A novel esterase gene, e69, was cloned from Erythrobacter seohaensis SW-135, which was isolated from a tidal flat sediment of the Yellow Sea in Korea. This gene is 825 bp in length and codes for a 29.54 kDa protein containing 274 amino acids. Phylogenetic analysis showed that E69 is a new member of the bacterial lipolytic enzyme family IV. This enzyme exhibited the highest level of activity toward p-nitrophenyl (NP) butyrate but little or no activity toward the other p-NP esters tested. The optimum temperature and pH of the catalytic activity of E69 were 60°C and pH 10.5, respectively. The enzyme exhibited stable activity over a wide range of alkaline pH values (7.5–9.5). In addition, E69 was found to be a halotolerant esterase as it exhibited the highest hydrolytic activity in the presence of 0.5 M NaCl and was still active in the presence of 3 M NaCl. Moreover, it possessed some degree of tolerance to Triton X-100 and several organic solvents. Through homology modeling and comparison with other esterases, it was suggested that the absence of the cap domain and its narrow substrate-binding pocket might be responsible for its narrow substrate specificity. Sequence and structural analysis results suggested that its high ratio of negatively to positively charged residues, large hydrophobic surface area, and negative electrostatic potential on the surface may be responsible for its alkaline adaptation. The results of this study provide insight into marine alkaliphilic esterases, and the unique properties of E69 make it a promising candidate as a biocatalyst for industrial applications.

BackgroundThe deep-sea environment harbors a vast pool of novel enzymes. Owing to the limitations of cultivation, cultivation-independent has become an effective method for mining novel enzymes from the environment. Based on a deep-sea sediment metagenomics library, lipolytic-positive clones were obtained by activity-based screening methods.ResultsTwo novel esterases, DMWf18-543 and DMWf18-558, were obtained from a deep-sea metagenomic library through activity-based screening and high-throughput sequencing methods. These esterases shared 80.7% amino acid identity with each other and were determined to be new members of bacterial lipolytic enzyme family IV. The two enzymes showed the highest activities toward p-nitrophenyl (p-NP) butyrate at pH 7.0 and 35–40 °C and were found to be resistant to some metal ions (Ba2+, Mg2+, and Sr2+) and detergents (Triton X-100, Tween 20, and Tween 80). DMWf18-543 and DMWf18-558 exhibited distinct substrate specificities and preferences. DMWf18-543 showed a catalytic range for substrates of C2–C8, whereas DMWf18-558 presented a wider range of C2–C14. Additionally, DMWf18-543 preferred p-NP butyrate, whereas DMWf18-558 preferred both p-NP butyrate and p-NP hexanoate. To investigate the mechanism underlying the phenotypic differences between the esterases, their three-dimensional structures were compared by using homology modeling. The results suggested that residue Leu199 of DMWf18-543 shortens and blocks the substrate-binding pocket. This hypothesis was confirmed by the finding that the DMWf18-558-A199L mutant showed a similar substrate specificity profile to that of DMWf18-543.ConclusionsThis study characterized two novel homologous esterases obtained from a deep-sea sediment metagenomic library. The structural modeling and mutagenesis analysis provided insight into the determinants of their substrate specificity and preference. The characterization and mechanistic analyses of these two novel enzymes should provide a basis for further exploration of their potential biotechnological applications.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0864-4) contains supplementary material, which is available to authorized users.

Two Gram-stain-negative, aerobic, motile by a single polar flagellum and rod-shaped strains, designated SCS-49T and SCS-111, were isolated from seawater of the South China Sea. The two strains grew at 4-35 °C, with 0.5-7.5 % (w/v) NaCl and at pH 6.5-9.0 and were able to reduce nitrate. Q-8 was the sole ubiquinone. The major fatty acids of the two strains were C16 : 0, C18 : 1ω7c and summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c). The polar lipids included diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phospoglycolipid, three unidentified glycolipids, five unidentified phospholipids and two to three unidentified lipids. The isolates formed a stable clade with Pseudohongiella acticola and Pseudohongiella spirulinae based on phylogenetic analysis of 16S rRNA gene sequences. Strains SCS-49T and SCS-111 exhibited 16S rRNA gene sequence similarity values of 97.2 and 96.0 % with respect to the type strains of P. acticola and P. spirulinae, respectively. The average nucleotide diversity and in silico DNA-DNA hybridization values between strain SCS-49T and P. acticola KCTC 42131T were 71.4 and 25.1 %, respectively and the values between strain SCS-49T and SCS-111 were 99.9 and 99.2 %, respectively. Based upon the phenotypic, chemotaxonomic and genetic data, strains SCS-49T and SCS-111 represent a novel species in the genus Pseudohongiella, for which the name Pseudohongiella nitratireducens sp. nov. is proposed. The type strain is SCS-49T (=CGMCC 1.15425T=KCTC 52155T=MCCC 1K03186T).

Strain Ery9T, isolated from surface seawater of the Atlantic Ocean, and strain Ery22T, isolated from deep-sea sediment of the Indian Ocean, were subjected to a taxonomic study using a polyphasic approach. Cells of the two strains were Gram-stain-negative, aerobic and rod-shaped. They produced yellow pigments and lacked bacteriochlorophyll a. On the basis of 16S rRNA gene sequence analysis, strain Ery9T was closely related to Croceicoccus naphthovorans PQ-2T (with 16S rRNA gene sequence similarity of 97.7 %), and strain Ery22T was closely related to Croceicoccusmarinus E4A9T (98.3 %). The 16S rRNA gene sequence similarity between strain Ery9T and strain Ery22T was 96.6 %. Phylogenetic analyses revealed that strains Ery9T and Ery22T fell within the cluster of the genus Croceicoccus and represented two independent lineages. The average nucleotide identity (ANI) values and the genome-to-genome distances between strains Ery9T and Ery22T and the type strains of species of the genus Croceicoccus with validly published names were 73.7-78.4 % and 20.1-22.3 %, respectively. The major respiratory quinone of the two isolates was ubiquinone-10 (Q-10). The DNA G+C contents of strains Ery9T and Ery22T were 62.8 and 62.5 mol%, respectively. Differential phylogenetic distinctiveness and chemotaxonomic differences, together with phenotypic properties, revealed that strains Ery9T and Ery22T could be differentiated from their closely related species. Therefore, it is concluded that strains Ery9T and Ery22T represent two novel species of the genus Croceicoccus, for which the names Croceicoccus pelagius sp. nov. (type strain Ery9T=CGMCC 1.15358T=DSM 101479T) and Croceicoccus mobilis sp. nov. (type strain Ery22T=CGMCC 1.15360T=DSM 101481T), are proposed.

A Gram-stain-positive, aerobic, chemo-organotrophic, rod-shaped, non-spore-forming strain, which produced convex, circular, pink-pigmented colonies, designated as DY32-46T, was isolated from seawater collected from the Pacific Ocean. DY32-46T was found to grow at 20–40 °C (optimum, 30–35 °C), pH 6.0–8.0 (optimum, pH 6.5) and with 0–5 % (w/v) NaCl (optimum, 1–2 %). The results of chemotaxonomic analysis indicated that the respiratory quinone of DY32-46T was MK-9(H4), and major fatty acids (>10 %) were C17 : 1 ω8c, summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), C16 : 0 and C15 : 1 ω6c. The polar lipids included diphosphatidylglycerol, phosphatidylglycerol, one unidentified aminophospholipid, three unidentified glycolipids, three unidentified phospholipids, one unidentified phosphoglycolipid and five unidentified lipids. The DNA G+C content of DY32-46T was 70.6 mol%. The results of phylogenetic analysis based on 16S rRNA gene sequences and genomic data indicated that DY32-46T should be assigned to the genus Euzebya . ANI and in silico DNA–DNA hybridization values between strain DY32-46T and type strains of Euzebya species were 73.1–87.2 % and 20.2–32.4 %, respectively. Different phenotypic properties, together with genetic distinctiveness, demonstrated that strain DY32-46T was clearly distinct from recognized species of the genus Euzebya . Therefore, DY32-46T represents a novel species within the genus Euzebya , for which the name Euzebya pacifica sp. nov is proposed. The type strain is DY32-46T (=MCCC 1K03476T=KCTC 49091T).