A Study On The Screening,Expression And Characterization Of Ethanol Tolerant Cellulase

Consolidated bioprocessing(CBP)is a promising technology used for cellulosic ethanol production.In this process,the hydrolysis of cellulose and ethanol fermentation are carried out simultaneously by the same microbe.Cellulase plays an important role in the hydrolysis of cellulose.However,with the continuous accumulation of ethanol,the higher concentration of ethanol in the fermentation solution will decrease the activity of cellulase.Therefore,the ethanol tolerance of cellulase is a problem needs to be solved in CBP.This study aimed to screen ethanol tolerant strains with highly active cellulase from Guizhou brewery soil samples and strains with high cellulase activity we isolated before.The gene of screened ethanol tolerant cellulase was expressed in both prokaryotic and eukaryotic cells.In the isolation and screening of brewery soil samples,224 strains belong to 36 genera were obtained,among which 63%strains can tolerate over 8%ethanol,21%strains showed cellulase activity under high ethanol concentration.Through further screening,three strains with high activity at the concentration of 10%ethanol were obtained.After preliminary expression and ethanol tolerance comparison,the most ethanol tolerant cellulase from Thermobifida halotolerans YIM 90462T was selected for further study.Prokaryotic expression,purification and characterization of the endoglucanase from strain Thermobifida halotolerans YIM 90462T were operated.The endoglucanase was proposed as ThCel6A,which was composed of 443 amino acids,with the molecular weight of 45.9 kDa.The protein structure contained a domain of glycosyl hydrolase family six and a cellulose binding domain of family two.The optimum reaction temperature and pH for recombinant ThCel6A enzyme were 55 ? and pH 8.5,respectively.The specific activity was up to 34.5U/mg,and more than 90%of the activity maintained at the presence of 15%NaCl and 20%ethanol,which indicated ThCel6A was tolerant to salt and ethanol.The ThCel6A gene was expressed in Pichia pastoris and the recombinant yeast was constructed successfully.The recombinant yeast Cl could hydrolyze carboxymethyl cellulose to reducing sugars in YPGC liquid medium,and the reducing sugar content reached 1.51g/l after 11 days fermentation.The enzyme activity of ThCel6A expressed in recombinant yeast reached the maximum 0.86IU/ml after 3 days,and the optimum reaction temperature and pH were 55 ? and pH 8.5,respectively,which were consistent with the optimum reaction condition expressed in E.coli.This study focused on the ethanol tolerance of cellulase,screened and expressed a high activity ethanol tolerant endoglucanase,and constructed a recombinant Pichia pastoris with endoglucanase activity.It provides a good enzymeresource for consolidated bioprocessing of cellulosic ethanol production.