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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  2019, Vol. 39 Issue (8): 59-65    DOI: 10.13523/j.cb.20190808
技术与方法     
利用短短芽孢杆菌进行酮还原酶CgKR2的高效表达与纯化 *
胡艳红1,2,龚雪梅1,2,丁柳柳1,2,高嵩1,2,李婷婷1,2,**()
1 淮海工学院江苏省海洋药物活性分子筛选重点实验室 连云港 222005
2 淮海工学院江苏省海洋生物产业技术协同创新中心 连云港 222005
Highly Efficient Expression and Purification of Ketoreductase CgKR2 Using Brevibacillus choshinensis SP3
HU Yan-hong1,2,GONG Xue-mei1,2,Ding Liu-liu1,2,GAO Song1,2,LI Ting-ting1,2,**()
1 Jiangsu Key Laboratory of Marine Pharmaceutical Compound Screening, Huaihai Institute of Technology, Lianyungang 222005, China
2 Co-Innovation Center of Jiangsu Marine Bio-industry Technology, Huaihai Institute of Technology, Lianyungang 222005, China
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摘要：

酮还原酶CgKR2能够一步还原前手性羰基化合物生成高附加值的手性醇,有望解决手性醇传统制备方法的步骤烦琐和高成本问题,具有很高的经济效益。研究表明,CgKR2催化底物2-氧代-4-苯基丁酸乙酯(OPBE)生成普利类降压药的重要中间体(R)-2-羟基-4-苯基丁酸乙酯[(R)-HPBE]具有良好效果。但CgKR2的生产成本高昂、过程烦琐。利用短短芽孢杆菌胞外分泌表达酮还原酶CgKR2,获得该酶的高效表达,并经简便的一步镍亲和层析纯化,即获得高纯度酮还原酶CgKR2,产率高达每升发酵7.8mg纯酶。以酶标板法测定其比活力、温度稳定性以及动力学参数等基本酶学性质,结果显示,CgKR2的比活力为(78.32±7.62)U/mg、Km为(0.2±0.02)mmol/L、Vmax为(117.64±3.6)μmol/(min·mg)、Kcat为73s -1,与以往报道的数据一致,并且获得的CgKR2纯酶在30℃下孵育72h依然保持80%的活性,酶活的稳定性远好于以往的制备方法。开发出的一套简便高效的酮还原酶CgKR2表达纯化工艺,降低了生产成本、简化了生产工艺,可推进手性醇生物催化制备的普及,对其他生物催化工程酶的制备方法研究也有借鉴作用。

关键词： 酮还原酶CgKR2;  短短芽孢杆菌;  分泌表达;  生物催化    
Abstract:

Ketone reductase CgKR2 transforms prochiral carbonyl compounds into high value-added chiral alcohols in one reaction step. Using this enzyme as a biocatalyst could solve the problems of tedious procedures and high costs in the traditional preparation of chiral alcohols, and has a good economic benefit potential. Biocatalysis of OPBE into (R)-HPBE, an important pharmaceutical intermediate of ACEI antihypertensive drugs, is a good application of CgKR2. However, extensive application of CgKR2 was hindered by its complicated and high-cost preparation. Highly efficient secretory expression of CgKR2 was obtained using Brevibacillus choshinensis. With a simple one-step Ni-affinity chromatography, CgKR2 was purified to high purity. The overall yield achieved 7.8mg per liter of fermentation. The purified CgKR2 was characterized in microplate assays, which indicated a specific activity of (78.32±7.62)U/mg, a Km of (0.2±0.02)mmol/L, a Vmax of (117.64±3.6)μmol/(min·mg), and a Kcat of 73s -1. These characterization data were consistent with the data previously reported on CgKR2. Moreover, CgKR2 was obtained ,it can be retained 80% of the activity after 72h of incubation under 30℃, showing a much better stability than the enzyme prepared by previous methods. A simple and highly efficient procedure for CgKR2 expression and purification were developed, which decreased the preparation cost and simplified the process. The new procedure not only would improve the usage of CgKR2 in chiral alcohol preparations, but also sets a good reference for preparation method developments of other biocatalytic enzymes.

Key words: Ketone reductase CgKR2    Brevibacillus choshinensis    Secretory expression    Biocatalysis
收稿日期: 2019-02-19 出版日期: 2019-09-18
ZTFLH:  Q55  
基金资助: *江苏省海洋药物活性分子筛选重点实验室开放课题(HY201801)
通讯作者: 李婷婷     E-mail: littly2006@163.com
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引用本文:

胡艳红,龚雪梅,丁柳柳,高嵩,李婷婷. 利用短短芽孢杆菌进行酮还原酶CgKR2的高效表达与纯化 *[J]. 中国生物工程杂志, 2019, 39(8): 59-65.

HU Yan-hong,GONG Xue-mei,Ding Liu-liu,GAO Song,LI Ting-ting. Highly Efficient Expression and Purification of Ketoreductase CgKR2 Using Brevibacillus choshinensis SP3. China Biotechnology, 2019, 39(8): 59-65.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20190808    或    https://manu60.magtech.com.cn/biotech/CN/Y2019/V39/I8/59

图1  重组CgKR2的质粒构建及在短短芽孢杆菌中的表达
图2  CgKR2蛋白的纯化流程与结果
图3  CgKR2催化OPBE底物的催化反应参数
图4  温度对CgKR2酶活的影响
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