Characterization of a Novel Moderately Thermophilic Solvent-Tolerant Esterase Isolated From a Compost Metagenome Library.

A novel esterase, EstCS1, was isolated from a compost metagenomics library. The EstCS1 protein, which consists of 309 amino acid residues with an anticipated molecular mass of 34 kDa, showed high amino acid sequence identities to predicted esterases and alpha/beta hydrolases (59%) from some cultured bacteria and to predicted lipases/esterases from uncultured bacteria. The phylogenetic analysis suggested that the EstCS1 belongs to the hormone-sensitive lipase family of lipolytic enzyme classification and contains a catalytic triad including Ser155-Asp255-His285.

Enzymatic characterization of a soluble aggregate induced by N-terminal extension to a lipolytic enzyme.

A self-assembling peptide (27PEP) was isolated from an open reading frame (ORF). The ORF consisted of an unknown functional domain and a catalytic (lipolytic and phospholipolytic) domain (MPlaG) on metagenomic fosmid clone. This extension of 27 amino acids prior to the N-terminus of the catalytic domain (27PEP-MPlaG), starting at Met, produced an aggregate of high molecular weight (> 700 kDa).

Engineered biosynthesis of milbemycins in the avermectin high-producing strain Streptomyces avermitilis.

Background: Milbemycins, produced from Streptomyces hygroscopicus subsp. aureolacrimosus and Streptomyces bingchenggensis, are 16-membered macrolides that share structural similarity with avermectin produced from Streptomyces avermitilis. Milbemycins possess strong acaricidal, insecticidal, and anthelmintic activities but low toxicity.

Quantitative analyses of individual sugars in mixture using FRET-based biosensors.

Molecular biosensors were developed and applied to measure individual sugars in biological mixtures such as bacterial culture broths. As the sensing units, four sugar-binding proteins (SBPs for allose, arabinose, ribose, and glucose) were selected from the Escherichia coli genome and connected to a cyan fluorescent protein and yellow fluorescent protein via dipeptide linkers (CFP-L-SBP-YFP). The putative sensors were randomized in the linker region (L) and then investigated with regard to the intensity of fluorescence resonance energy transfer on the binding of the respective sugars.

Novel metagenome-derived, cold-adapted alkaline phospholipase with superior lipase activity as an intermediate between phospholipase and lipase.

A novel lipolytic enzyme was isolated from a metagenomic library obtained from tidal flat sediments on the Korean west coast. Its putative functional domain, designated MPlaG, showed the highest similarity to phospholipase A from Grimontia hollisae CIP 101886, though it was screened from an emulsified tricaprylin plate. Phylogenetic analysis showed that MPlaG is far from family I.

Cellulophaga geojensis sp. nov., a member of the family Flavobacteriaceae isolated from marine sand.

A Gram-stain-negative, aerobic, non-flagellated, non-spore-forming, motile (by gliding) bacterial strain, designated M-M6(T), was isolated from marine sand of Geoje island, Korea. Strain M-M6(T) grew optimally at 25 °C, at pH 7.0-8.

A novel family VII esterase with industrial potential from compost metagenomic library.

Background: Among 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.

Tropicimonas aquimaris sp. nov., isolated from seawater, and emended description of the genus Tropicimonas Harwati et al. 2009.

A Gram-staining-negative, aerobic, non-motile and rod-shaped bacterial strain, designated DPG-21(T), was isolated from seawater from the South Sea in Korea, and investigated using a polyphasic taxonomic approach. Strain DPG-21(T) grew optimally at pH 7.0-8.

Algoriphagus namhaensis sp. nov., isolated from seawater.

A Gram-staining-negative, non-motile, non-spore-forming bacterial strain, DPG-3(T), was isolated from seawater from the South Sea in Korea, and its taxonomic position was investigated using a polyphasic approach. Strain DPG-3(T) grew optimally at 30 °C and in the presence of 2% (w/v) NaCl. In a neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, strain DPG-3(T) fell within a clade comprising Algoriphagus species and appeared most closely related to Algoriphagus halophilus JC 2051(T) (96.

Pseudorhodobacter aquimaris sp. nov., isolated from seawater, and emended description of the genus Pseudorhodobacter Uchino et al. 2002.

A Gram-stain-negative, aerobic, motile, ovoid bacterial strain, designated HDW-19(T), was isolated from seawater of the west coast of Korea and subjected to a polyphasic taxonomic study. Strain HDW-19(T) grew optimally at pH 7.0-8.

Mucilaginibacter boryungensis sp. nov., isolated from soil.

A Gram-stain-negative, non-motile, non-spore-forming bacterial strain, BDR-9(T), was isolated from soil collected from Boryung on the west coast of the Korean peninsula, and its taxonomic position was investigated by using a polyphasic study. Strain BDR-9(T) grew optimally at 25 °C, at pH 6.0-7.

Paraperlucidibaca baekdonensis gen. nov., sp. nov., isolated from seawater.

A Gram-negative, non-spore-forming, rod-shaped bacterial strain, RL-2(T), was isolated from seawater of the East Sea in Korea and was subjected to a polyphasic taxonomic study. Strain RL-2(T) grew optimally at pH 7.5-8.

Ruegeria faecimaris sp. nov., isolated from a tidal flat sediment.

A Gram-negative-staining, non-motile and rod-shaped bacterial strain, HD-28(T), was isolated from a tidal flat of the Yellow Sea, Korea. Strain HD-28(T) grew optimally at pH 7.0-8.

Lysobacter dokdonensis sp. nov., isolated from soil.

A Gram-negative, non-motile, rod-shaped bacterial strain, DS-58(T), was isolated from a soil sample from Dokdo, an island of Korea, and its taxonomic position was investigated. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain DS-58(T) fell within the family Xanthomonadaceae. The isolate showed 96.

Roseovarius marinus sp. nov., isolated from seawater.

A Gram-stain-negative, non-motile, ovoid- to rod-shaped bacterium, designated HDW-9(T), belonging to the class Alphaproteobacteria, was isolated from seawater of the Yellow Sea, Korea. Strain HDW-9(T) grew optimally at pH 7.0-8.

Planococcus salinarum sp. nov., isolated from a marine solar saltern, and emended description of the genus Planococcus.

A Gram-positive, non-motile and coccoid-, short rod- or rod-shaped bacterial strain, ISL-16(T), was isolated from a marine solar saltern in Korea and its taxonomic position was investigated using a polyphasic taxonomic approach. Strain ISL-16(T) grew optimally at pH 7.0-8.

Algoriphagus lutimaris sp. nov., isolated from a tidal flat sediment.

A Gram-negative, non-motile, non-spore-forming bacterial strain, S1-3(T), was isolated from a tidal flat sediment on the west coast of Korea and its taxonomic position was investigated. Strain S1-3(T) grew optimally at 30 degrees C and in the presence of 2 % (w/v) NaCl. Strain S1-3(T) contained MK-7 as the predominant menaquinone and C(16 : 1)omega7c and/or iso-C(15 : 0) 2-OH and iso-C(15 : 0) as the major fatty acids.

Jannaschia seohaensis sp. nov., isolated from a tidal flat sediment.

A Gram-negative, motile and pleomorphic bacterial strain, SMK-146(T), was isolated from a tidal flat sediment of the Yellow Sea, Korea, and its taxonomic position was investigated. Strain SMK-146(T) grew optimally at pH 7.0-8.

Salimicrobium flavidum sp. nov., isolated from a marine solar saltern.

A Gram-variable-staining, motile and coccoid-, ovoid- or rod-shaped bacterium, strain ISL-25(T), was isolated from a marine solar saltern of the Yellow Sea, Korea, and its taxonomic position was investigated by means of a polyphasic study. Strain ISL-25(T) grew optimally at pH 7.0-8.

Seohaeicola saemankumensis gen. nov., sp. nov., isolated from a tidal flat.

A Gram-negative, non-motile and rod-, oval- or coccoid-shaped bacterial strain, SD-15(T), was isolated from a tidal flat of the Yellow Sea, Korea. The novel strain, which was phylogenetically closely related to the genera Phaeobacter, Leisingera and Marinovum, was studied using a polyphasic taxonomic approach. Strain SD-15(T) grew optimally at pH 7.

Novel cold-adapted alkaline lipase from an intertidal flat metagenome and proposal for a new family of bacterial lipases.

A new lipase, LipEH166, isolated from an intertidal flat metagenome, showed no amino acid similarity to any known lipolytic enzyme except in the consensus region. This suggested that LipEH166 and its homologues belong to a new family of lipolytic enzymes. Partial characterization indicated that LipEH166 is a novel cold-adapted alkaline lipase.

Functional tuning of a salvaged green fluorescent protein variant with a new sequence space by directed evolution.

We previously reported a method, designated functional salvage screen (FSS), to generate protein lineages with new sequence spaces through the functional or structural salvage of a defective protein by employing green fluorescent protein (GFP) as a model protein. Here, in an attempt to mimic a step in the natural evolution process of proteins, the functionally salvaged mutant GFP-I5 with new sequence space, but showing low fluorescence intensity and stability, was selected and fine-tuned by directed evolution. During a course of functional tuning, GFP-I5 was found to evolve rapidly, recovering the spectral traits to those of the parent GFPuv.

Improvement of oxidative and thermostability of N-carbamyl-d-amino Acid amidohydrolase by directed evolution.

N-Carbamyl-D-amino acid amidohydrolase (N-carbamoylase), which is currently employed in the industrial production of unnatural D-amino acid in conjunction with D-hydantoinase, has low oxidative and thermostability. We attempted the simultaneous improvement of the oxidative and thermostability of N-carbamoylase from Agrobacterium tumefaciens NRRL B11291 by directed evolution using DNA shuffling. In a second generation of evolution, the best mutant 2S3 with improved oxidative and thermostability was selected, purified and characterized.

Directed evolution of N-carbamyl-D-amino acid amidohydrolase for simultaneous improvement of oxidative and thermal stability.

Directed evolution of N-carbamyl-D-amino acid amidohydrolase from Agrobacterium tumefaciens NRRL B11291 was attempted in order to simultaneously improve oxidative and thermal stability. A mutant library was generated by DNA shuffling, and positive clones with improved oxidative and thermal stability were screened on the basis of the activity staining method on a solid agar plate containing pH indicator (phenol red) and substrate (N-carbamyl-D-p-hydroxyphenylglycine). Two rounds of directed evolution resulted in the best mutant 2S3 with a significantly improved stability.