共表达PPK和GMAS全细胞催化合成L-茶氨酸

Synthesis of L-theanine by Whole Cell Catalysis with co-expression of PPK and GMAS

摘要:

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 oC，pH=7.0，谷氨酸钠300 mmol/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，转化率达到66.34%。

Abstract:

Polyphosphate kinase (PPK) and γ-glutamylmethylamide synthetase (GMAS) were recombinant expressed using pETDuet-1 plasmid in Escherichia coli BL21(DE3), and L-theanine was synthesized by whole cell catalysis with co-expression of PPK and GMAS. The parameters of reaction were investigated and 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: 35 oC, pH=7.0, 300 mmol/L monosodium glutamate, 420 mmol/L ethylamine hydrochloride, 100 mmol/L polyP, 2 mmol/L ATP. After 24 h in a 100 mL reaction system, under the optimal conditions the L-theanine yield can reach 199 mmol/L, and the conversion rate reaches 66.34%.