高等学校化学学报 ›› 2019, Vol. 40 ›› Issue (4): 698-704.doi: 10.7503/cjcu20180698

• 有机化学 • 上一篇    下一篇

亮氨酸脱氢酶催化底物偶联法合成α-酮异己酸

丰险1,2, 穆晓清1,2(), 聂尧1, 徐岩1   

  1. 1. 江南大学工业生物技术教育部重点实验室, 无锡 214122
    2. 宿迁市江南大学产业技术研究院, 宿迁 223800
  • 收稿日期:2018-10-16 出版日期:2019-01-24 发布日期:2019-01-24
  • 作者简介:

    联系人简介: 穆晓清, 男, 博士, 副教授, 主要从事生物化工及酶工程方面的研究. E-mail: xqmu@jiangnan.edu.cn

  • 基金资助:
    国家自然科学基金(批准号:21336009, 21176103)、 国家“八六三”计划项目(批准号:2015AA021004)和高等学校学科创新引智计划(111计划)项目(批准号:111-2-06)资助.

Synthesis of α-Ketoisocaproate Through Substrate Coupling Reaction Catalyzed by Leucine Dehydrogenase

FENG Xian1,2, MU Xiaoqing1,2,*(), NIE Yao1, XU Yan1   

  1. 1. Key Laboratory of Industrial Biotechnology of Ministry of Education, Jiangnan University, Wuxi 214122, China
    2. Suqian Jiangnan University Institute of Industrial Technology, Suqian 223800, China
  • Received:2018-10-16 Online:2019-01-24 Published:2019-01-24
  • Contact: MU Xiaoqing E-mail:xqmu@jiangnan.edu.cn
  • Supported by:
    † Supported by the National Natural Science Foundation of China(Nos.21336009, 21176103), the National High Technology Research and Development Program of China(No.2015AA021004) and the Overseas Expertise Introduction Project for Discipline Innovation, China(No.111-2-06).

摘要:

构建了亮氨酸脱氢酶(LeuDH)催化的底物偶联反应体系, 打破氧化脱氨反应平衡, 同时制得高附加值的α-酮异己酸(α-KIC)和L-2-氨基丁酸, 并实现辅酶NAD+的高效循环再生. 基于LeuDH的底物专一性和催化动力学参数, 考察了不同酮酸底物对于底物偶联反应效率的影响, 选择转化率最高的2-丁酮酸作为偶联底物, 使α-KIC产率由单步氧化反应的2.75%提高至66.82%. 通过考察底物浓度、 pH值、 NH4+浓度和辅酶NAD+浓度等反应条件对偶联反应效率的影响, 使α-KIC产率进一步提高至83.25%, 同时辅酶NAD+的总转化数(TTN)达到5.88×105. 通过改变底物L-亮氨酸和2-丁酮酸的摩尔比, 能够将α-KIC的产率进一步提高至92.74%.

关键词: 亮氨酸脱氢酶, 底物偶联, 辅酶再生, α-酮异己酸;, L-2-氨基丁酸

Abstract:

In order to shift the balance of oxidative deamination, the substrate coupling reaction system catalyzed byleucine dehydrogenase(LeuDH) was constructed. The α-KIC and L-2-aminobutyrate with high valuable were synthesized. Meanwhile, the coenzyme was efficiently regenerated. Based on the studies of the substrates specificity and kinetic parameters of LeuDH, the efficiency of substrate coupling reaction was compared using a series of ketonic acids. The result showed that 2-oxobutyrate could be used as the best coupling substrate. The yield of α-KIC was improved from 2.75% of the single-step oxidation reaction to 66.82% of the coupling reaction. The reaction conditions were optimized, including pH values, concentrations of substrate, NH4+ and coenzyme. The yield of α-KIC and the total turnover number(TTN) of ketonic acid reached 83.25% and 5.88×105, respectively. By adapting the molar ratios of the L-leucine and 2-oxobutyrate, the yield of α-KIC was increased to 92.74%. The substrate coupling reaction system was suggested to be the effective way for the production of α-KIC.

Key words: Leucine dehydrogenase, Substrate coupling, Coenzyme regeneration, α-Ketoisocaprate;, L-2-Aminobutyrate

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