Expression of Pyranose Oxidase with Optimized Codon in Pichia pastoris

doi:10.13560/j.cnki.biotech.bull.1985.2021-0148

Abstract

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

WANG Yue, GAO Qing-hua, DONG Cong, LUO Tong-yang, WANG Qing-qing. Expression of Pyranose Oxidase with Optimized Codon in Pichia pastoris[J]. Biotechnology Bulletin, 2022, 38(4): 269-277.

Figures/Tables 11

Fig. 1 Gene sequence after the optimization of codons The boxed sequences indicate restriction sites EcoR Iand Not I. The underlined sequences show 6×His-tag

Fig. 2 Verification of pPIC9K-P2O digested by double enzymes M：DNA marker. 1：Digestion of pPIC9K-P2O by EcoR I and Not I.2：pPIC9K-P2O plasmid

Fig. 3 Acquirement of transformants

Fig. 4 Screening of recombinant P. pastoris with high-yield P2O

Fig. 5 SDS-PAGE result of recombinant protein M：Protein marker. 1：Supernatant of negative control. 2-8：Supernatant of positive transformants

Fig. 6 Purification results of recombinant protein M：Protein marker. 1-8：Purification of recombinant protein

Fig. 7 Production of P2O by high-density fermentation in 10 L bioreactor A：Growth curve of P2O activity. B：SDS-PAGE result. M：Protein marker. 1-12：Accumulation of recombinant protein after 0,12,24,36,48,60,72,84,96,108,120 and 132 h

Fig. 8 Optimal pH and pH stability A：Relative activity at different pH conditions at 30℃. B：The relative enzyme activity of P2O kept in the same temperature for 24 h at different pH conditions at 30℃. The used buffers were 100 mmol/L sodium citrate（■,black line）,potassium phosphate（●,red line）,Tris-HCl（▲,blue line）and glycine-NaOH（▼,pink line）

Fig. 9 Optimal temperature and temperature stability A：Relative activity of P2O at different temperatures at pH 6.5. B：The relative enzyme activity of P2O kept in the different temperatures for 30 min at pH 6.5

Fig. 10 Effects of metal ions on the activity of P2O

Fig. 11 Substrate specificity of P2O

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