Cloning And Expression Of Endoglucanase ? Gene In Pichia Pastoris From Trichoderma Viride

Cellulose is the most abundant and potential renewable resource on the earth.Microbial degradation of cellulose is a green and environment-friendly method for the effective utilization of cellulose.Trichoderma viride can produce a large amount of cellulase.In this study,T.viride strain AS3.3711 was used as experimental material,primers were designed according to the eg VII gene sequence of T.viride AS3.3711 with accession number EU518927 on the GenBank database.DNA and RNA were used as templates for PCR,the obtained sequences were compared and the endoglucanase gene had no intron,the length of eg VII gene was782 bp,the open reading frame was 750 bp,it encoded 249 amino acids,the theoretical molecular weight was 26.8002 KD and the theoretical isoelectric point was 7.62.The signal peptide was N-terminal 19 amino acids and belonged to an extracellular enzyme of the glycoside hydrolase family 61.To efficiently express endoglucanase the cloned eg VII gene was ligated into expressionvector pPIC9K to obtain recombinant plasmids pPIC9K-?1-eg VII,pPIC9K-?2-eg VII and pPIC9K-?3-eg VII.The recombinant plasmid was transformed into Pichia pastoris GS115 strain by electroporation.After the endoglucanase activity was determined,the activity of pPIC9K-05 was found to be the highest.The expression of yeast transformant was induced by methanol,the target protein was successfully identified by SDS-PAGE and its size was basically consistent with the theoretical value.The expression level of P.pastoris transformants induced by methanol was detected by qRT-PCR,the results showed that the maximum expression of the transformant occurred on the third day.Optimization of the fermentation conditions of the recombinant P.pastoris by orthogonal experiment was as follows:methanol content was 1%,YNB content was1.34%,pH was 6 and incubation temperature was 29?.The highest enzyme yield was 106.91 U/g.Enzymatic analysis showed that the enzyme activity was stable at 40-60?and 60?was the best temperature.The pH was in the range of 6.0-8.0 and the best was 6.0.Among the effects of metal cations on enzyme activity,Ag⁺activated and Mn²⁺inhibited.MCC was the best of the five substrates selected,the endoglucanase:Vmax was 0.5635 mg/?mL·min?,Km was 0.2623mg/mL,Kcat=Vmax/E was 0.1702 S^-1,Kcat/Km was 0.6489 mL/?S·mg?.