Cloning and expression of a serine racemase gene homologue of the green alga Chlamydomonas reinhardtii and characterization of the gene product

A unicellular green alga Chlamydomonas reinhardtii (C. reinhardtii) has served as a model system to study many fundamental biological processes. We demonstrated that some D-amino acids have no inhibitory effect on the growth of C. reinhardtii and the green alga has alanine racemase and D-threonine aldolase. The homologous gene of serine racemase was found on the genome sequence of C. reinhardtii. In this study, a homologous gene of serine racemase on the genome of C. reinhardtii was cloned and expressed in E. coli cells, and the gene product was purified and characterized. Total RNA was extracted from C. reinhardtii cells. Sense and antisense primers were designed for PCR based on the upstream and downstream regions of the putative gene for serine racemase. First strand cDNA was synthesized from the mRNA and the antisense primer. Amplification of nucleotides between the two primers was performed with the cDNA. The fragment (ser-h) was sequenced. The deduced protein consisted of 340 amino acids with a molecular weight of 35,300. The amino acid sequence of the protein showed similarities to the reported serine racemases; Oryza sativa, 55%; Mus musculus, 52%; Schizosaccharomyces pombe, 39%. A modified serine racemase homologous (ser-h\u27) whose codons were optimized for E. coli was synthesized and used to construct pET24/ser-h\u27 and to transform BL21 (DE3). SDS-PAGE of the crude extract revealed that the gene product was overexpressed. The gene product was purified to electrophoretic homogeneity from the recombinant cells using ammonium sulfate fractionation and Column chromatography. Further characterization and crystallization of the enzyme are currently under study

Purification and pressure dependence of alanine racemase from the psychro- piezophilic bacterium shewanella violacea DSS 12

Shewanella violacea DSS12 (S. violacea) is a psychrophilic and piezophilic bacterium, isolated from mud of the Ryukyu Trench in Japan. The bacterium displays optimal growth at 8°C and 30 MPa. Alanine racemase is an enzyme which catalyses the interconversion of l-alanine and d-alanine, and is responsible for the synthesis of d-alanine contained in the peptidoglycan of bacterial cell wall. In this study, we purified alanine racemase from S. violacea and investigated the enzymological characteristics of alanine racemase. The bacterium was aerobically cultured using marine broth 2216 in a 5-liter medium bottle at 4°C for 3 days. The bacterial cells were lysed by applying of 100 MPa pressure using a French press, and the lysate was centrifuged. The supernatant obtained was ultracentrifuged at 141,000 g, and the supernatant obtained was applied to ammonium sulfate fractionation. The active fraction was dissolved and passed through a butyl-Toyopearl, phenyl-Sepharose, and shodex KW-200 columns to obtain a partially purified enzyme. Consequently, the enzyme was purified 540-fold and showed a specific activity of 2.68 μmol/min/mg. Alanine racemase exhibited high activity against l-Ala and l-Ser as substrates. The optimal pH and temperature of alanine racemase were 9.0 and 25°C, respectively. Please click Additional Files below to see the full abstract