谷氨酸棒杆菌YILW苏氨酸脱水酶基因的克隆表达及酶学性质

摘要

将L-异亮氨酸生产菌谷氨酸棒杆菌(Corynebacterium glutamicum YILW) 苏氨酸脱水酶(threonine dehydratase,TD)的编码基因ilvA在大肠杆菌中进行异源表达及进行初步的酶学性质研究.分别以C.glutamicum ATCC13032、YILW的基因组DNA为模板,利用PCR技术扩增出苏氨酸脱水酶的编码基因ilvA,测序获得编码序列.利用质粒PET-His将该基因在大肠杆菌BL21(DE3)中进行重组表达、金属螯合纯化,对其酶学性质进行初步研究.结果显示C. glutamicum YILW编码基因序列与已报道的ilvA序列相差5个碱基,相似度为99.6%,第383位氨基酸由苯丙氨酸突变为缬氨酸.酶学性质研究表明:重组酶YilwTD最适反应温度为32 ℃,在20~55 ℃范围内该酶较稳定,最适pH为6.7,该酶底物专一性强,对最适底物苏氨酸的米氏常数Km=8.32 mmol/L, 最大反应速度Vmax= 3.18×104 U/mg,与野生型酶相比,突变(F383V)后可显著降低终产物对酶的反馈抑制作用.为揭示突变对苏氨酸脱水酶活性的影响及进一步利用基因工程技术改造L-异亮氨酸生产菌,提高L-异亮氨酸产量奠定了基础.%The threonine dehydratase gene ilvA from Corynebacterium glutamicum YILW was expressed in Escherichia coli and the enzymatic properties were characterized.The two ilvA genes amplified from the C.glutamicum YILW and ATCC13032 genome by PCR with specific primers, sequenced and expressed at E.coli BL21 (DE3) by using expression vector pET-His.The recombinant threonine dehydrates enzyme was purified by metalchelate chromatography and its several characteristics were determined.According to the alignment of amino acid seqences, it was found that the comparability between the cloned ilvA sequence and the reported sequence was up to 99.6％.Only the 383th amino acid Phe mutated to Val (Phe→Val),the others were not.The optimal pH and the temperature of recombinant enzyme YilwTD were pH 6.7 and 32 ℃, respectively.The recombinant enzyme YilwTD showed the highest activity toward threonine (Km =8.32 mmol/L, Vmax =3.18 × 104 U/mg,at 32 ℃ ).Furthermore, the mutation (F383V) could significantly reduce the end-product feedback inhibition of the enzyme.The enzymatic properties of threonine dehydratase from C.glutamicum YILW were characterized for providing the fundament to the revealing the impact of mutations on the enzyme and increasing L-isoleucine production.