No abstract

Salinisphaera sp. P7-4 was isolated from the intestine of silver whiting, Sillago japonicas caught in the Pacific Ocean, and the esterase gene was cloned using the shotgun method. The amino acid sequence deduced from the nucleotide sequence (951 bp) corresponded to a protein of 316 amino acid residues with a molecular weight of 34,443. The esterase had 46 and 44% identities with the esterase enzymes of Ralstonia eutropha JMP134 and Rhodopseudomonas palustris HaA2, respectively. The primary structure of P7-4 esterase showed the conserved catalytic triad (Ser, Asp, His), consensus pentapeptide GXSXG, and oxyanion hole sequence (HG). The protein P7-4 was successfully expressed in Escherichia coli in a biologically active form. The enzyme showed high catalytic activity at low temperatures (5-25° C) with an activation energy of 2.18 kcal/mol. This result indicated that the esterase from Salinisphaera sp. P7-4 is a new cold-adapted enzyme. The enzyme preferentially hydrolyzed acyl-group chains with short chain lengths of ≤10 carbon. Metal ions such as Cd2(+), Co2(+), Cu2(+), Hg2(+), Ni2(+) and Zn2(+) inhibited enzymatic activity. Additionally, EDTA has no effect on its activity, whereas inhibition was observed with PMSF, a serine hydrolase inhibitor.

Citrobacter braakii produced an intracellular acid glucose phosphatase (AgpC) which was purifi ed 986 fold to homogeneity with the specifi c activity of 286 units/mg. AgpC hydrolyzed a wide variety of phosphorylated compounds with high activity for glucose-1-phosphate and glucose-6-phosphate. The optimum pH and temperature for the enzyme activity was pH 5.0 and 45 C, respectively. The Km value for glucose-1-phosphate was 5.12 mM with a Vmax 27.8 U mg − −1 . Its molecular weight was 46 kDa by SDS-PAGE gel and the sequence of N-terminal amino acid residues identifi ed was Gln-Thr-Ala-Pro-Glu-Gly-Tyr-Gln-Leu-Gln. The glucose-1-phosphatase gene (agpC) was cloned from the C. braakii genomic library. This gene comprised 1,242 nucleotides and encoded a polypeptide of 413 amino acids. The result of its BLAST search showed a significant similarity with glucose-1-phosphatase from enterobacteria such as E. coli, Enterobacter, Shigella, and Salmonella.