Expression, purification, and characterization of alanine racemase from Pseudomonas putida YZ-26 |
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Authors: | Jun-Lin Liu Xiao-Qin Liu Ya-Wei Shi |
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Institution: | (1) 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; |
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Abstract: | 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 His6-tagged alanine racemase. The recombinant alanine racemase was efficiently purified to homogeneity using Ni2+–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 Sr2+, Mn2+, Co2+, and Ni2+ obviously enhanced enzymatic activity, while the Cu2+ ion showed inhibitory effects. |
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