产酸克雷伯氏菌赖氨酸脱羧酶的异源表达及粗酶性质

Heterologous expression and characterization of Klebsiella oxytoca lysine decarboxylase

摘要:

赖氨酸脱羧酶，可以催化赖氨酸脱羧生成戊二胺。戊二胺是重要的平台化合物，可以合成新型聚酰胺材料、脂肪族异氰酸酯等新材料。本研究对来自于产酸克雷伯氏菌的赖氨酸脱羧酶进行异源表达。以pUC18质粒为载体，将来源于产酸克雷伯氏菌的赖氨酸脱羧酶基因ldc克隆到大肠杆菌，得到菌株LN18。在添加0.5 mmol/L IPTG的LB培养基中，对LN18进行摇瓶培养，发酵液酶活可达到35 U/g发酵液，从发酵液制备的赖氨酸脱羧酶粗酶蛋白的酶活可以达到30 000 U/g粗蛋白。产酸克雷伯氏菌赖氨酸脱羧粗酶蛋白大小约80 kDa，粗酶的最适温度和pH值分别为55 ℃和5.5，与文献中报道的大肠杆菌的赖氨酸脱羧酶CadA在pH 8.0几乎没有酶活不同，产酸克雷伯氏菌的赖氨酸脱羧酶在pH 8.0的酶活达到最优pH下酶活的30%以上。金属离子对酶活有一定的影响，Mg2+对酶活有促进作用，Fe2+ 、Zn2+、Ca2+有一定的抑制作用。

Abstract:

Cadaverine is a biogenic amine that has the potential to become an important platform chemical for the production of industrial polymers, such as polyamides and polyurethanes. We reported here a lysine decarboxylase from Klebsiella oxytoca. The lysine decarboxylase from Klebsiella oxytoca was cloned to Escherichia coli to get the strain LN18. The specific activity of the crude protein from LN18 reached 30 000 U. The molecular weight was about 80 kDa. The optimum temperature and pH of the crude protein were 55 ℃ and 5.5 respectively. The specific activity could keep over 30% at pH 8.0 compared the one at ph 5.5, much difference from Escherichia coli lysine decarboxylase cadA. Mg2+ was positive to the specific activity, whereas Fe2+, Zn2+ and Ca2+ were negative.