• Korean Journal of Microbiology
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• 제52권2호
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• pp.192-201
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• 2016

Recently, Psychrobacter sp. ArcL13 strain showing the extracellular lipase activity was isolated from the Chuckchi Sea of the Arctic Ocean. However, due to the low expression levels of the enzyme in the natural strain, the production of recombinant lipase is crucial for various applications. Identification of the gene for the enzyme is prerequisite for the production of the recombinant protein. Therefore, in the present study, a novel lipase gene (ArcL13-Lip) was isolated from Psychrobacter sp. ArcL13 strain by gene prospecting using PCR, and its complete nucleotide sequence was determined. Sequence analysis showed that ArcL13-Lip has high amino acid sequence similarity to lipases from bacteria of some Psychrobacter genus (84-90%) despite low nucleotide sequence similarity. The lipase gene was cloned into the bacterial expression plasmid and expressed in E. coli. SDS-PAGE analysis of the cells showed that ArcL13-Lip was expressed as inclusion bodies with a molecular mass of about 35 kDa. Refolding was achieved by diluting the unfolded protein into refolding buffers containing various additives, and the highest refolding efficiency was seen in the glucose-containing buffer. Refolded ArcL13-Lip showed high hydrolytic activity toward p-nitrophenyl caprylate and p-nitrophenyl decanoate among different p-nitrophenyl esters. Recombinant ArcL13-Lip displayed maximal activity at $40^{\circ}C$ and pH 8.0 with p-nitrophenyl caprylate as a substrate. Activity assays performed at various temperatures showed that ArcL13-Lip is a cold-active lipase with about 40% and 73% of enzymatic activity at $10^{\circ}C$ and $20^{\circ}C$, respectively, compared to its maximal activity at $40^{\circ}C$.

• Microbiology and Biotechnology Letters
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• 제33권1호
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• pp.29-34
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• 2005

Lipase-producing bacteria (S3) were isolated from intertidal flat at Saemanguem. A isolated strain was identified as Psychrobacter species by physiological and fermentational characterization as well as 16S rRNA analysis. The strain was then named as Psychrobacter sp. S3. P. sp. S3 grew most rapidly at $30^{\circ}C$, but grew well even at $10^{\circ}C$ and its lipase activity was most high when cultivated at $20^{\circ}C$. Lipase S3 had optimum temperature of $30^{\circ}C$ for the hydrolysis of p-nitrophenyl caproate and had more than $80^{\circ}C$ activity even at $10^{\circ}C$. The activation energy was calculated to be 1.5 kcal/mol, which showed that it was a typical cold-adapted enzyme. It was an alkaline enzyme with optimum pH of $9.0\~9.5$. It could hydrolyze various length of triglycerides. Among them, it hydrolyzed most rapidly $C_4,\;C_{14},\; C_{16}-length$ triglycerides. When added to tributyrin-agarose gel, lipase S3 hydrolyzed tributyrin most rapidly at 30 and $40^{\circ}C$, but it could hydrolyze well even at $4^{\circ}C$.

• Journal of Microbiology and Biotechnology
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• 제17권4호
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• pp.604-610
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• 2007

A psychrotrophic strain 7195 showing extracellular lipolytic activity towards tributyrin was isolated from deep-sea sediment of Prydz Bay and identified as a Psychrobacter species. By screening a genomic DNA library of Psychrobacter sp. 7195, an open reading frame of 954 bp coding for a lipase gene, lipA1, was identified, cloned, and sequenced. The deduced LipA1 consisted of 317 amino acids with a molecular mass of 35,210 kDa. It had one consensus motif, G-N-S-M-G (GXSXG), containing the putative active-site serine, which was conserved in other cold-adapted lipolytic enzymes. The recombinant LipA1 was purified by column chromatography with DEAE Sepharose CL-4B, and Sephadex G-75, and preparative polyacrylamide gel electrophoresis, in sequence. The purified enzyme showed highest activity at $30^{\circ}C$, and was unstable at temperatures higher than $30^{\circ}C$, indicating that it was a typical cold-adapted enzyme. The optimal pH for activity was 9.0, and the enzyme was stable between pH 7.0-10.0 after 24h incubation at $4^{\circ}C$. The addition of $Ca^{2+}\;and\;Mg^{2+}$ enhanced the enzyme activity of LipA1, whereas the $Cd^{2+},\;Zn^{2+},\;CO^{2+},\;Fe^{3+},\;Hg^{2+},\;Fe^{2+},\;Rb^{2+}$, and EDTA strongly inhibited the activity. The LipA1 was activated by various detergents, such as Triton X-100, Tween 80, Tween 40, Span 60, Span 40, CHAPS, and SDS, and showed better resistance towards them. Substrate specificity analysis showed that there was a preference for trimyristin and p-nitrophenyl myristate $(C_{14}\;acyl\; groups)$.

• Biomolecules & Therapeutics
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• 제16권4호
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• pp.356-363
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• 2008

A bacterial strain with good antibacterial activities against Staphylococus aureus and Escherichia coli was isolated from a marine sponge Stelleta sp., and it was identified as a Psychrobacter sp. by comparative 16S rDNA sequence analysis. In our search for bioactive secondary metabolites from this psychrophillic and halotolerent bacterium, sixteen cyclic dipeptides (1-16) were isolated and their structures were identified on the basis of NMR analysis. In the test of the compounds for the protective effect against Vibrio vulnificusinduced cytotoxicity in human intestinal epithelial cells, cyclo-(L-Pro-L-Phe) (5) exhibited significant protective effect. Compounds 2, 6, and 11, which contain D-amino acid, were first isolated from bacteria.

• Journal of Life Science
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• 제20권4호
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• pp.487-495
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• 2010

A microorganism hydrolyzing carboxymethylcellulose (CMC) was isolated from seawater, identified as Psychrobacter aquimaris by analysis of 16S rDNA sequences, and named P. aquimari LBH-10. This strain produced an acidic carboxymethylcellulase (CMCase), which hydrolyzed carboxymethylcellulose (CMC), cellobiose, curdlan, filter paper, p-nitrophenyl-$\beta$-D-glucopyranoside (pNPG), pullulan, and xylan, but there was no detectable activity on avicel and cellulose. The optimal temperature for CMCase produced by P. aquimari LBH-10 was $50^{\circ}C$ and more than 90% of its original activity was maintained at broad temperatures ranging from 20 to $50^{\circ}C$ after 24 hr. The optimal pH of the CMCase was 3.5, and more than 70% of its original activity was maintained under acidic conditions between pH 2.5 and 7.0 at $50^{\circ}C$ after 24 hr. The optimal pH of CMCase produced by P. aquimaris LBH-10 seems to be lower than those produced by any other bacterial and fungal strain. $CoCl_2$, EDTA, and $PbCl_2$ at a concentration of 0.1 M enhanced CMCase-produced P. aquimaris LBH-10, whereas $HgCl_2$, KCl, $MnCl_2$, $NiCl_2$, and $SrCl_2$ inhibited it.

• Journal of Microbiology
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• 제44권6호
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• pp.694-698
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• 2006

Several marine bacterial strains were isolated from Undaria pinnatifida (Miyok in Korean). Sixty-six strains were isolated on R2A agar media at $10^{\circ}C$ and identified by a phylogenetic analysis of the 16S rRNA gene sequences. They were grouped into 10 different sequence types based on the initial sequence analysis of the 5' domain of the gene (approximately 500 bp). Full sequences of 16S rRNA gene, were obtained from one strain in each sequence type and the species-affiliation was determined using phylogenetic and sequence similarity analyses. The results of the analyses indicated that they were closely related to Psychrobacter aquimaris, P. celer, P. nivimaris, P. pulmonis, Psychromonas arctica or Bacillus psychrodurans. These bacteria are marine or psychrotrophic bacteria. Because the sporophytes of U. pinnatifida are cultured on the costal area during winter, the U. pinnatifida-associated bacteria appeared to grow at low temperatures. U. pinnatifida sporophytes can be a good source for the isolation of psychrotrophic bacteria.

• Journal of Life Science
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• 제28권12호
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• pp.1477-1488
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• 2018

This study investigated the muscles, intestines, and gonads of Sulculus diversicolor supertexta to examine the diversity of microbial communities within examples collected from the Jeju Coast. Using different media, initial pure isolation in MA, 1% BHIA, and 1% TSA indicated that the muscles, intestines, and gonads supported more communities, respectively. In analysis of relative similarity with 16s rRNA sequencing, 190 pure colonies were isolated, and further analysis with NBLAST identified 71 species, 39 genera, 25 families, and five phyla. Homogeny with the reference strain was 91-100%. Microbial communities in S. supertexta consisted of gamma and alpha Proteobacteria (48%), Actinobacteria (32.5%), Firmicutes (16.9%), Deinococcus-Thermus (1.3%), and Bacteroides (1.3%). In all tissue, Psychrobacter cibarius in Moraxellaceae was dominant. Alteromonadaceae, Enterobacteriaceae, Pasturellaceae, Moraxellaceae, Rhodobacteraceae, Geminicoccaceae, Dietziaceae, Intrasporangiaceae, Microbacteriaceae, Micrococcaceae, Micromonosporaceae, Streptomycetaceae, Aerococcaceae, Bacillaceae, Paenibacillaceae, Planococcaceae, and Staphylcoccaceae were commonly isolated across all tissues, and Flavobacteriaceae, Corynebacteriaceae, Yesiniaceae, Vibrionaceae, Hahellaceae, Pseudomonadaceae were also identified from the intestines. In microbial monitoring of four harmful bacteria, Streptomyces albus (96%) showed antibacterial activity against all four strains, and Agrococcus baldri (99%) and Psychrobacter nivimaris (99%) presented against E. Coli and E. aerogens. In addition, some strains with low homogeny were isolated and further experiments are therefore required, for example to refine the antimicrobial substances including new strain investigations. These additional experiments would aim to establish generic resources for the microbial communities in S. Supertexta and provide basic data for applied microbiological research.

• Journal of Microbiology and Biotechnology
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• 제14권5호
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• pp.952-958
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• 2004

Of 23 psychrotrophic bacteria isolated from the west Pacific deep-sea sediments, 19 were assigned to the $\gamma$-Proteobacteria, 3 to the <$\beta$-Proteobacteria, and 1 to the Gram-positive bacteria, as determined by their 16S rDNA sequences. Ten psychrotrophs, affiliated to the Psychrobacter, Pseudoalteromonas, and Pseudomonas genera in the $\gamma$-Proteobacteria group, were screened for lipolytic bacteria. The majority of the lipolytic isolates had growth temperatures between 4-$30^\circ{C}$, and all of them were neutrophilic, aerobic, or facultatively anaerobic, and some were able to produce multiple kinds of ectohydrolytic enzymes. The deep-sea strains Psychrobacter sp. wp37 and Pseudoalteromonas sp. wp27 were chosen for further lipase production analysis. Both strains had the highest lipase production when grown at 10 to $20^\circ{C}$; their highest lipase production occurred at the late-exponential growth stage; and the majority of the enzymes were excreted to the outside of the cells. Lipases from both strains had the same optimal reaction temperature and pH (20-$30^\circ{C}$, pH 7-8) and could retain about 60% of their highest activity at $4^\circ{C}$. Furthermore, SDS-PAGE and an in-gel activity test showed that they had the same high molecular mass of about 85 kDa.

• Journal of Life Science
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• 제16권1호
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• pp.120-125
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• 2006

To isolate and identify histamine degrading bacteria from Kwamegi, bacteria were screened with restriction media containing histamine. Ten strains were selected through morphological and biochemical identification procedure followed by comparison with DNA sequence of 16 rRNA gene. And also, these strains were confirmed by the histamine degrading assay such as turbidity and enzymatic assay. The results of identification are as followings : Ewingella americana B791, Arthrobacter sp. R45S, Halomonas marisflava, Psychrobacter sp. 9B-7, Bacillus sp. LMC 21002, Psychrohacter cibarius BC-220, Bacillus megaterium KL-197 were identified showing homology of $99\%,\;95\%,\;98\%,\;99\%,\;99\%,\;99\%\;and\;98\%$, respectively. Three strains remain unidentified. Arthrobacter sp. R45S, H. marisflava, Bacillus sp. LMG 21002, B. megaterium KL-197 showed histamine degrading activity, whereas, Psychrobacter sp. 9B-7 only showed weak activity. Three unidentified strains also have histamine degrading activity. In contrast, E. american B791 and p. cibarius JG-220 did not show any significant activity of histamine degradation. The strains isolated from this study showed relatively fast growth rate and histamine degrading rate as compared to those from salted mackerel.

• Proceedings of the Korean Society of Life Science Conference
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• 한국생명과학회 2008년도 제49회 학술심포지움 및 국제학술대회
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• pp.55.2-55.2
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• 2008