Restriction endonuclease is one of the most important enzymes in modern biomedicine.Due to the cytotoxicity of restriction endonucleases in digesting host DNA during recombinant expression,the production of restriction endonucleases was difficult,and foreign companies had monopolized their supply.Previously,our laboratory had produced several restriction endonucleases successfully through broad methylation protection.However,the production process relied on low-density shake flask culture,and the yield was unable to meet the market demand.In this study,we optimized the fermentation conditions of recombinant restriction endonuclease Pst I.Several fermentation conditions such as induction temperature,induction time,induction agent concentration,supplementary carbon source,pH,and induction timing were screened by single-factor test.The results showed that the highest expression of Pst I was achieved at the following conditions:fermentation temperature at 37 ℃,6 h after the start of fermentation with the addition of a final concentration of 1.4 mmol/L IPTG for 8 h,pH of the fermentation system at 8.0,and using glycerol as a supplemental carbon source.The yield of Pst I protein was 0.0129 g/L,2.15 times as much as before optimization,while active enzyme yield was 1.926×106 U/L,3.74 times as much as before optimization.
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