L-茶氨酸是茶叶中一种独特的非蛋白质氨基酸。γ-谷氨基甲酰胺合成酶可以催化L-谷氨酸、乙胺和ATP合成L-茶氨酸,但反应成本较高。为了提高L-茶氨酸的生产效率,该研究开发了直接发酵生产L-茶氨酸的新方法。首先,在大肠杆菌基因组上双拷贝Methylovorus mays来源的γ-谷氨酰甲胺合成酶基因gmas,获得了一株遗传稳定的用于L-茶氨酸生产的重组菌株。其次,在5 L罐中采用流加乙胺的方式发酵20 h, L-茶氨酸产量达到30.45 g/L,糖酸转化率达到20.17%。最后,根据L-茶氨酸的物化性质拟定出合适的分离提取路线,从发酵液中获得了98.51%纯度的L-茶氨酸成品,总提取收率可达到70.34%。该方法操作简单,原料成本较低,具有较好的工业应用前景。
L-theaine is a unique non-protein amino acid in tea. The L-theaine can be synthesized from L-glutamate and ethylamine by γ-glutamylmethylamide synthetase with assistance of ATP, but the cost of process is high. In order to improve the production efficiency of L-theanine, a new direct fermentation method was developed. Firstly, a genetically stable recombinant Escherichia coli strain was constructed by integrating double copy of the γ-glutamylmethylamine synthase gene (gmas), which derived from Methylovorus mays, into the genome. Subsequently, fed-batch fermentation was carried out in a 5 L fermenter with ethylamine supplement. The yield of L-theanine reached 30.45 g/L, and the conversion rate of glucose to L-theanine reached 20.17% after 20 h fermentation. Additionally, a suitable separation and extraction route was established according to the knowledge about the physicochemical properties of L-theanine. The whole extraction yield of L-theanine from the fermentation broth was 70.34% with a purity of 98.51%. The method has the advantages of simple operation and cheap raw materials, and has broad prospects for industrial application.
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