KPC-2型碳青霉烯酶的表达纯化及其催化抗生素水解的动力学

目的：表达纯化KPC-2型碳青霉烯酶，并研究其催化抗生素水解的酶动力学活性。方法：将KPC-2基因与pET-22b（＋）原核表达载体连接后转化大肠杆菌BL21（DE3），IPTG诱导表达，表达产物经FF镍柱纯化后，SDS-PAGE检测蛋白的纯度；用BioTek酶联仪进一步检测其抗生素水解范围及动力学特性。结果：构建了高效表达KPC-2的工程菌，得到了纯度在90%以上的KPC-2蛋白，该蛋白能水解几乎所有β内酰胺类抗生素，其水解美罗培南等碳青霉烯类抗生素的能力较强，最适温度约为40℃，最适pH值约为6.5。结论：为进一步了解最常见的KPC功能与活性提供了重要信息。

Expression, Purification of Klebsiella pneumoniae Carbapenemase-2 and its Catalytic Kinetic of Antibiotics Hydrolysis

Objective： To express and purify Klebsiella pneumoniae carbapenemase-2（KPC-2） and study its en-zyme kinetics characteristic. Methods： The KPC-2 gene was amplified by PCR from the genomic DNA of K. pneu-moniae ATCC BAA-1705 strain, then it was cloned into pET-22b（＋） vector and transformed into the host cells E. coli BL21（DE3） strain. The KPC-2 protein was expressed following IPTG induction, and was purified by affinity chromatography. The purity of purified proteins were determined by SDS-PAGE. Explore and BioTek Gen5 was used to investigate its antibiotic hydrolyzation spectrum and enzyme kinetics characteristic. Results： The KPC-2 en-gineering bacteria was constructed. The purity of the target protein with the molecular weight about 30 kD was higher than 90%. The protein could hydrolyze almost all β-lactam antibiotics especially carbapenems, and its opti-mum temperature was about 40℃, optimum pH was about 6.5. Conclusion： The experiment results provide valu-able information for further understanding of most common PKC function and activity.