干酪乳杆菌L-乳酸脱氢酶在大肠杆菌中的表达、纯化及酶学性质

Overexpression, purification and properties of ldhL gene from Lactobacillus casei in Escherichia coli

摘要:

L-乳酸脱氢酶(L-lactate dehydrogenase, L-LDH)是发酵生产L-乳酸中催化丙酮酸转化成L-乳酸的关键酶。以干酪乳杆菌G-02 (Lactobacillus casei G-02)基因组DNA为模板, 克隆得到L-LDH基因(ldhL), 经序列分析后将其连接到表达载体pET-28a(+)上, 构建成重组质粒pET-ldhL转化到大肠杆菌BL21(DE3)中, 实现ldhL基因的表达。30 °C加入IPTG诱导表达后, 经镍柱亲和层析纯化的重组蛋白样品通过SDS-PAGE分析, 约在40 kD处出现显著的特异性条带。对表达的L-LDH生物学特异性研究显示: 重组L-LDH的比酶活为1 722 U/mg, 最适反应温度为40 °C?45 °C; 果糖-1,6-二磷酸(FBP)为别构激活剂, 使最适pH向中性方向偏移(pH为6.6?6.8), Mn2+可拓宽最适酶活pH范围; Mn2+、Ca2+和Mg2+对L-LDH有激活作用, 而Zn2+对L-LDH有抑制作用。

Abstract:

L-lactate dehydrogenase (L-LDH) is a key enzyme which catalyzes the formation of L-lactate from pyruvate in the Lactobacillus sp.. The gene ldhL encoding L-LDH was amplified from genome DNA of Lactobacillus casei using PCR technique. The PCR product was cloned into pUcm-T vector and double digested with restriction endonucleases, and then the DNA fragment of ldhL was inserted into pET-28a(+). The recombinants expression plasmid pET-ldhL was obtained, and was transformed into E. coli BL21. After it was induced to express L-LDH with IPTG, and purified by affinity chromatography. SDS-PAGE showed that the molecular weight of specific fusion protein was 40 kD. The biochemical properties of L-LDH showed that the specific activity were up to 1 722 U/mg with optimum catalysis temperature of 40 °C?45 °C and pH of 6.6?6.8. Fructose-1,6-bisphosphate (FBP) is a positive allosteric modifier of the enzyme, the addition of Mn2+ to the assay in the presence of FBP broadens the pH profile, particularly towards neutral pH values. Mn2+, Ca2+ and Mg2+ increases the activity of L-LDH, but Zn2+ decreases its activity.