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

Abstract:

[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 (k_cat/K_m) towards L/D-serine compared to the wild type StAlr, while the mutant A3V showed a distinct reduction in K_m value and dramatic increase in k_cat and k_cat/K_m values towards L/D-alanine and L/D-serine. For substrate L-alanine and D-alanine, the k_cat/K_m values of A3V were 105.51 and 97.36-fold of that of wild type StAlr, whereas for L-serine and D-serine, the k_cat/K_m 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.