 53.8 fold purification.Sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)was used to obtain a single band,and protein spectrum identification and amino acid sequence alignment showed that the thermophilic ethanol-resistant beta-glucosidase molecular weight was 76740 Dalton,and pI is 6.01,which is close to the expected enzyme molecular weight 76.5 kDa.Compared with the beta-glucosidase from original strain,the characteristics of the recombinant enzyme did not change.The optimum hydrolysis temperature and pH value is 70? and 5.0,respectively.The ethanol concentration in the reaction system from 10?20%(v/v)for promoting beta-glycosidase activity is the strongest with 86.29%increase in beta-glycosidase activity,indicating the integrity and stability of the cloned beta-glucosidase.Because the beta-glucosidase activity was stimulated by ethanol,the beta-glucosidase was applied in high concentration of partial filling sugarcane bagasse for hydrolysis and simultaneous saccharification and fermentation(SSF).By fed-batch strategy and synergistic hydrolysis of enzyme solution and beta-glucosidase,the concentration of 35%bagasse can be hydrolyzed in 120 hours,and the highest sugar concentration of 225.84 g/L was obtained with hydrolysis conversion efficiency of 55.17%.When crude broth of beta-glucosidase was added to the SSF process,tof the highest ethanol production reached 41.25 g/L in 120 hours.Compared with the control without beta-glucosidase addition,ethanol production was increased nearly 2 folds.When the maximum concentration of the substrate was increased to 35%(w/v),the highest ethanol concentration of 49.07 g/L was reached in 48 hours.When adding epB-BGL in alkali pretreatment bagasse with high temperature(45?)SSF,the glucose was consumed faster in 24 hour and the ethanol concentration reached 28.2 g/L,with a yield of 0.43 g ethanol/g glucose.The final ethanol concentration was improved by about 39%.