摘要
从谷氨酸棒杆菌(Corynebacterium glutamicum)中克隆得到L-天冬氨酸α-脱羧酶基因panD。将该基因连接到pET-24a载体上,并在Escherichia coli BL21(DE3)中实现了成功表达。蛋白电泳检测显示,PanD自裂解后形成的α亚基和β亚基,即PanD重组酶具有自身加工功能。重组酶的比酶活高达2.60 U/mg(156 mmol/g·h)。酶学性质研究表明,其最适温度为55℃,最适pH为7.0。80℃条件下,PanD的半衰期约为40 min。在37℃,pH7.5条件下,K_m值为4.26 mmol/L,V_(max)为35.97 nmol/min,k_(cat)为1.02/s,催化效率k_(cat)/K_m为0.24 L/mmol·s。
L-aspartate-α-decarboxylase gene panD from Corynebacterium glutamicum was cloned and efficiently expressed in Escherichia coli BL21 （ DE3 ） . The a and β subunits of PanD were observed by Tris-tricine SDS-PAGE, indicating that the PanD was successful processed by self cleavage. The specific activity of the recombinant enzyme was as high as 2.60 U/mg （ 156 mmol/g . h ） . The optimum temperature was 55℃ and the optimum pH was 7.0. Under the condition of 80℃, the half-life of PanD was approximately 40 min. Under the condition of 37℃ and pH7.5, the K）m value was 4.26 mmol/L, the V_max value was 35.97 nmol/min, the k_cat value was 1.02/s and the catalytic efficiency （ k_cat/K_m ） was 0.24 L/mmol. s.
出处
《生物技术通报》
CAS
CSCD
北大核心
2013年第4期110-115,共6页
Biotechnology Bulletin
基金
国家自然科学基金项目(31070711)
新世纪优秀人才项目(NCET-10-0461)
关键词
L-天冬氨酸α-脱羧酶
谷氨酸棒杆菌
表达
纯化
酶学性质
L-aspartate-α-decarboxylase
Corynebacterium glutamicum
Expression
Purification
Enzymatic properties
