We cloned lipG, which encoded a lipolytic enzyme, from a Korean tidal flat metagenomic library. LipG was related to six putative lipases previously identified only in bacterial genome sequences. These enzymes comprise a new family. We partially characterized LipG, providing the first experimental data for a member of this family.

A Gram-negative, red-pigment-producing marine bacterial strain, designated S1-1, was isolated from the tidal flat sediment of the Yellow Sea, Korea. On the basis of phenotypic, phylogenetic, and genetic data, strain S1-1 (KCTC 11448BP) represented a new species of the genus Zooshikella. Thus, we propose the name Zooshikella rubidus sp. nov. Liquid chromatography and mass spectrometry of the red pigments produced by strain S1-1 revealed that the major metabolic compounds were prodigiosin and cycloprodigiosin. In addition, this organism produced six minor prodigiosin analogues, including two new structures that were previously unknown. To our knowledge, this is the first description of a microorganism that simultaneously produces prodigiosin and cycloprodigiosin as two major metabolites. Both prodigiosin and cycloprodigiosin showed antimicrobial activity against several microbial species. These bacteria were approximately 1.5-fold more sensitive to cycloprodigiosin than to prodigiosin. The metabolites also showed anticancer activity against human melanoma cells, which showed significantly more sensitivity to prodigiosin than to cycloprodigiosin. The secondary metabolite profiles of strain S1-1 and two reference bacterial strains were compared by liquid chromatography-mass spectrometry. Multivariate statistical analyses based on secondary metabolite profiles by liquid chromatography-mass spectrometry indicated that the metabolite profile of strain S1-1 could clearly be distinguished from those of two phylogenetically related, prodigiosin-producing bacterial strains.

Three Gram-negative, yellow-pigmented, rod-shaped bacterial strains, SMK-12T, SMK-36 and SMK-45, were isolated from a tidal flat sediment of the Yellow Sea in Korea, and their taxonomic positions were investigated by a polyphasic approach. The three strains grew optimally at 25–30 °C and in the presence of 2–3 % (w/v) NaCl. They contained MK-6 as the predominant menaquinone. The major cellular fatty acids were iso-C15 : 0, iso-C17 : 0 3-OH, iso-C15 : 1, anteiso-C15 : 0, iso-C15 : 0 3-OH and C16 : 1 ω7c and/or iso-C15 : 0 2-OH. The DNA G+C contents of the three strains were 34·7–34·9 mol%. The phylogenetic tree based on 16S rRNA gene sequences revealed that the three strains form one distinct evolutionary lineage supported by a bootstrap value of 100 % within the family Flavobacteriaceae. The three strains exhibited 16S rRNA gene sequence similarity levels of 93·8–94·9 % to the nearest phylogenetic neighbours, the genera Algibacter, Bizionia and Formosa. On the basis of differences in phenotypic characteristics and phylogenetic distinctiveness, strains SMK-12T, SMK-36 and SMK-45 were classified in a novel genus and species, for which the name Gaetbulibacter saemankumensis gen. nov., sp. nov. is proposed. The type strain for the novel species is SMK-12T (=KCTC 12379T=DSM 17032T).

BackgroundAmong the vast microbial genomic resources now available, most microbes are unculturable in the laboratory. A culture-independent metagenomic approach is a novel technique that circumvents this culture limitation. For the screening of novel lipolytic enzymes, a metagenomic library was constructed from compost, and the clone of estCS2 was selected for lipolytic properties on a tributyrin-containing medium.ResultsThe estCS2 sequence encodes a protein of 570 amino acid residues, with a predicted molecular mass of 63 kDa, and based on amino acid identity it most closely matches (45%) the carboxylesterase from Haliangium ochraceum DSM 14365. EstCS2 belong to family VII, according to the lipolytic enzyme classification proposed by Arpigny and Jaeger, and it retains the catalytic triad Ser245-Glu363-His466 that is typical of an α/β hydrolase. The Ser245 residue in the catalytic triad of EstCS2 is located in the consensus active site motif GXSXG. The EstCS2 exhibits strong activity toward p-nitrophenyl caproate (C6), and it is stable up to 60°C with an optimal enzymatic activity at 55°C. The maximal activity is observed at pH 9, and it remains active between pH 6-10. EstCS2 shows remarkable stability in up to 50% (v/v) dimethyl sulfoxide (DMSO) or dimethylformamide (DMF). The enzyme has the ability to cleave sterically hindered esters of tertiary alcohol, as well as to degrade polyurethanes, which are widely used in various industries.ConclusionsThe high stability of EstCS2 in organic solvents and its activity towards esters of ketoprofen and tertiary alcohols, and in polyurethane suggests that it has potential uses for many applications in biotransformation and bioremediation.