Expression of site-directed mutant glutamine synthetase and enzymatic glutamine production

HUANG Xing; LIU Ming; GONG Bo; ZENG Xing; ZHANG Songyi; CAO Zhu’an

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Expression of site-directed mutant glutamine synthetase and enzymatic glutamine production

更新时间：2025-12-10

- Expression of site-directed mutant glutamine synthetase and enzymatic glutamine production
- CIESC Journal Issue 6, Pages: 1479-1484(2008)
- 作者机构：
  
  清华大学化学工程系
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- Online First：05 June 2008，
  
  Published：05 June 2008
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HUANG Xing, LIU Ming, GONG Bo, et al. Expression of site-directed mutant glutamine synthetase and enzymatic glutamine production[J]. CIESC Journal, 2008, (6): 1479-1484.
DOI：

HUANG Xing, LIU Ming, GONG Bo, et al. Expression of site-directed mutant glutamine synthetase and enzymatic glutamine production[J]. CIESC Journal, 2008, (6): 1479-1484. DOI：

摘要

针对酶法生产谷氨酰胺中高浓度铵盐条件下的谷氨酰胺合成酶（GS）腺苷酰化问题，从谷氨酸棒杆菌Corynebacterium glutamicum ATCC 14067调取编码GS的基因glnA，将GS的腺苷酰化位点Tyr405定点突变为Phe405，并在大肠杆菌中表达突变后的GS，优化产酶条件。用突变的GS在摇瓶中进行酶催化过程，通过补加酶催化底物谷氨酸钠和氯化铵，可以提高定点突变的GS生产谷氨酰胺的能力，谷氨酰胺产量达到16.8 g·L-1;在5 L反应器规模的酶催化生产谷氨酰胺过程中，通过调控底物补加方案和反应条件，谷氨酰胺的产量达到34.2 g·L-1，谷氨酰胺对谷氨酸的摩尔转化率为96.3%。

Abstract

To solve putative adenylation of glutamine synthetase (GS) in the enzymatic production of glutamine at a high concentration of ammonium salt

glnA encoding GS was cloned from Corynebacterium glutamicum ATCC 14067 and mutated by overlapping primer to replace the putative adenylation site of GS

Tyr405 by Phe405.The mutant GS was expressed in E.coli and the conditions of GS production were optimized.In the process catalyzed by the mutant GS

glutamine production was increased by adding monosodium glutamate and ammonium chloride in shaking flasks and glutamine yield reached 16.8 g·L-1.By controlling the enzymatic conditions in 5 L bioreactor

glutamine yield reached 34.2 g·L-1 with a molar conversion of 96.3% to glutamate.

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