| Pichia yeast expression system,one of the eukaryotic expression systems to express heterologous proteins, use methanol as the sole carbon and energy source. Methanol which is toxic, inflammable and explosive is not safe in large-scale production process and also limits its application in production safety, food safety, etc.Pichia constitutive expression system under the regulation of glyceraldehyde 3-phosphate dehydrogenase promoter(p GAP) has no need to use methanol when expressing exogenous proteins, and the recombinants can culture continuously under conditions of same carbon sources(glucose, glycerol, methanol or oleic acid, etc.), meanwhile, the constitutive expression can reached a high level under appropriate conditions. Therefore, it is becoming a focus of research on using glyceraldehyde-3-phosphate dehydrogenase promoter to replace alcohol oxidase promoter in recent years.As a biocatalyst, cellulase can degrade cellulose, which is widely used in terms of the food industry, and environmental protection, etc. In recent years, more and more basic researches on cellulose have made significant progress. The enzyme with higher activity were obtained through the technology of gene cloning and gene expression regulation, and applied more and more widely. With the development of enzyme preparation industry, Amounts of microbial enzymes tend to product large-scalely liquid enzyme preparation. However liquid enzyme is unstable and easy to inactivation, and thus in industrial productions, the stability of liquid enzyme are maintained generally by adding stabilizers and preservatives. Tthe screening and optimizing the complex stabilizer to improve thermal stability of cellulase has a very important significance.In this study, we used the promotor GAP to replace promotor AOX, constructed a constitutive expression vector p PIC9K-gap. The gap gene was cloned from P. pastoris GS115 genome by PCR and ligated to p PIC9 K. GUS gene was used as a reporter gene to verify the feasibility of p PIC9K-gap. Then the gus gene was replaced with the eg gene to produce the recombinant vector p PIC9K-gap-eg, then transformed plasmid p PIC9K-gap-eg into Pichia pastoris GS115 for constitutive expression. The feasible bioactive recombinant was obtained by histamine-absence screening and activity screening. At last, the constitutive expression level of the recombinants named as PPGAP-eg4 was reached 19 U/m L, which were no significant different to the inducible expression level of the control strain( containing p PIC9K-eg)of 16 U/m L.The maximum activity of endocellulase of the recombinants PPGAP-eg4 was 40 U/m L, which was 2.2 times production of the original strain, under the conditions with 0.5% inoculum, 0.5% glycerol, and continuous induction to the seventh days.To improve storage stability of endocellulase, the effect of some stabilizers on the stability of endocellulase were detected in this experiment. It was found that galactose, dextrin, gelatin had more obvious influences on stability. The reagent was studied via single factor test and orthogonal design of the three factors and three levels. The reagent was determined as follows: galactose 4%, gelatin 1%, dextrin 8%. Under the protection of the combined protective agent, the enzyme activity remained 250.67% after being incubated at 65 ℃ for 2 h, significantly improves the endocellulase thermal stability. Morever, when the combined stabilier were added into liquid enzyme preparations, the residual enzyme activity could remain above 50% in 3 months, which were laid the foundation for wider application of endocellulas. |
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