Search

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 Met261, produced an aggregate of high molecular weight (> 700 kDa). Compared with MPlaG, 27PEP-MPlaG showed the same temperature and pH effect for maximum activity but was stable in the presence of inhibitors such as EDTA and PMSF. The 27PEP-MPlaG exhibited lower specific activity than that of MPlaG, but when pre-incubated for 30 min at temperatures between 4 and 100 °C, its activity increased at temperatures greater than 40 °C under alkaline conditions and eventually reached the specific activity level of MPlaG at 60 °C. We experimentally determined that the aggregate caused by 27PEP was dissociated at elevated temperatures resulting in an active catalytic monomer. The 27PEP-indued aggregation may be attractive as application tool for improving or engineering of biocatalysts and biomaterials.

Additional file 6 : Figure S5. MS/MS spectra of milbemycins produced from S. avermitilis mutant strains.

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. Due to the high commercial value of the milbemycins and increasing resistance to the avermectins and their derivatives, it is imperative to develop an efficient combinatorial biosynthesis system exploiting an overproduction host strain to produce the milbemycins and novel analogs in large quantities. Results The respective replacement of AveA1 and AveA3 (or module 7 in AveA3) of the avermectin polyketide synthase (PKS) in the avermectin high-producing strain S. avermitilis SA-01 with MilA1 and MilA3 (or module 7 in MilA3) of the milbemycin PKS resulted in the production of milbemycins A3, A4, and D in small amounts and their respective C5-O-methylated congener milbemycins B2, B3, and G as major products with total titers of approximately 292 mg/l. Subsequent inactivation of the C5-O-methyltransferase AveD led to a production of milbemycins A3/A4 (the main components of the commercial product milbemectin) in approximately 225 and 377 mg/l in the flask and 5 l fermenter culture, respectively, along with trace amounts of milbemycin D. Conclusions We demonstrated that milbemycin biosynthesis can be engineered in the avermectin-producing S. avermitilis by combinatorial biosynthesis with only a slight decrease in its production level. Application of a similar strategy utilizing higher producing industrial strains will provide a more efficient combinatorial biosynthesis system based on S. avermitilis for further enhanced production of the milbemycins and their novel analogs with improved insecticidal potential. Electronic supplementary material The online version of this article (doi:10.1186/s12934-017-0626-8) contains supplementary material, which is available to authorized users.

A gene coding for an esterase (EstEH112) was isolated from metagenome originated from Korean intertidal flat sediment. The putative esterase gene encoded a 340 amino acids protein with characteristic residues of lipolytic enzymes such as a conserved pentapeptide (GXSXG), the typical catalytic S-D-H triad, and a GGG(A)X-motif in the oxyanion hole near the active site similar to the hormone sensitive lipase (HSL) family. p-Nitrophenyl butyrate was the best substrate for the enzyme among the other p-nitrophenyl esters investigated. The apparent optimal temperature and pH for EstEH112 was 35 °C and at pH 8.0, respectively. EstEH112 efficiently catalyzed the hydrolysis of various large tertiary alcohol esters. These characteristics of EstEH112 make it a potential candidate for application in biocatalysis.

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.6 lipases, including Staphylococcus hyicus lipase, a unique lipase which can hydrolyze phospholipids, and is more evolutionarily related to the bacterial phospholipase A 1 family. The specific activities of MPlaG against olive oil and phosphatidylcholine were determined to be 2,957 ± 144 and 1,735 ± 147 U mg −1 , respectively, which means that MPlaG is a lipid-preferred phospholipase. Among different synthetic esters, triglycerides, and phosphatidylcholine, purified MPlaG exhibited the highest activity toward p -nitrophenyl palmitate (C 16 ), tributyrin (C 4 ), and 1,2-dihexanoyl-phosphatidylcholine (C 8 ). Finally, MPlaG was identified as a phospholipase A 1 with lipase activity by cleavage of the sn -1 position of OPPC, interfacial activity, and triolein hydrolysis. These findings suggest that MPlaG is the first experimentally characterized phospholipase A 1 with lipase activity obtained from a metagenomic library. Our study provides an opportunity to improve our insight into the evolution of lipases and phospholipases.

A Gram-stain-negative, aerobic, non-flagellated, non-spore-forming, motile (by gliding) bacterial strain, designated M-M6T, was isolated from marine sand of Geoje island, Korea. Strain M-M6T grew optimally at 25 °C, at pH 7.0–8.0 and in the presence of 2 % (w/v) NaCl. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain M-M6T fell within the clade comprising Cellulophaga species, forming a coherent cluster with Cellulophaga lytica ATCC 23178T and Cellulophaga fucicola NN015860T, with which it shared 16S rRNA gene sequence similarities of 98.1 and 98.2 %, respectively. Sequence similarities between strain M-M6T and the type strains of other recognized Cellulophaga species were in the range 92.4–93.8 %. Strain M-M6T contained MK-6 as the predominant menaquinone and iso-C15 : 0, iso-C15 : 1 G, iso-C17 : 0 3-OH, and C16 : 1ω7c and/or iso-C15 : 0 2-OH as the major fatty acids. The major polar lipids detected in strain M-M6T and the type strains of C. lytica and C. fucicola were two unidentified lipids, one unidentified aminolipid and one unidentified aminophospholipid. The DNA G+C content of strain M-M6T was 35.4 mol%. Levels of DNA–DNA relatedness between strain M-M6T and C. lytica JCM 8516T and C. fucicola JCM 21778T were 33 and 35 %, respectively. Differential phenotypic properties and phylogenetic and genetic distinctiveness distinguished strain M-M6T from all recognized Cellulophaga species. On the basis of the data presented, strain M-M6T is considered to represent a novel species of the genus Cellulophaga , for which the name Cellulophaga geojensis sp. nov. is proposed. The type strain is M-M6T ( = KCTC 23498T = CCUG 60801T).

A Gram-stain-negative, aerobic, motile, ovoid bacterial strain, designated HDW-19T, was isolated from seawater of the west coast of Korea and subjected to a polyphasic taxonomic study. Strain HDW-19T grew optimally at pH 7.0–8.0, at 30 °C and in the presence of 2–3 % (w/v) NaCl. Bacteriochlorophyll a was not produced by strain HDW-19T. Neighbour-joining, maximum-likelihood and maximum-parsimony phylogenetic trees based on 16S rRNA gene sequences showed that strain HDW-19T clustered with Pseudorhodobacter ferrugineus IAM 12616T, with which it shared 96.4 % similarity. A neighbour-joining phylogenetic tree based on gyrB gene sequences showed that strain HDW-19T also clustered with the type strain of P. ferrugineus, sharing 83.0 % similarity. Strain HDW-19T contained Q-10 as the predominant ubiquinone and C18 : 1ω7c as the major fatty acid. The major polar lipids were phosphatidylcholine, phosphatidylglycerol, three unidentified aminophospholipids and two unidentified aminolipids. The DNA G+C content of strain HDW-19T was 60.9 mol%. Differential phenotypic properties, together with phylogenetic distinctiveness, showed that strain HDW-19T can be differentiated from P. ferrugineus. On this basis, strain HDW-19T is considered to represent a novel species of the genus Pseudorhodobacter, for which the name Pseudorhodobacter aquimaris sp. nov. is proposed. The type strain is HDW-19T ( = KCTC 23043T = CCUG 58879T).

A Gram-negative, non-spore-forming, rod-shaped bacterial strain, RL-2T, was isolated from seawater of the East Sea in Korea and was subjected to a polyphasic taxonomic study. Strain RL-2T grew optimally at pH 7.5–8.0, at 20 °C and in the absence of NaCl. Phylogenetic trees based on 16S rRNA gene sequence analysis showed that strain RL-2T forms a cluster with Perlucidibaca piscinae IMCC1704T and various uncultured and unidentified gammaproteobacteria. Strain RL-2T exhibited 16S rRNA gene sequence similarity values of 96.1 % to Perlucidibaca piscinae IMCC1704T and 93.7–99.7 % to the uncultured bacterial clones belonging to the cluster and an unidentified gammaproteobacterium. The fatty acid profile of strain RL-2T was similar to that of Perlucidibaca piscinae IMCC1704T, but the predominant ubiquinone type (Q-11) of strain RL-2T was different from that (Q-8) of Perlucidibaca piscinae IMCC1704T. The DNA G+C content of strain RL-2T was 61.3 mol%. On the basis of phylogenetic, chemotaxonomic and phenotypic data, strain RL-2T is considered to represent a novel species of a new genus in the family Moraxellaceae, for which the name Paraperlucidibaca baekdonensis gen. nov., sp. nov. is proposed. The type strain of Paraperlucidibaca baekdonensis is RL-2T ( = KCTC 23145T = CCUG 59307T).

A Gram-positive, non-motile and coccoid-, short rod- or rod-shaped bacterial strain, ISL-16T, was isolated from a marine solar saltern in Korea and its taxonomic position was investigated using a polyphasic taxonomic approach. Strain ISL-16T grew optimally at pH 7.0–8.0, at 30 °C and in the presence of 2 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain ISL-16T joined the cluster comprising species of the genus Planococcus. Its 16S rRNA gene sequence contained the same signature nucleotides as those defined for the genus Planococcus. Strain ISL-16T exhibited 16S rRNA gene sequence similarity values of 96.9–98.2 % to the type strains of species of the genus Planococcus. Strain ISL-16T contained MK-8 and MK-7 as the predominant menaquinones and anteiso-C15 : 0, C16 : 1 ω7c alcohol and anteiso-C17 : 0 as the major fatty acids. The DNA G+C content was 48.3 mol%. DNA–DNA relatedness values between strain ISL-16T and the type strains of species of the genus Planococcus were 15–28 %. Differential phenotypic properties, together with its phylogenetic and genetic distinctiveness, enabled strain ISL-16T to be differentiated from recognized species of the genus Planococcus. On the basis of the data presented, strain ISL-16T is considered to represent a novel species of the genus Planococcus, for which the name Planococcus salinarum sp. nov. is proposed. The type strain is ISL-16T (=KCTC 13584T=CCUG 57753T). An emended description of the genus Planococcus is also given.

A Gram-negative, motile and pleomorphic bacterial strain, SMK-146T, was isolated from a tidal flat sediment of the Yellow Sea, Korea, and its taxonomic position was investigated. Strain SMK-146T grew optimally at pH 7.0–8.0 and 30 °C. It contained Q-10 as the predominant ubiquinone and C18 : 1 ω7c and 11-methyl C18 : 1 ω7c as the major fatty acids. The major polar lipids were phosphatidylcholine, phosphatidylglycerol and phosphatidylethanolamine. The DNA G+C content was 68.4 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain SMK-146T belongs to the genus Jannaschia. Strain SMK-146T exhibited 16S rRNA gene sequence similarity values of 95.3–97.0 % to the type strains of the five recognized Jannaschia species. The mean DNA–DNA relatedness value between strain SMK-146T and Jannaschia seosinensis KCCM 42114T, the closest phylogenetic neighbour, was 17 %. Differential phenotypic properties also revealed that strain SMK-146T differs from the recognized Jannaschia species. On the basis of phenotypic, phylogenetic and genetic data, strain SMK-146T represents a novel species of the genus Jannaschia, for which the name Jannaschia seohaensis sp. nov. is proposed. The type strain is SMK-146T (=KCTC 22172T =CCUG 55326T).

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 7.0-8.0 and 30 degrees C in the presence of 2 % (w/v) NaCl. It contained Q-10 as the predominant ubiquinone and C(18 : 1)omega7c and 11-methyl C(18 : 1)omega7c as the major fatty acids. The major polar lipids were phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine and an unidentified lipid. The DNA G+C content was 63.4 mol%. Strain SD-15(T) exhibited the highest 16S rRNA gene sequence similarity values (95.1-96.4 %) to the type strains of species of the genus Phaeobacter, Leisingera methylohalidivorans MB2(T) and Marinovum algicola ATCC 51440(T). Strain SD-15(T) could be differentiated from members of the genera Phaeobacter, Leisingera and Marinovum by differences in the contents of some fatty acids, by the absence of aminolipid and by differences in some phenotypic properties. On the basis of phenotypic, chemotaxonomic and phylogenetic data, strain SD-15(T) represents a new genus and novel species, for which the name Seohaeicola saemankumensis gen. nov., sp. nov. is proposed. The type strain of the type species is Seohaeicola saemankumensis SD-15(T) (=KCTC 22175(T)=CCUG 55328(T)).

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. The mutant 3E4 from the third round of directed evolution possessed four substitutions; three (F64L, E111V and K166Q) were at the original GFP gene and the other (K8N) at the inserted segment. The fluorescence intensity of 3E4 was approximately 28-fold stronger than GFP-I5, and other spectral properties were retained. Biochemical and biophysical investigations suggested that the fine-tuning by directed evolution led the salvaged variant GFP-I5 to a functionally favorable structure, resulting in recovery of stability and fluorescence. Site-directed mutagenesis of the mutated amino acid residues in both GFPuv and GFP-I5 revealed that each amino acid residue has a different effect on the fluorescence intensity, which implies that 3E4 adopted a new evolutionary path with respect to fluorescence characteristics compared with the parent GFPuv. Directed evolution in conjunction with FSS is expected to be used for generating protein lineages with new fitness landscapes.

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. As a result, four representatives were selected from each library and examined for their specificity using 16 available sugars. The apparent dissociation constants of the allose, arabinose, ribose, and glucose sensors were estimated to be 0.35, 0.36, 0.17, and 0.18 μM. Finally, the sugar sensors were applied to monitor the consumption rate of individual sugars in an E. coli culture broth. The individual sugar profiles exhibited a good correlation with those obtained using an HPLC method, confirming that the biosensors offer a rapid and easy-to-use method for monitoring individual sugars in mixed compositions.

A Gram-staining-negative, aerobic, non-motile and rod-shaped bacterial strain, designated DPG-21T, was isolated from seawater from the South Sea in Korea, and investigated using a polyphasic taxonomic approach. Strain DPG-21T grew optimally at pH 7.0–8.0, 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-21T clustered with Tropicimonas isoalkanivorans B51T (with a sequence similarity of 97.1 %); the novel strain showed lower 16S rRNA gene sequence similarities (T and T. isoalkanivorans DSM 19548T was 12 %. The predominant ubiquinones of strain DPG-21T were Q-10 and Q-9 while C18 : 1ω7c was the strain’s major fatty acid. The polar lipid profile of strain DPG-21T was similar to that of T. isoalkanivorans DSM 19548T. The genomic DNA G+C content of the novel strain was 69.6 mol%. Some phenotypic properties and the phylogenetic and genetic data indicated that strain DPG-21T was distinct from T. isoalkanivorans and represents a novel species of the genus Tropicimonas, for which the name Tropicimonas aquimaris sp. nov. is proposed. The type strain is DPG-21T ( = KCTC 23424T = CCUG 60524T).

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. Results The 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. Conclusions The 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.

A Gram-staining-negative, non-motile, non-spore-forming bacterial strain, DPG-3T, was isolated from seawater from the South Sea in Korea, and its taxonomic position was investigated using a polyphasic approach. Strain DPG-3T 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-3T fell within a clade comprising Algoriphagus species and appeared most closely related to Algoriphagus halophilus JC 2051T (96.1 %16S rRNA gene sequence similarity) and Algoriphagus lutimaris S1-3T (96.4 %). The type strains of other Algoriphagus species showed 16S rRNA gene sequence similarities of 92.9–96.0 % with strain DPG-3T. The predominant menaquinone of strain DPG-3T was MK-7. The major fatty acids were iso-C15 : 0 and iso-C15 : 0 2-OH and/or C16 : 1ω7c (summed feature 3). The major polar lipids detected in strain DPG-3T were phosphatidylcholine, phosphatidylethanolamine and an unidentified lipid. The genomic DNA G+C content was 44.8 mol%. On the basis of phenotypic, chemotaxonomic and phylogenetic data, strain DPG-3T is considered to represent a novel species of the genus Algoriphagus, for which the name Algoriphagus namhaensis sp. nov. is proposed. The type strain is DPG-3T ( = KCTC 23419T = CCUG 60523T).

A Gram-stain-negative, non-motile, non-spore-forming bacterial strain, BDR-9T, 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-9T grew optimally at 25 °C, at pH 6.0–7.5 and in the absence of NaCl. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain BDR-9T fell within the clade comprising species of the genus Mucilaginibacter within the phylum Bacteroidetes. 16S rRNA gene sequence similarity values between strain BDR-9T and the type strains of species of the genus Mucilaginibacter were in the range 94.0–95.6 %. Strain BDR-9T contained MK-7 as the predominant menaquinone and iso-C15 : 0 and C16 : 1ω7c and/or iso-C15 : 0 2-OH as the major fatty acids. The DNA G+C content was 44.3 mol%. Differential phenotypic properties and phylogenetic distinctiveness of strain BDR-9T demonstrated that this strain is distinguishable from species of the genus Mucilaginibacter. On the basis of phenotypic, chemotaxonomic and phylogenetic data, strain BDR-9T is considered to represent a novel species of the genus Mucilaginibacter, for which the name Mucilaginibacter boryungensis sp. nov. is proposed. The type strain is BDR-9T ( = KCTC 23157T = CCUG 59599T).

A Gram-negative-staining, non-motile and rod-shaped bacterial strain, HD-28T, was isolated from a tidal flat of the Yellow Sea, Korea. Strain HD-28T grew optimally at pH 7.0–8.0 and 30 °C in the presence of 2–3 % (w/v) NaCl. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain HD-28T was most closely related to species of the genus Ruegeria and exhibited 95.5–96.9 % 16S rRNA gene sequence similarity to the type strains of Ruegeria species. A neighbour-joining phylogenetic tree based on gyrB gene sequences also showed that strain HD-28T fell within the cluster comprising recognized species of the genus Ruegeria, showing 77.5–83.9 % sequence similarity. Strain HD-28T contained Q-10 as the predominant ubiquinone and C18 : 1ω7c as the major fatty acid. The major polar lipids detected in strain HD-28T were phosphatidylcholine, phosphatidylglycerol, an unidentified aminolipid and two unidentified lipids. The DNA G+C content was 57.9 mol%. Differential phenotypic properties, together with phylogenetic distinctiveness, demonstrated that strain HD-28T could be distinguished from recognized species of the genus Ruegeria. On the basis of phenotypic, chemotaxonomic and phylogenetic data, strain HD-28T is considered to represent a novel species of the genus Ruegeria, for which the name Ruegeria faecimaris sp. nov. is proposed. The type strain is HD-28T ( = KCTC 23044T = CCUG 58878T).

A Gram-negative, non-motile, rod-shaped bacterial strain, DS-58T, 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-58T fell within the family Xanthomonadaceae. The isolate showed 96.9 % 16S rRNA gene sequence similarity with its closest phylogenetic neighbour, Lysobacter niastensis GH41-7T, and 93.4–95.7 % 16S rRNA gene sequence similarity with other members of the genus Lysobacter. Strain DS-58T contained Q-8 as the predominant ubiquinone and iso-C16 : 0, iso-C15 : 0 and iso-C17 : 1ω9c as the major fatty acids. The DNA G+C content was 68.1 mol%. Strain DS-58T could be distinguished phenotypically from type strains of closely related species of the genus Lysobacter and phylogenetically from all members of the genus Lysobacter. On the basis of phenotypic, chemotaxonomic and phylogenetic data, strain DS-58T is considered to represent a novel species of the genus Lysobacter, for which the name Lysobacter dokdonensis sp. nov. is proposed. The type strain is DS-58T ( = KCTC 12822T = DSM 17958T).

A Gram-stain-negative, non-motile, ovoid- to rod-shaped bacterium, designated HDW-9T, belonging to the class Alphaproteobacteria, was isolated from seawater of the Yellow Sea, Korea. Strain HDW-9T grew optimally at pH 7.0–8.0, at 30 °C and with 2–3 % (w/v) NaCl. Neighbour-joining, maximum-likelihood and maximum-parsimony phylogenetic trees based on 16S rRNA gene sequences showed that strain HDW-9T clustered with Roseovarius crassostreae CV919-312T, with which it exhibited 95.5 % 16S rRNA gene sequence similarity. Strain HDW-9T exhibited 92.5–94.7 % 16S rRNA gene sequence similarity with the other type strains of species of the genus Roseovarius. Strain HDW-9T contained Q-10 as the predominant ubiquinone and C18 : 1 ω7c as the major fatty acid. The DNA G+C content was 58.3 mol%. Differential phenotypic properties distinguished strain HDW-9T from the other members of the genus Roseovarius. Strain HDW-9T is considered to represent a novel species of the genus Roseovarius, for which the name Roseovarius marinus sp. nov. is proposed. The type strain is HDW-9T (=KCTC 22805T =CCUG 58403T).

A Gram-negative, non-motile, non-spore-forming bacterial strain, S1-3T, was isolated from a tidal flat sediment on the west coast of Korea and its taxonomic position was investigated. Strain S1-3T grew optimally at 30 °C and in the presence of 2 % (w/v) NaCl. Strain S1-3T contained MK-7 as the predominant menaquinone and C16 : 1 ω7c and/or iso-C15 : 0 2-OH and iso-C15 : 0 as the major fatty acids. The DNA G+C content was 41.4 mol%. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain S1-3T fell within the clade comprising Algoriphagus species, clustering with Algoriphagus halophilus IMSNU 14013T, with which it exhibited 99.6 % 16S rRNA gene sequence similarity. The 16S rRNA gene sequence similarity between strain S1-3T and the type strains of other Algoriphagus species was 94.0–97.1 %. Differential phenotypic properties and phylogenetic and genetic distinctiveness of strain S1-3T demonstrated that this strain is distinguishable from the other Algoriphagus species as well as A. halophilus. On the basis of phenotypic, chemotaxonomic, phylogenetic and genetic data, strain S1-3T is considered to represent a novel species of the genus Algoriphagus, for which the name Algoriphagus lutimaris sp. nov. is proposed. The type strain is S1-3T (=KCTC 22630T =CCUG 57608T).

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.

Publisher Summary This chapter analyzes the improvement of the functional expression of N-carbamoylase by directed evolution using the green fluorescent protein (GFP) fusion reporter system. A simple and efficient method used to improve the functional expression of N-carbamoylase in E. coli using directed evolution is presented. GFP was used as a reporter protein for screening of the N-carbamoylase variant, showing a higher level of functional expression or correct folding. It is found that for the construction of the fusion protein with GFP, the N-carbamoylase gene from A. tumefaciens NRRL B11291 is amplified and digested with NcoI and Eco RI, and is then ligated into pTrc99A containing the GFP gene. It is observed that to avoid steric hindrance during folding, 10 amino acids are incorporated between N-carbamoylase and GFP as a linker. It is found that to evaluate the correlation between the fluorescence intensity of GFP and the functional expression of N-carbamoylase among wild-type and mutant N-carbamoylase/GFP fusions, selected clones were cultivated and induced under identical conditions. A screening system using a fusion reporter protein such as d -hydantoinase or GFP can be used effectively for selecting proteins with higher expression or correct folding in directed molecular 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. The temperature at which 50% of the initial activity remains after incubation for 30 min was 73 degrees C for 2S3, whereas it was 61 degrees C for wild-type enzyme. Treatment of wild-type enzyme with 0.2 mM hydrogen peroxide for 30 min at 25 degrees C resulted in a complete loss of activity, but 2S3 retained about 79% of the initial activity under the same conditions. The K(m) value of 2S3 was estimated to be similar to that of wild-type enzyme; however k(cat) was decreased, leading to a slightly reduced value of k(cat)/K(m), compared with wild-type enzyme. DNA sequence analysis revealed that six amino acid residues were changed in 2S3 and substitutions included Q23L, V40A, H58Y, G75S, M184L and T262A. The stabilizing effects of each amino acid residue were investigated by incorporating mutations individually into wild-type enzyme. Q23L, H58Y, M184L and T262A were found to enhance both oxidative and thermostability of the enzyme and of them, T262A showed the most significant effect. V40A and G75S gave rise to an increase only in oxidative stability. The positions of the mutated amino acid residues were identified in the structure of N-carbamoylase from Agrobacterium sp. KNK 712 and structural analysis of the stabilizing effects of each amino acid substitution was also carried out.

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. Oxidative stability of the evolved enzyme 2S3 was about 18-fold higher than that of the wild type, and it also showed an 8-fold increased thermostability. The K(m) value of 2S3 was comparable to that of wild-type enzyme, but k(cat) was slightly decreased. DNA sequence analysis revealed that six amino acid residues (Q23L, V40A, H58Y, G75S, M184L, and T262A) were substituted in 2S3. From the mutational analysis, four mutations (Q23L, H58Y, M184L, and T262A) were found to lead to an improvement of both oxidative and thermal stability. Of them, T262A had the most significant effect, and V40A and G75S only increased the oxidative stability.

We developed a kinetic model that describes a heterogeneous reaction system for the production of D-p-hydroxyphenylglycine from D,L-p-hydroxyphenyl-hydantoin using D-hydantoinase of Bacillus stearothermophilus SD1 and N-carbamoylase of Agrobacterium tumefaciens NRRL B11291. As a biocatalyst, whole cells with separately or co-expressed enzymes were used. The reaction system involves dissolution of substrate particles, enzymatic conversion, racemization of the L-form substrate, and transfer of the dissolved substrate, intermediate, and product through the cell membrane. Because the two enzymes have different pH optimum, kinetic parameters were evaluated at different pH for the reaction systems. The model was simulated using the kinetic parameters and compared with experimental data, and it was found that the kinetic model well describes the behavior of the reaction systems using whole cells with separately and co-expressed enzymes. Factors affecting the kinetics of the reaction systems were analyzed on the basis of the kinetic model. In the reaction system with separately expressed enzymes, racemization rate and transport of the reaction intermediate (N-carbamoyl-D-p-hydroxyphenylglycine) were revealed to be the limiting factors at neutral pH, resulting in accumulation of intermediate in the reaction medium. At alkaline condition, on the other hand, inhibition of N-carbamoylase by ammonia was severe, and thereby the reaction rate significantly reduced. In the co-expressed enzyme system, accumulation of intermediate was negligible in the reaction medium, and the improved performance was observed compared to that with separately expressed enzymes. The present model might be applied for the optimization and development of the reaction system using two sequential enzymes.