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| Expression and Characteristics of Apis mellifera NADPH-cytochrome P450 Reductase Gene in Escherichia coli |
| WU Xue-long, YANG Xiao-hui, WANG Jun-qing, WANG Rui-ming |
| Shandong Provincial Key Laboratory of Microbial Engineering, Department of Biology Engineering, Qilu University of Technology, Jinan 250353, China |
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Abstract 10-Hydroxy-2-decenoic acid (10-HDA),a major fatty acid component from royal jelly,is known to have various biologic activities in antibacteria, antitumour and lifespan-extending.However, it is unclear about the underlying molecular mechanism of its biosynthesis. A NADPH-cytochrome P450 reductase (EC 1.6.2.4, NADPH-cytochrome P450 reductase, CPR) gene from Apis mellifera was cloned and expressed in Escherichia coli. Subsequently, the enzymatic properties of CPR were analyzed. Results showed that the target protein was successfully expressed in E. coli after induced by IPTG, and its molecular weight was 86.29 kDa. The CPR protein was then purified by Ni-NTA protein affinity chromatography and its specific activity was 77.33 (EU of CPR)/μg.The purified CPR protein displayed the maximum activity at pH 8.0 and 40℃. Its activity was not significantly affected by several metal ions except Ni2+, Zn2+ and Co2+ which caused a strong inhibition. The Km and kcat, towards cytochrome C at 30℃, were 76μM and 268/min, respectively. It lay a foundation for researching the function of CPR in 10-HDA biosynthesis pathway.
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Received: 25 May 2016
Published: 25 December 2016
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