改造大肠杆菌卟啉代谢途径对重组过氧化物酶活性的影响

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

摘要:

[目的]原核表达某些需辅因子的外源蛋白时往往酶活偏低，为提高酶活和减少外加辅因子的成本，我们尝试在大肠杆菌中表达外源过氧化氢-过氧化物酶的同时提高大肠杆菌中与该酶辅因子相关的合成代谢。[方法]本研究克隆了中度嗜盐菌Halomonas elongata DSM2581的过氧化氢-过氧化物酶CAT-POD（catalase-peroxidase）编码基因katG的ORF，构建原核表达载体pET28a-katG，实现了CAT-POD在大肠杆菌中的重组表达。由于CAT-POD活性依赖其活性中心血红素，而血卟啉是血红素的骨架，通过构建原核表达载体pUC19-tac-hemA，将编码5-氨基乙酰丙酸合成酶的hemA基因在大肠杆菌中过量表达，提高卟啉的含量，从而提高重组蛋白CAT-POD的酶活。[结果]最终的CAT酶活达到了377 U/mL，为对照组的7.5倍。[结论]本研究为工业生产高活性CAT-POD提供了有效的方案，也为体外重组表达含辅因子的蛋白提供可借鉴的思路。

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.