利用重组枯草芽孢杆菌生产γ-氨基丁酸的研究
Study on Utilizing a Recombinant B.subtilis 168/pHT01-gadA-pdxH for Synthesis of γ-aminobutyric Acid
张六六 1毛连山2
作者信息
- 1. 江苏纳克生物工程有限公司,江苏淮安211700
- 2. 南京林业大学化学工程学院,江苏南京210037
- 折叠
摘要
[目的]构建一株高产L-谷氨酸脱羧酶的重组枯芽孢杆菌B.subtilis 168/pHT01-gadA-pdxH.[方法]以枯草芽孢杆菌(Bacillus subtilis)为宿主细胞,将大肠杆菌脱羧酶基因(gadA)与其辅酶磷酸吡哆醛的再生基因(pdxH)在质粒pHT01上串联表达,构建一株高产L-谷氨酸脱羧酶的重组枯草芽孢杆菌B.subtilis 168/pHT01-gadA-pdxH.[结果]带有pdxH基因的重组菌B.subtilis 168/p-HT01-gadA-pdxH催化谷氨酸生成γ-氨基丁酸(GABA)的效率明显高于B.subtilis 168/pHT01-gadA.催化反应24h时,生成的GABA浓度达252 g/L,较对照菌株提高了61 g/L.进一步对重组菌B.subtilis 168/ pHT01-gadA-pdxH全细胞转化谷氨酸生成GABA的条件进行了优化,其最优条件:转化缓冲液为pH 5.0的0.1 mol/L Tris-HC1,转化温度40℃,激活剂为5 mmol/L Ca2与5 mmol/L Mg2+.在上述最优条件下,催化24h生成的GABA浓度这327 g/L.[结论]所获得的重组菌转化效率较高,具有一定的工业化应用前景.
Abstract
[Objective] The aim was to construct a recombinant B.subtilis 168/pHT01-gadA-pdxH for producing high yield of L-glutamate decarboxylase.[Method] The gadA and pdxH gene from E.coli was cloned to the plasmid pHT01,resulted in the two recombinant plasmids pHT01-gadA and pHT01-gadA-pdxH,and then separately transformed to Bacillus subtilis 168.[Result] The recombinant B.subtilis 168/pHT01-gadA-pdxH can produce 252 g/L γ-aminobutyric acid (GABA) after catalytic reaction 24 h,compared with the B.subtilis 168/pHT01-gadA increased by about 61 g/L.The whole cell transformation glutamate to GABA conditions were optimized,and the optimum conditions were 0.1 mol/L Tris-HCl pH 5.0 buffer,40 ℃,5 mmol/L Ca2+ and 5 mmol/L Mg2+.Under optimum conditions,the GABA concentration reached 327 g/L by the recombinant B.subtilis 168/pHT01-gadA-pdxH.[Conclusion] The obtained recombinant bacteria has higher transformation efficiency,with a certain industrialization prospect.
关键词
枯草芽孢杆菌/L-谷氨酸脱羧酶/磷酸吡哆醛的再生/L-谷氨酸/-γ-氨基丁酸Key words
Bacillus subtilis/L-glutamate decarboxylase/Regeneration of pyridoxal phosphate/L-glutamic acid/γ-aminobutyric acid引用本文复制引用
出版年
2016
国家科技期刊平台
NSTL
维普
万方数据