谷氨酸脱羧酶基因的挖掘、表征及全细胞制备γ-氨基丁酸的研究
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TS201.3

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Mining and Characterization of Glutamate Decarboxylase Gene for Whole Cell Preparation of γ-Aminobutyric Acid
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    摘要:

    γ-氨基丁酸是一种重要的生物活性因子,通过谷氨酸脱羧酶(GAD)使L-谷氨酸脱羧而合成。作者首先将酿酒酵母谷氨酸脱羧酶基因进行克隆并实现其在大肠杆菌中表达。通过亲和层析纯化获得了比活力高达66.55 U/mg的重组酶ScGAD。进一步酶学性质分析结果表明,ScGAD最适反应温度为60 ℃,最适反应pH 为4.0,且在30~50 ℃、pH 4.0~9.0时表现出优越的稳定性;其动力学参数Km为14.28 mmol/L,对L-谷氨酸具有较好的亲和力。最后通过全细胞制备γ-氨基丁酸(GABA)的最适条件探究,得到GABA生成效率最高的条件为60 ℃、pH 4.0,在此条件下,100 mmol/L底物L-谷氨酸经全细胞催化可合成GABA 35.9 g/(g·h)。该研究为GABA高效生产提供依据。

    Abstract:

    γ-Gminobutyric acid is an important biologically active factor, which is synthesized though the decarboxylation of L-glutamic acid by glutamate decarboxylase (GAD). The author firstly cloned and expressed the glutamate decarboxylase-encoding gene from Saccharomyces cerevisiae in E. coli. The specific activity of the recombinant ScGAD purified by affinity chromatography reached a maximum value of 66.55 U/mg. Further enzymatic property analysis results indicated that ScGAD exhibited an optimum reaction temperature of 60 ℃, an optimum reaction pH of 4.0, excellent stability in the range of 30~50 ℃ and pH 4.0~9.0, and the value of 14.28 mmol/L for kinetic constant Km indicating ScGAD of a good affinity to L-glutamic acid. Finally, through the investigation of the optimal conditions for GABA preparation by whole-cell catalysis using ScGAD, the highest generation efficiency of GABA was achieved at the conditions of 60 ℃ and pH 4.0. On this basis, 100 mmol/L of the substrate (sodium L-glutamate) could be converted to 35.9 g/(g·h) of GABA through whole-cell catalysis. This study provides a basis for efficient production of GABA.

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冯骁,池慧兵,孟凡强,陆兆新,朱萍,吕凤霞.谷氨酸脱羧酶基因的挖掘、表征及全细胞制备γ-氨基丁酸的研究[J].食品与生物技术学报,2022,41(10):58-66.

FENG Xiao, CHI Huibing, MENG Fanqiang, LU Zhaoxin, ZHU Ping, LYU Fengxia. Mining and Characterization of Glutamate Decarboxylase Gene for Whole Cell Preparation of γ-Aminobutyric Acid[J]. Journal of Food Science and Biotechnology,2022,41(10):58-66.

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  • 在线发布日期: 2022-11-16
  • 出版日期: 2022-10-25

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