原玻璃蝇节杆菌D-氨基酸氧化酶及突变体的酶学特性

Characterization of D-amino acid oxidase and its mutants from Arthrobacter protophormiae

摘要:

摘要:【目的】研究原玻璃蝇节杆菌(DSM 20168)中D-氨基酸氧化酶的酶学特性。【方法】通过PCR从原玻璃蝇节杆菌(DSM 15035，20168)中克隆获得D-氨基酸氧化酶基因apdaao-1和apdaao-2，构建原核表达载体，以表达质粒pET-ApDAAO-2为模板，采用QuickChange Site-Directed Mutagenesis技术构建定点突变体，经过原核表达及纯化获得重组型和突变体酶蛋白，分析其酶学特性。【结果】通过原核表达及纯化成功获得了2个重组蛋白和4个突变体酶蛋白，SDS-PAGE检测显示其分子量均约为36 kDa;酶学特性分析表明，ApDAAO-2和突变体蛋白的最适反应温度为30℃;ApDAAO-2和T286A的最适反应pH范围为7.0－11.0，其它突变体为8.0－11.0;ApDAAO-2和突变体都具有较广泛的底物特异性，除T256K的最适底物为D-Phe外，其余均为D-Met;动力学参数测定结果显示，以二级表观常数kcat/Km表示，对于底物D-Met或D-Phe，ApDAAO-2和4个突变体的kcat /Km值均比ApDAAO-1和pKDAAO高数倍以上。【结论】ApDAAO-2及突变体具有比ApDAAO-1和pKDAAO更广泛的底物特异性和较高的催化效率，有一定的商业应用价值。

Abstract:

Abstract:［Objective］ To characterize D-amino acid oxidase from Arthrobacter protophormiae (DSM 20168)．［Methods］ Genes apdaao-1 and apdaao-2 from A．protophormiae (DSM 15035 ＆ 20168) were cloned by PCR;expression vectors were constructed and expressed in E.coli BL21 (DE3)．The mutant was constructed by site-directed mutagenesis using plasmid pET-ApDAAO-2 as the template．After Ni-NTA column chromatography purification，the protein was characterized.［Results］ Protein ApDAAO-1，ApDAAO-2 and 4 mutants were expressed and purified successfully．The apparent molecular masses of all purified proteins were about 36 kDa by SDS-PAGE．The optimum temperature of ApDAAO-2 and 4 mutants was 30℃ similar to ApDAAO-1． ApDAAO-2 and its mutants exhibited much broader optimal pH than ApDAAO-1，and they revealed broad substrate specificity and high specificity to D-Met (100%) except T256K，which showed the substrate preference for D-Phe (108%)．For substrates D-Met and D-Phe，the secondorder rate constants kcat/Km of ApDAAO-2 and 4 mutants were several-fold higher than ApDAAO-1 and pKDAAO，respectively.［Conclusion］Comparing with ApDAAO-1 and pKDAAO，ApDAAO-2 and its mutants had much broader substrate specificity and higher catalytic efficiency，which suggested that they might have much higher commercial value.