A cold-adapted esterase of a novel marine isolate, Pseudoalteromonas arctica: gene cloning, enzyme purification and characterization.

A gene encoding an esterase (estO) was identified and sequenced from a gene library screen of the psychrotolerant bacterium Pseudoalteromonas arctica. Analysis of the 1,203 bp coding region revealed that the deduced peptide sequence is composed of 400 amino acids with a predicted molecular mass of 44.1 kDa. EstO contains a N-terminal esterase domain and an additional OsmC domain at the C-terminus (osmotically induced family of proteins). The highly conserved five-residue motif typical for all alpha/beta hydrolases (G x S x G) was detected from position 104 to 108 together with a putative catalytic triad consisting of Ser(106), Asp(196), and His(225). Sequence comparison showed that EstO exhibits 90% amino acid identity with hypothetical proteins containing similar esterase and OsmC domains but only around 10% identity to the amino acid sequences of known esterases. EstO variants with and without the OsmC domain were produced and purified as His-tag fusion proteins in E. coli. EstO displayed an optimum pH of 7.5 and optimum temperature of 25 degrees C with more than 50% retained activity at the freezing point of water. The thermostability of EstO (50% activity after 5 h at 40 degrees C) dramatically increased in the truncated variant (50% activity after 2.5 h at 90 degrees C). Furthermore, the esterase displays broad substrate specificity for esters of short-chain fatty acids (C(2)-C(8)).

Pseudoalteromonas arctica sp. nov., an aerobic, psychrotolerant, marine bacterium isolated from Spitzbergen.

A novel aerobic, psychrotolerant marine bacterium was isolated at 4 degrees C from seawater samples collected from Spitzbergen in the Arctic. The strain was a polar-flagellated, Gram-negative bacterium that grew optimally at 10-15 degrees C and pH 7-8 in media containing 2-3% NaCl (w/v), using various carbohydrates and organic acids as substrates. The main fatty acid components included 16:0 (12.7% of total fatty acids), straight-chain saturated fatty acid methyl ester (FAME) and 16:1omega7c (40.2%) monounsaturated FAME. Phylogenetic analysis revealed a close relationship (99% 16S rRNA gene sequence similarity) between the novel isolate and Pseudoalteromonas elyakovii KMM 162T and some other species of the genus Pseudoalteromonas. The DNA G+C content of the novel strain was 39 mol%. DNA-DNA hybridization showed only 47.6% DNA-DNA relatedness with P. elyakovii KMM 162T, 44.2% with Pseudoalteromonas distincta KMM 638T and 22.6% with Pseudoalteromonas nigrifaciens NCIMB 8614T. Based on phylogenetic and phenotypic characteristics, this isolate represents a novel species of the genus Pseudoalteromonas for which the name Pseudoalteromonas arctica is proposed; the type strain is A 37-1-2T (=LMG 23753T=DSM 18437T).