重组表达天冬氨酸-β-脱羧酶及其酶学性质

Enzymatic Properties of Recombinant Aspartate-β-decarboxylase

摘要:

天冬氨酸-β-脱羧酶能以L-天冬氨酸为底物脱羧生成L-丙氨酸。本文利用pETDuet-1为载体在宿主细胞E. coli BL21(DE3)中重组表达了粪产碱菌（Alcaligenes faecalis）来源的天冬氨酸-β-脱羧酶（Asd），研究了其酶学性质，考察了pH、pH稳定性、温度、热稳定性、底物浓度对酶活的影响。结果表明：天冬氨酸-β-脱羧酶重组表达成功；最适反应温度和pH为45℃和6.0；动力学常数Km和Vmax分别为0.72 mmol/L和10.52 mol/(L?min?g)。0.1 g菌体细胞在10 mL 0.2 mol/L磷酸缓冲溶液中催化2.0 g L-天冬氨酸，40℃，pH=6.0反应15 h，L-天冬氨酸的摩尔转化率达到99%。

Abstract:

Aspartate-β-decarboxylase is an enzyme that catalyzes the decarboxylation of L-aspartate to produce L-alanine. The gene coding and expressing this enzyme was cloned from Alcaligenes faecalis. In this study, the vector pETDuet-1 was used to recombinant express the aspartate-β-decarboxylase in Escherichia coli BL21(DE3). Several influencing factors of the enzyme reaction, such as pH, pH stability, temperature, thermal stability, concentration of substrate, were all investigated. The results indicated that the recombinant aspartate-β-decarboxylase was successfully expressed and the reaction was optimal at pH 6.0 and 45癈. The Km and Vmax values of aspartate-β-decarboxylase were 0.72 mmol/L and 10.52 mol/(L?min?g). 10 ml of 0.2 mol/L phosphate buffer included 0.1 g wet cells, 0.2 mmol/L 5'-pyridoxal phosphate, and 0.2 g L-aspartate, after 15 hours, the mole conversion rate of L-aspartate was up to 99%.