Effect of porphyrin metabolism pathway modification of Escherichia coli on recombinant catalase-peroxidase activity

Abstract:

[Objective] The cofactor-required prokaryotic expressions of extrinsic protein often show low activity. To improve the enzyme activity and reduce the cost of adding cofactors, we tried to express CAT-POD in Escherichia coli and promote the related synthetic metabolism of enzymes cofactors.[Methods] We cloned katG, the CAT-POD coding gene of Halomonas elongata DSM2581, and constructed it into the prokaryotic expression vector pet28a-katG, to achieve the recombinant expression of CAT-POD in E. coli. Additionally, CAT-POD's activity depends on its active centre, heme. And hematoporphyrin is the main structure of the heme. We cloned hemA, a gene coding 5-amino levulinic acid synthetase, and constructed it into the prokaryotic expression vector pUC19-tac-hemA. By overexpressing hemA, we raised the content of the porphyrin in E. coli, and improved the enzyme activity of recombinant protein CAT-POD.[Results] Ultimately, the CAT enzyme activity of katG reached 377 U/mL, 7.5 times of the control group.[Conclusion] This study provided an effective method for the industrial production of high-active CAT-POD, and a reference for the expression of protein that contains prosthetic group.