Study on optimal expression of protein glutaminase in Bacillus subtilis
Protein-glutaminase(PG)acts on glutamine in large proteins or their small side chains to deamidify it to glutamate to improve protein solubility and emulsification.Therefore,this enzyme has a wide range of applications in the food industry.PG is derived from Chryseobacterium proteolyticum,but the production of PG expressed by the original Chryseobacterium proteolyticum is very low.In order to improve the expression level of protein glutaminase,the protein glutaminase gene derived from Bacillus subtilis was codon optimized in this study,and the codon was replaced with a Bacillus subtilis preferred codon,after which the PG genes before and after codon optimization were ligated into the expression vector pBSA43,and finally transformed into Bacillus subtilis WB600 for high efficiency expression.The results showed that the PG enzyme activity of the recombinant strain before codon optimization was 1.83 U·mL-1,and that of the recombinant strain after codon optimization was 2.91 U·mL-1.In order to further improve the expression level of PG,the optimal fermentation conditions were determined after optimization by response surface analysis:fermentation time of 62.3 h,fermentation temperature of 32.5℃,and an initial pH of 7.24 for the medium.Under these conditions,the PG enzyme activity of the recombinant strain reached 5.76 U·mL-1 after codon optimization.The protein glutaminase gene derived from Bacillus subtilis was codon optimized to significantly increase the expression level,which provided a reference for the efficient expression of protein glutaminase in Bacillus subtilis.
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