[1]阚延泽,孙文秀,李伟.黄鳝醛酮还原酶的定点突变及对其酶活性的影响[J].江苏农业学报,2018,(03):617-622.[doi:doi:10.3969/j.issn.1000-4440.2018.03.020]
　KAN Yan-ze,SUN Wen-xiu,LI Wei.Site-directed mutagenesis and effects on enzymatic activity of swamp eel aldo-keto reductase, Eakr[J].,2018,(03):617-622.[doi:doi:10.3969/j.issn.1000-4440.2018.03.020]
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黄鳝醛酮还原酶的定点突变及对其酶活性的影响()
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江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2018年03期
页码:
617-622
栏目:
畜牧兽医·水产养殖
出版日期:
2018-06-25

文章信息/Info

Title:
Site-directed mutagenesis and effects on enzymatic activity of swamp eel aldo-keto reductase, Eakr
作者:
阚延泽; 孙文秀; 李伟
（长江大学生命科学学院,湖北荆州434025）
Author(s):
KAN Yan-ze; SUN Wen-xiu; LI Wei
(College of Life Science，Yangtze University， Jingzhou 434025，China)
关键词:
醛酮还原酶; 定点突变; 催化四联体; 底物识别
Keywords:
aldo-keto reductase; site-directed mutagenesis; catalytic tetrad; substrate recognition
分类号:
Q554
DOI:
doi:10.3969/j.issn.1000-4440.2018.03.020
文献标志码:
A
摘要:
为分析催化四联体中关键氨基酸突变对黄鳝醛酮还原酶活性的影响，本研究利用重叠PCR技术对黄鳝Eakr基因进行了A81K定点突变，将突变基因亚克隆至pET-28a (+)构建了重组表达载体。重组载体转化BL21(DE3)菌株后经IPTG诱导,镍离子柱亲和层析获得了突变型蛋白（EakrA81K）；以NADPH为辅酶，分析了突变型蛋白EakrA81K的底物谱，并比较了野生型（Eakr）和突变型蛋白（EakrA81K）对不同底物的还原活性；最后，以甲醛为底物比较了野生型和突变型蛋白的最适反应温度和最适pH。结果表明：EakrA81K对醛类物质以及中长链酮类物质具有较高的还原活性，而对醇、糖、酸类物质没有明显活性；A81K位点特异突变使得黄鳝醛酮还原酶和底物之间的亲和力显著增加，并提高了酶对大部分底物的还原活性；突变稍降低了黄鳝醛酮还原酶的酸度耐受能力但提升了该酶的温度耐受能力。这暗示着黄鳝醛酮还原酶Eakr的81位氨基酸在底物识别上具有重要作用。
Abstract:
In order to probe the effects of mutation of the key amino acid in the catalytic tetrad on enzymatic activity of Eakr, SOE PCR was performed to clone and introduce an A81K mutation into the aldo-keto reductase Eakr gene. Subsequently, recombinant mutants were constructed and verified. The constructed recombinant plasmid pET-28a-EakrA81K was transformed to Escherichia coli BL21 (DE3) and induced with IPTG, the expressed protein was purified by nickel ion affinity chromatography. Aldehydes and ketones were chosen to determine the substrate specificity of EakrA81K, NADPH was utilized as a cofactor. Moreover, the enzymatic activities between Eakr and EakrA81K were compared. In addition, the optimal reaction temperature and pH of the two proteins were also analyzed. The results showed that EakrA81K displayed efficient activities to aldehydes and medium long chain ketones. Moreover, A81K mutation resulted in higher affinity between substrates and the enzyme. The acidity tolerance ability of swamp eel aldo-keto reductase was reduced, and temperature tolerance ability of the enzyme was improved. All above results suggest that Eakr amino acid at position 81 plays important roles in substrate recognition.

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备注/Memo

备注/Memo:
收稿日期：2017-10-10 基金项目：湿地生态与农业利用教育部工程研究中心开放课题（KF2015016）；湖北省教育厅基金项目（Q20131206） 作者简介：阚延泽（1993-）,男， 江苏徐州人，硕士研究生，主要从事生化与分子生物学研究。(Tel)13545817727；(E-mail)378053841@qq.com 通讯作者：李伟， ( E-mail)wetli@yangtzeu.edu.cn
更新日期/Last Update: 2018-07-04