L-氨基酸连接酶偶联乙酸激酶催化合成丙谷二肽

Enzymatic production of L-alanyl-L-glutamine by L-amino acid ligasecoupled with acetate kinase

枯草芽孢杆菌来源的L-氨基酸连接酶(YwfE)是一种ATP依赖酶,可以催化L-丙氨酸(Ala)和L-谷氨酰胺(Gln)合成丙谷二肽。为了降低反应中ATP的使用量,研究使用不同微生物来源的乙酸激酶(Ack)用于丙谷二肽合成反应中ATP的再生,发现丙酮丁醇梭菌来源的乙酸激酶Ack3和YwfE的偶联效果最好。确定最优的催化反应体系包括Ala 30 mmol/L,Gln 30 mmol/L,MgSO 450 mmol/L,ATP 5 mmol/L,ACP-Li 60 mmol/L,纯化的YwfE蛋白200 mg/L,纯化的Ack3蛋白80 mg/L。在37℃,pH 9条件下反应16 h生成24.3 mmol/L的丙谷二肽,摩尔转化率为81%,同时产生2.3 mmol/L的副产物丙丙二肽。研究表明:ATP再生系统的引入有助于提高丙谷二肽的摩尔转化率,降低催化反应的成本。

YwfE derived from Bacillus subtilis ATCC 15245 is an ATP-dependent enzyme,which can catalyze the synthesis of L-alanyl-L-glutamine(Ala-Gln)using L-alanine and L-glutamine as the substrate.To decrease the amount of ATP used in the reaction,acetate kinase(Ack)from different microorganisms were used for the of regenerate ATP.Ack3 derived from Clostridium acetobutylicum showed the best coupling performance with YwfE.The optimal reaction system for Ala-Gln production contained 30 mmol/L Ala,30 mmol/L Gln,50 mmol/L MgSO 4,5 mmol/L ATP 60 mmol/L ACP-Li,200 mg/L purified YwfE,and 80 mg/L purified Ack3.Under the condition of 37℃and pH 9 in 16 h,24.3 mmol/L Ala-Gln and 2.3 mmol/L by-product L-alanyl-L-alanine were obtained,with a maximum molar yield of 81%.This study demonstrated that the introduction of ATP regeneration system can improve the molar conversion rate of Ala-Gln and reduce the cost of catalytic reaction.