棒状链霉菌扩环酶R306位点的定点突变

目的:通过对棒状链霉菌的脱乙酰氧基头孢菌素C合酶(DAOCS)C末端R306位点突变,改变酶的活力和底物专一性。方法:利用生物信息学和空间结构分析推测,利用定点突变技术,对DAOCS的C-末端R306位点进行定点突变。结果:将DAOCS的C末端R306位点突变为其它3种性质相异的氨基酸,显示酶的活力和底物专一性都有一定改变。结论:DAOCS的C末端修饰对于提高酶活力或改变酶的底物专一性是一个非常有效的策略。

Study on site -directed mutagenesis of Streptomyces clavuligerus deacetoxycephalosporin C synthase

Aim: R306 of Streptomyces clavuligerus deacetoxycephalosporin C synthase was mutagenized to alter its catalytic efficiency and substrate specificity.Methods:The residue R306 located in close proximity to the C-terminus of DAOCS was mutated into three different characters amino acid residues with site-directed mutagenesis which was based on the analysis of biological information and space structure.Results:After replacing R306 of scDAOCS with various aliphatic amino acids,the activities of the expressed enzymes were investigated.The R306L mutant scDAOCS showed improvements for all of the penicillin analogue conversions(124% to 190%).The changing of arginine to leucine is instrumental in enhancing interaction with hydrophobic substrates,thereby contributing to the improvement of its enzymatic activity.However,the catalytic ability of the R306F mutant scDAOCS was diminished,while that of the R306K mutant scDAOCS was abolished.Conclusion:Modification of C-terminus of DAOCS is an effective strategy for changing both catalytic efficiency and substrate specificity.