Overexpression of HAC1 enhances the expression of glucose oxidase in Pichia pastoris
[Objective]To increase the expression of glucose oxidase in Pichia pastoris.[Method]The constructed expression vectors pPICZB-HAC1,pPICZB-Hrd1 and pPIC3.5K-Ubc1 were linearized and integrated into the genome of Penicillium glucose oxidase receptor cells of Pichia pastoris by electroporation.The recombinant yeasts were screened with 50 μg/mL zeocin or 200 µg/mL G418.After the transformants were fermented in testtube and a high-yield recombinant strain was obtained.On this basis,the expression of glucose oxidase in Pichia pastoris was increased by exogenous addition of hemin in 10 L fermentor.[Result]The enzyme activity of recombinant strains overexpressing transcriptional activator HAC1,downstream chaperone Hrd1 and Ubc1 was increased by 56.46％,43.51％and 48.02％,respectively,compared with that of the original strains.Among them,the enzyme activity of recombinant Penicillium glucose oxidase strain overexpressing HAC1 reached 1308 U/mL in 10 L fermenter.[Conclusion]Overexpression of HAC1 or chaperones can help the protein to fold correctly,and the overexpression of HAC1 strains has the highest expression of glucose oxidase enzyme activity,increasing the enzyme activity by 86％.
Pichia pastorisglucose oxidaseUPRHAC1chaperoneheminheterologous expressionsecretory protein
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