α-氨基酸酯酰基转移酶表达纯化、酶学性质及其催化应用

Expression, purification, characterization and application of α-amino acid ester acyltransferase from recombinant Escherichia coli

摘要:

α-氨基酸酯酰基转移酶 (α-amino acid ester acyltransferase，AET) 能够催化底物L-丙氨酸甲酯盐酸盐、L-谷氨酰胺合成L-丙氨酰-L-谷氨酰胺 (L-alanyl-L-glutamine，丙谷二肽)。利用重组大肠杆菌saet-QC01表达α-氨基酸酯酰基转移酶，对其表达条件进行了优化，通过Ni-NTA亲和层析法分离纯化重组蛋白，并对其酶学性质、催化应用进行了研究。适合酶表达的诱导条件：温度20 ℃，诱导阶段 (OD600=2.0?2.5)，IPTG浓度0.6 mmol/L，诱导时间12 h。α-氨基酸酯酰基转移酶的最适反应温度27 ℃，最适pH 8.5，在pH 7.0?8.0很稳定，在酸性条件下相对稳定，低浓度的Co2+、低浓度的EDTA对酶活有促进作用。在底物浓度丙氨酸甲酯盐酸盐600 mmol/L、谷氨酰胺480 mmol/L，丙谷二肽的产量达到78.2 g/L，生产速率达到1.955 g/(L·min)，转化率达到75.0%。α-氨基酸酯酰基转移酶具有良好的酸碱耐受性，催化效率高的优良特性，在工业生产中具有较好的应用潜力。

Abstract:

α-Amino acid ester acyltransferase (Aet) catalyzes the L-alanyl-L-glutamine forming reaction from L-alaine methylester hydrochloride and L-glutamine. In this study, the recombinant Escherichia coli saet-QC01 was used to express the α-amino acid acyltransferase, and its expression conditions were optimized. The recombinant protein was separated and purified by Ni-NTA affinity chromatography, and its enzymatic properties and catalytic applications were studied. The induction conditions suitable for enzyme production optimized were as follows: The temperature was 20 ℃, the induction stage (OD600=2.0?2.5), IPTG concentration was 0.6 mmol/L, induction time was 12 h. The optimal reaction conditions of α-amino acid acyltransferase were 27 ℃, pH 8.5, it was most stable between pH 7.0 and 8.0 and relatively stable in an acidic environment, and low concentration of Co2+ or EDTA could promote the enzyme activity. Under optimal reaction conditions, 600 mmol/L of L-alaine methylester hydrochloride and 480 mmol/L of L-glutamine, the yield of L-alanyl-L-glutamine reached 78.2 g/L and productivity of 1.955 g/L/min, the conversion rate reached 75.0%. α-Amino acid ester acyltransferase has excellent acid-basei resistance, high catalytic efficiency. These characteristics suggest its application prospects in the industrial production.