Expression, purification, and characterization of alanine racemase from Pseudomonas putida YZ-26.
Alanine racemase catalyzes the interconversion of D: - and L: -alanine and plays an important role in supplying D: -alanine, a component of peptidoglycan biosynthesis, to most bacteria. Alanine racemase exists mostly in prokaryotes and is generally absent in higher eukaryotes; this makes it an attractive target for the design of new antibacterial drugs. Here, we present the cloning and characterization of a new gene-encoding alanine racemase from Pseudomonas putida YZ-26. An open reading frame (ORF) of 1,230 bp, encoding a protein of 410 amino acids with a calculated molecular weight of 44,217.3 Da, was cloned into modified vector pET32M to form the recombinant plasmid pET-alr. After introduction into E.coli BL21, the strain pET-alr/E.coli BL21 expressed His(6)-tagged alanine racemase. The recombinant alanine racemase was efficiently purified to homogeneity using Ni(2+)-NTA and a gel filtration column, with 82.5% activity recovery. The amino acid sequence deduced from the alanine racemase gene revealed identity similarities of 97.0, 93, 23, and 22.0% with from P. putida F1, P. putida200, P. aeruginosa, and Salmonella typhimurium, respectively. The recombinant alanine racemase is a monomeric protein with a molecular mass of 43 kDa. The enzyme exhibited activity with L: -alanine and L: -isoleucine, and showed higher specificity for the former compared with the latter. The enzyme was stable from pH 7.0-11.0; its optimum pH was at 9.0. The optimum temperature for the enzyme was 37°C, and its activity was rapidly lost at temperatures above 40°C. Divalent metals, including Sr(2+), Mn(2+), Co(2+), and Ni(2+) obviously enhanced enzymatic activity, while the Cu(2+) ion showed inhibitory effects.
Institute of Biotechnology, Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Shanxi University, 92 Wucheng Road, Taiyuan, People's Republic of China.
Jun-Lin
JL
Liu
Xiao-Qin
XQ
Liu
Ya-Wei
YW
Shi
D000411:Alanine Racemase / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D002413:Cations, Divalent / Q000494:pharmacology; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004795:Enzyme Stability; D015870:Gene Expression; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
D002413:Cations, Divalent; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D000411:Alanine Racemase