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| Vitamin D3 Hydroxylase and Its Electronic Transfer Chain in vitro Construction and Activity Analysis |
| KE Xia, DING Guan-jun, SUN Jun, Wang Lu, ZHENG Yu-guo |
| Key Laboratory of Bioorgainc Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, China |
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Abstract Active vitamin D3 has a wide range of physiological activity and phamaceutical value. Construction of key enzyme system for the selective hydroxylation of VD3 in Escherichia coli cell is an effective mean to realize the biological conformation of active vitamin D3 through molecular manipulation. The recombinant expression vector pET28b-FdR-Fdx and pET28b-Vdh were constructed, VD3 hydroxylase (Vdh, EC 1.14.13.159) originated from Pseudonocardia, ferredoxin and ferredoxin reductase originated from Acinetobacter sp. OC4 were actively expressed in Escherichia coli as the host cell and purified by nickel column. Reduced CO difference spectra assay was carried out to evaluate the activity of VD3 hydroxylase in vitro. 2, 6-dichlorobenzenone-indophenol sodium salt and cytochrome c, serving as an electron acceptor, were used to evaluate both the oxidation activity of the electron transfer chain for NADH/NADPH and the coupling effect with the hydroxylase Vdh. Finally, 25(OH)VD3 is selectively hydroxylated by Vdh and its electron transport chain using vitamin D3 as the substrate.
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Received: 28 December 2015
Published: 26 January 2016
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