Biotechnology Bulletin ›› 2022, Vol. 38 ›› Issue (4): 269-277.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0148

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Expression of Pyranose Oxidase with Optimized Codon in Pichia pastoris

WANG Yue(), GAO Qing-hua, DONG Cong, LUO Tong-yang, WANG Qing-qing   

  1. Hebei Research Institute of Microbiology Co.,Ltd.,Baoding 071051
  • Received:2021-02-04 Online:2022-04-26 Published:2022-05-06

Abstract:

Pyranose oxidase(P2O)plays an important role in lignin degradation and carbohydrate biosynthesis. P2O is also used in biological fuel cell,biosensors and clinical diagnostic analysis. The expression of pyranose oxidase in Pichia pastoris and enzymatic characters were carried out in our laboratory,which will provide a theoretical basis for the industrial and efficient production of pyranose oxidase in the future. Based on the codon preference of P. pastoris,the codon of pyranose oxidase gene was optimized by biotechnology. The recombinant P. pastoris GS115 was constructed via gene exogenous expression technology for achieving the efficient expression of P2O. The enzymatic properties of the recombinant P2O were studied. After optimizing the fermentation conditions of high-yielding recombinant strains,large-scale culture was conducted in 10 L fermenter. As results,P2O was highly expressed in P. pastoris after codon optimization. After 132 h induction culture in 10 L fermenter,enzyme activity reached 220 U/mL. The optimal temperature of recombinant pyranose oxidase was 55℃,and its thermal stability was good under the condition of no more than 60℃. The optimal pH for this enzyme was 6.5. In the range of pH 5-9,the relative enzyme activity was higher than 50%. P2O showed high stability in a wide range of pH,especially in alkaline conditions. Cu2+ presented a greater inhibition on enzyme activity. Assay of substrate specificity showed that the optimal substrate for the recombinant enzyme was D-glucose. In conclusion,the codon-optimized recombinant expression plasmid pPIC9K-P2O is successfully constructed in this study and highly expressed in P. pastoris GS115.

Key words: pyranose oxidase, codon optimization, Pichia pastoris, heterogeneous expression