通过易错PCR提高鼠伤寒沙门氏菌丙氨酸消旋酶催化活性

Improving catalytic activity of alanine racemase from Salmonella typhimurium by error-prone PCR

【目的】通过易错PCR技术提高鼠伤寒沙门氏菌中丙氨酸消旋酶的催化活性。【方法】利用易错PCR技术构建丙氨酸消旋酶基因alrSt的突变体文库,采用缺陷菌株UT5028筛选突变体基因,以D-氨基酸氧化酶偶联法检测各突变蛋白的活性,通过凝胶过滤层析法分析酶蛋白寡聚化状态,并采用HPLC检测酶蛋白的动力学参数。【结果】经过易错PCR及定点突变技术最终获得了3个催化活性有所提高的突变体A3V、Y343H和A3VY343H,酶学特性分析发现,与野生型蛋白StAlr相比,突变体Y343H仅对底物L/D-丝氨酸的催化效率略有提高,kcat/Km值分别是StAlr的2.01和3.68倍;而突变体A3V则对底物L/D-丙氨酸或L/D-丝氨酸的Km、kcat和kcat/Km值均有较大幅度的改变,其kcat/Km值分别是StAlr的105.51、97.36、4.63和10.73倍。凝胶过滤层析结果显示,突变体A3V在蛋白含量极低时就呈现出单体和二聚体共存状态,且随着蛋白含量的增加,其向二聚体状态迁移的速率最为明显。【结论】丙氨酸消旋酶StAlr的第3位点是影响其催化活性和低聚合状态的关键位点。

[Objective]To enhance the catalytic activity of alanine racemase from Salmonella typhimurium by error-prone PCR.[Methods]A mutant library of alanine racemase from S.typhimurium was constructed by error-prone PCR using plasmid pTrc99A-StAlr or pTrc99A-Y343H as template,and DNA recombination with improved catalytic activity were screened by serine auxotroph strain UT5028.Racemase activities for converting both L-alanine to D-alanine and L-serine to D-serine were calculated based on the absorbance at 550 nm using Epoch Microplate Spectrophotometer.The cell lysate was separated by gel filtration chromatography,and each fraction was detected for the alanine racemase activity to analyze the oligomerization states.Kinetic parameters of StAlr and mutants were determined by measuring the total amount of L/D-alanine or L/D-serine by high performance liquid chromatography with a spectrofluorometer.[Results]Three mutants Y343H,A3VY343H and A3V with improved catalytic activities were obtained by two rounds of error-prone PCR and site-directed mutagenesis,separately.Based on the kinetic parameters,the mutant Y343H only displayed a 2.01 and 3.68-fold improvement in catalytic efficiency(kcat/Km)towards L/D-serine compared to the wild type StAlr,while the mutant A3V showed a distinct reduction in Km value and dramatic increase in kcat and kcat/Km values towards L/D-alanine and L/D-serine.For substrate L-alanine and D-alanine,the kcat/Km values of A3V were 105.51 and 97.36-fold of that of wild type StAlr,whereas for L-serine and D-serine,the kcat/Km values of A3V were 4.63 and 10.73-fold of that of wild type StAlr.Gel filtration chromatography revealed that only the mutant A3V eluted as two distinct peaks at a very low amount of protein,which may correspond to the dimeric and monomeric form,respectively.As the amount of protein increased,the oligomerization states of all proteins were gradually shifted from monomeric to dimeric form.These indicated that the monomer-dimer transition of mutant A3V might be much faster than that of protein StAlr and A3VY343H.[Conclusion]The residue A3 located at N-terminus of StAlr might be a key residue for its catalytic activity and oligomerization state.