[Cloning, sequence analysis and expression of alanine racemase gene in Pseudomonas putida]

Two distinct alanine racemase genes from Pseudomonas putida 200 were cloned and sequenced. DadX encodes a peptide of 357 amino acids with a calculated molecular weight of 38.82kDa. The putative product of alr gene is a peptide of 409 amino acids with molecular weight of 44.182kDa. A homology comparison revealed identities of 96.64%, 71.99%, 44.88% and 47.37% of the DadX alanine racemase to those from P. putida KT2440, Pseudomonas aeruginosa, Salmonella typhimurium and Escherichia coli, respectively. The amino acids sequence deduced from alr gene showed the homologies of 94.38%, 22.89%, 25.72% and 26.44% to those from the microorganisms above, respectively. Two motifs believed essential to the enzyme activity are found both in DadX and Alr, such as pyridoxal-5'-phosphate binding site. Both dadX and alr were expressed in E. coli TG1. Neither alanine racemase activity or serine racemase activity was detected in the host strain. Only alanine racemase activity was found in E. coli TG1/pCTD. But both E. coli TG1/pCTA and TG1/pCBA exhibit activity toward L-alanine and L-serine. Transcription of alr gene in E. coli is independent from extraneous promoter, a result confirmed by the significant enzyme activity observed in the E. coli TG1/pCBA, which indicates the presence of a possible promoter upstream the structure gene.