红曲霉丙酮酸脱羧酶基因克隆及酶学性质分析

Enzymatic Characterization of Recombinant Pyruvate Decarboxylase from Monascus anka CICC 5031

目的:研究生物乙醇发酵关键酶丙酮酸脱羧酶(Pyruvate decarboxylase,PDC)的性质和功能。方法:从SMART技术构建的红曲霉(Monascus anka CICC 5031)cDNA文库中筛选pdc基因,亚克隆到表达载体pET-21b(+),原核表达、亲和层析纯化重组PDC。分析重组PDC和野生型PDC的酶学性质。结果:pdc基因的开放阅读框长1 713bp,编码一个570个氨基酸残基的蛋白质。重组PDC在E.coli BL21(DE3)中的表达量为菌体总蛋白的32.7%。重组PDC与野生型PDC比活分别为20.2U/mg和30.1U/mg。二者的最适反应条件均为pH6.0、30℃。重组PDC和野生型PDC的Km值分别为2.6mmol/L和0.56mmol/L。结论:红曲霉pdc基因可在大肠杆菌中高效表达,重组PDC稳定性较好,可用作生物乙醇生产的候选资源。

Objective:To study the nature and function of pyruvate decarboxylase(PDC),the key enzyme produce ethanol by fermentation.Method:Monascus anka(CICC 5031) pyruvate decarboxylase gene(pdc) was obtained by screening the constructed full-length cDNA library which was constructed with SMART technique,and was subcloned to expression vector pET-21b(+).Recombinant PDC was expressed in E.coli and purified by affinity chromatography.Native PDC was extracted and purified from M.anka through Sephadex G-25 and DEAE-anion exchange resin.Enzymatic characterization of both recombinant and native PDC were studied,respectively.Result:The entire open reading frame of M.anka pdc gene was 1713bp,encoding a 570 amino acid protein.M.anka pdc gene was succussfully heterologously expressed in E.coli BL21(DE3),accounting for 32.7% of total cellular proteins.The specific activity of recombinant and native PDC was 20.2 and 30.11U/mg.Kinetic analysis indicated that recombinant and native PDC had the same optimum conditions:pH6.0,30℃,the Km value of the former was 2.6mmol/L and the later was 0.56mmol/L,separately.Conclusion:The high activity and stable PDC from M.anka accounts for the new candidate resources of fuel ethanol production.