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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2016, Vol. 36 Issue (5): 89-96    DOI: 10.13523/j.cb.20160513
技术与方法     
VD3羟化酶及其电子传递链的体外构建及活性研究
柯霞, 丁冠军, 孙骏, 王露, 郑裕国
浙江省生物有机合成技术研究重点实验室 浙江工业大学生物工程学院 杭州 310014
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
 全文: PDF(2460 KB)   HTML
摘要：

活性维生素D3具有广泛生理活性及药用价值,利用分子操作技术,在大肠杆菌细胞中重组表达VD3羟化过程的关键酶体系,是实现活性维生素D3生物法合成的有效手段。构建了来源于自养无枝酸菌(Pseudonocardia autotrophica)VD3羟化酶(Vdh, EC 1.14.13.159)及来源于不动杆菌(Acinetobacter sp. OC4)的铁氧还蛋白Fdx及铁氧还蛋白还原酶FdR的重组表达载体pET28b-Vdh、pET28b-FdR-Fdx,以大肠杆菌为宿主细胞,体外诱导表达并通过镍柱纯化三种蛋白质,通过CO差光谱法评价羟化酶Vdh体外活性,并利用2,6-二氯靛酚钠(DCIP)和细胞色素c作为电子受体评价电子传递链FdR-Fdx对NADH和NADPH的氧化活性及与羟化酶Vdh的偶联作用,最后利用Vdh及其电子传递链催化维生素D3的选择性羟化合成25(OH)VD3。
关键词： 电子传递链;  维生素D3羟化酶;  生物催化    
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.
Key words: Vitamin D3 hydroxylase    Biocatalysis    Electron transfer chain
收稿日期: 2015-12-28 出版日期: 2016-01-26
ZTFLH:  Q554+.1  
基金资助:

国家自然科学基金(31400978),浙江省教育厅科研项目(Y201329109)资助项目
通讯作者: 郑裕国     E-mail: zhengyg@zjut.edu.cn
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柯霞
丁冠军
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引用本文:

柯霞, 丁冠军, 孙骏, 王露, 郑裕国. VD3羟化酶及其电子传递链的体外构建及活性研究[J]. 中国生物工程杂志, 2016, 36(5): 89-96.

KE Xia, DING Guan-jun, SUN Jun, Wang Lu, ZHENG Yu-guo. Vitamin D3 Hydroxylase and Its Electronic Transfer Chain in vitro Construction and Activity Analysis. China Biotechnology, 2016, 36(5): 89-96.

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https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20160513    或    https://manu60.magtech.com.cn/biotech/CN/Y2016/V36/I5/89

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