The Research On Enzymatic Properties Of Alanine Racemase From Bacillus Pseudofirmus OF4

As an important natural Amino acid, L-Alanine was existed in nature but nor do the D-Alanine. D-Alanine was existed widely in Peptidoglycan of the bacterium cell wall, Crosslinking with the Peptidoglycan unit in the form of D-Alanyl-D-Alanine. Alanine racemase (EC 5.1.1.1) can be used to catalyse the Optical Structure changing of L-Alanine and D-Alanine so as to mediate the transformation between each other. It was depended on 5’-PLP and belonged to Isomerase family. Alanine racemase provided necessary Pre-D-Alanine for the form of bacterium cell wall.It was an essential enzyme in Prokaryotes. Additional, Alanine racemase played a key role in inhibiting the Bacillus spores Germination. Alanine racemase was also associated with human diseases, such as tuberculosis, anthrax, and otitis. Therefore, accord to the molecular mechanism of Alanine racemase, exploring for Enzyme inhibitor could be regarded as an potential target for the design of new antibacterial drugs.In this study, An open reading frame of 1100 bp in the partially genome sequence of Alkaliphilic Bacillus pseudofirmus OF4 was identified as a putative alanine racemase gene (dadXOF4), which was cloned and expressed in Escherichia coli BL21 (DE3). The encoded protein DadXOF4 was purified to homogeneity by His-tag affinity column, gel filtration and ion-exchange chromatography. The racematic was demonstrated by complement of a D-alanine auxotroph strain MB2795.The molecular weight comfirmed by gel filtration was 41 KD. The optimal pH was 10.5 and the racemization temperature optimum was 40℃. The kinetic parameters Km and Vmax of alanine racemase determined by HPLC analysis, were 41.79 mM,10,500 units/mg for L-alanine and 14.91 mM,3,708 units/mg for D-alanine at 40℃, respectively.OF4dadx was compared with alanine racemases of Thermoanaerobacter tengcongensis MB4Tand B. stearothermophilus,According to the amino acid sequence alignment,four mutants N175E,V237E,I350S,Y352F were designed and constructed by site-direct mutagenesis.Compared the results of Enzymatic properties, the optimal pH of OFdadX-N175E was 9.6 and the racemization temperature optimum was 30℃. The optimal pH of OFdadX-V237E was 11 and the racemization temperature optimum was 30℃. The optimal pH of OFdadX-I350S was 12 and the racemization temperature optimum was 40℃. Mutation N175E and I350S had lower thermal stability and pH stability. Y352F had different substrate specificity exclude D-alanine.N175X saturation mutagenesis library was constructed by whole-plamid PCR and screened to get a serial of mutants with further improved activity. Sequencing of the improved mutants showed that hydrophobic mutant increase catalytic efficiency most effectively. The large hydrophobic mutants(R, S, and D) increased the catalytic efficiency by almost 5-fold.