化工进展 ›› 2019, Vol. 38 ›› Issue (12): 5504-5511.DOI: 10.16085/j.issn.1000-6613.2019-0472

• 生物与医药化工 • 上一篇    下一篇

醇脱氢酶KpADH的127位点对催化活性和对映选择性的影响

朱诚(),许国超,戴威,周婕妤,倪晔()   

  1. 江南大学生物工程学院,江苏 无锡 214122
  • 收稿日期:2019-03-28 出版日期:2019-12-05 发布日期:2019-12-05
  • 通讯作者: 倪晔
  • 作者简介:朱诚(1994—),男,硕士研究生,研究方向为生物催化与酶工程。E-mail: 6160206048@vip.jiangnan.edu.cn
  • 基金资助:
    国家轻工技术与工程一流学科计划(LITE2018-07);国家自然科学基金(21506073)

Effect of position 127 on the activity and enantioselectivity of alcohol dehydrogenase KpADH

Cheng ZHU(),Guochao XU,Wei DAI,Jieyu ZHOU,Ye NI()   

  1. School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China
  • Received:2019-03-28 Online:2019-12-05 Published:2019-12-05
  • Contact: Ye NI

摘要:

醇脱氢酶可用于合成手性化合物,在医药、材料等领域应用广泛。来源于多孢克鲁维酵母(Kluyveromyces polysporus)的醇脱氢酶KpADH同时具有还原(4-氯苯基)-(吡啶-2-基)-甲酮(CPMK)和氧化异丙醇、1,4-丁二醇的活性。通过分子对接和结构分析,发现Y127是紧靠底物的关键位点。本文通过对127位点定点饱和突变来提高催化活性和对映选择性,突变体Y127V和Y127I还原CPMK的比活力达到95.0U/mg和84.0U/mg,分别是野生型的6.5倍和5.8倍,突变体Y127L催化CPMK生成(R)-(4-氯苯基)-(吡啶-2-基)-甲醇[(R)-CPMA]的e.e.值由82%提高到99.2%。同时,127位点的突变提高了对醇类底物的氧化活性,突变体Y127I对异丙醇的比活力是野生型的1.46倍,而Y127C更青睐于对1,4-丁二醇的催化氧化,其比活力是野生型的3.00倍。分子间作用力分析表明,Y127L与底物CPMK间增加的氢键和π-π作用力稳定了底物构象,进一步提高了还原CPMK的对映选择性。本文为醇脱氢酶KpADH的分子改造和机制解析提供了指导,提高了该酶的工业应用潜力。

关键词: 醇脱氢酶, 定点饱和突变, 比活力, 对映选择性, 生物催化

Abstract:

Alcohol dehydrogenases can be used to synthesize chiral compounds, which are widely applied in pharmaceuticals, materials and other fields. Alcohol dehydrogenase from Kluyveromyces polysporus (KpADH) exhibited both reducing activity toward (4-chlorophenyl)-(pyridin-2-yl)-methanone (CPMK) and oxidizing activity toward isopropanol and 1,4-butanediol. Based on molecular docking and structural analysis, Y127 was identified as the key site close to the substrate. To improve the catalytic performance and enantioselectivity, site-saturation mutagenesis was applied at Y127. Specific activity toward CPMK of variants Y127V and Y127I increased to 95.0U/mg and 84.0U/mg, which were 6.5- and 5.8-fold of wild type KpADH (WTKpADH), respectively. The enantioselectivity toward CPMK of variant Y127L increased from 82% to 99.2% e.e. (R). Mutations at 127 also enhanced the oxidizing activity toward alcohol substrates. Specific activity toward isopropanol of Y127I was 1.46-fold of WTKpADH. Variant Y127C displayed higher activity in the oxidation of 1,4-butanediol, whose specific activity was 3.00-fold of WTKpADH. Intermolecular forces analysis indicated that an increased hydrogen bond and extra π-π interactions stabilized the conformation, which resulted in enhanced enantioselectivity. This study provides guidance for molecular engineering and mechanism analysis of alcohol dehydrogenase KpADH, and extends its application potential in industry.

Key words: alcohol dehydrogenase, site-saturation mutagenesis, specific activity, enantioselectivity, biocatalysis

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