共表达PPK和GMAS全细胞催化合成L-茶氨酸

Synthesis of L-Theanine by Whole Cell Catalyst Co-expressing PPK and GMAS

利用pETDuet-1质粒在大肠杆菌BL21(DE3)中重组表达了多聚磷酸盐激酶(PPK)和γ-谷氨酰甲胺合成酶(GMAS),并以共表达PPK和GMAS的重组菌全细胞催化合成L-茶氨酸,优化了反应参数。SDS-PAGE结果表明,PPK和GMAS共表达成功;最佳全细胞催化反应条件为:35℃,pH=7.0,谷氨酸钠300mmol/L,乙胺盐酸盐420 mmol/L,六偏磷酸钠100 mmol/L,添加2 mmol/L起始量ATP,在100 mL反应体系中转化24 h,L-茶氨酸浓度达到199 mmol/L,谷氨酸钠的转化率达到66.34%。

Polyphosphate kinase(PPK)andγ-glutamylmethylamide synthetase(GMAS)were recombinant expressed using pETDuet-1 plasmid in Escherichia coli BL21(DE3).L-theanine was synthesized by whole cell catalyst co-expression of PPK and GMAS and the reaction parameters were optimized.The SDS-PAGE analysis showed that PPK and GMAS were successfully co-expressed in the recombinant strains.The optimal conditions for whole cell biocatalytic reaction were as follows:temperature 35℃,pH=7.0,300 mmol/L monosodium glutamate,420 mmol/L ethylamine hydrochloride,100 mmol/L sodium hexametaphosphate 2 mmol/L ATP.Under the optimal conditions,the L-theanine yield reached 199 mmol/L,and the conversion rate of monosodium glutamate was 66.34%after 24 h in a 100 mL reaction system.