• Journal of Microbiology
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• 제41권1호
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• pp.58-62
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• 2003

An extracellular pretense of Bacillus amyloliquefaciens S94 was purified to apparent homogeneity. The enzyme activity was strongly inhibited by general inhibitor for serine protease, PMSF, suggesting that the enzyme is a serine pretense. The purified enzyme activity was inhibited by leucine peptidase inhibitor, bestatin, suggesting that the enzyme is a leucine endopeptidase. The maximum proteolytic activity against different protein substrates occurred at pH 10, 45$^{\circ}C$ (protein substrate) and pH 8, 45$^{\circ}C$ (synthetic substrate). The purified enzyme was specific in that it readily hydrolyBed substrates with Leu or Lys residues at P$_1$ site. The pretense had characteristics of a cold-adapted protein, which was more active for the hydrolysis of synthetic substrate in the range of 15$^{\circ}C$ to 45$^{\circ}C$, specially at low temperature.

• Journal of Applied Biological Chemistry
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• 제60권3호
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• pp.257-263
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• 2017

A cellulolytic strain Y2 was isolated from soil obtained in the Canadian Alpine region. The isolate was identified as Paenibacillus sp. Y2 by 16S rRNA sequencing. When grown in LB medium supplemented with carboxymethyl-cellulose (CMC), CMCase production increased to 122.0% of that observed in LB without CMC. Culture supernatant was concentrated by ultrafiltration and 80% ammonium sulfate precipitates were separated by Hi-Trap Q and CHT-II chromatography. The purified enzyme (EG-PY2) showed a homogeneous single band and the molecular mass was estimated to be 38 kDa by SDS-PAGE. Optimum pH and temperature of the enzyme were 4.5 and $30^{\circ}C$, respectively. The half-life of enzyme activity at 50 was 140.7 min, but the enzyme was drastically inactivated within 5 min at $55^{\circ}C$. The enzyme was highly activated to 135.7 and 126.7% by 5.0 mM of $Cu^{2+}$ or $Mg^{2+}$ ions, respectively, and moderately activated by $Ba^{2+}$ and $Ca^{2+}$ ions, whereas it was inhibited to 76.8% by $Fe^{2+}$, and to ${\leq}50%$ by $Mn^{2+}$, $Co^{2+}$, $Zn^{2+}$, and EDTA. The enzyme was activated to 211.5% in the presence of 0.5 M of NaCl and greatly tolerant to 3.15M of NaCl. The enzyme showed 2.98 times higher ${\beta}$-glucanase activity than CMCase activity. Based on these results, it can be concluded that EG-PY2 is an acidophilic, cold-active, and halotolerant endoglucanase. The authors suggest it is considered to be useful for various industrial applications, such as, fruit juice clarification, acidic deinking processes, high-salt food processing, textile and pulp industries, and for biofuel production from seaweeds.

• Journal of Microbiology and Biotechnology
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• 제24권9호
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• pp.1260-1268
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• 2014

Screening of a gene library from Paenibacillus sp. PBS-2 generated in Escherichia coli led to the identification of a clone with lipolytic activity. Sequence analysis showed an open reading frame encoding a polypeptide of 378 amino acid residues with a predicted molecular mass of 42 kDa. The esterase displayed 69% and 42% identity with the putative ${\beta}$-lactamases from Paenibacillus sp. JDR-2 and Clostridium sp. BNL1100, respectively. The esterase contained a Ser-x-x-Lys motif that is conserved among all ${\beta}$-lactamases found to date. The protein PBS-2 was produced in both soluble and insoluble forms when E. coli cells harboring the gene were cultured at $18^{\circ}C$. The enzyme is a serine protein and was active against p-nitrophenyl esters of $C_2$, $C_4$, $C_8$, and $C_{10}$. The optimum pH and temperature for enzyme activity were pH 9.0 and $30^{\circ}C$, respectively. Relative activity of 55% remained at up to $5^{\circ}C$ with an activation energy of 5.84 kcal/mol, which indicates that the enzyme is cold-adapted. Enzyme activity was inhibited by $Cd^{2+}$, $Cu^{2+}$, and $Hg^{2+}$ ions. As expected for a serine esterase, activity was inhibited by phenylmethylsulfonyl fluoride. The enzyme was remarkably active and stable in the presence of commercial detergents and organic solvents. This cold-adapted esterase has potential as a biocatalyst and detergent additive for use at low temperatures.

• Journal of Microbiology and Biotechnology
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• 제25권5호
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• pp.681-686
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• 2015

The purpose of this study was to find a cold-adapted and surfactant-stable alginate lyase as a candidate for biotechnological and industrial applications. The gene for a new alginate lyase, AlyL1, from Agarivorans sp. L11 was cloned and expressed in Escherichia coli. The recombinant AlyL1 was most active at 40℃ (1,370 U/mg). It was a cold-adapted alginate lyase, which showed 54.5% and 72.1% of maximum activity at 15℃ and 20℃, respectively. AlyL1 was an alkaliphilic enzyme and most active at pH 8.6. In addition, it showed high stability in the presence of various surfactants at a high concentration (from 0.1% to 1% (w/v)). AlyL1 was an endo-type alginate lyase that degraded both polyM and polyG blocks, yielding disaccharides and trisaccharides as the main products. This is the first report of the cloning and functional expression of a cold-adapted and surfactant-stable alginate lyase. AlyL1 might be an interesting candidate for biotechnological and industrial applications.

• 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 $(C_{14}\;acyl\; groups)$.

• 한국미생물·생명공학회지
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• 제50권1호
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• pp.71-80
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• 2022

Lipases (triacylglycerol acylhydrolases [EC 3.1.1.3]) are water-soluble enzymes. They catalyze the hydrolysis of fats and oils. A cold-active lipase from marine Bacillus cereus HSS, isolated from the Mediterranean Sea, Alexandria, Egypt, was purified and characterized. The total purification depending on lipase activity was 438.9 fold purification recording 632 U/mg protein. The molecular weight of the purified lipase was estimated to be 65 kDa using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The optimum substrate concentration, enzyme concentration, pH, and temperature were 1.5 mM, 100 µl, pH 6 and 10℃, respectively. The lipase was tolerant to NaCl concentrations ranging from 1.5 to 4.5%. The lipase was affected by the tested metal ions, and its activity was inhibited by 16% in the presence of 0.05 M SDS. The application of the cold-active lipase for the removal of an oil stain from a white cotton cloth showed that it is a promising biological agent for the treatment of oily wastes and other related applications. To the best of our knowledge, this is the first report of the purification and characterization of a lipase from marine B. cereus HSS isolated from the Mediterranean Sea.

• Fisheries and Aquatic Sciences
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• 제16권4호
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• pp.311-318
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• 2013

The gene encoding an esterase from Photobacterium sp. MA1-3 was cloned in Escherichia coli using the shotgun method. The amino acid sequence deduced from the nucleotide sequence (948 bp) corresponded to a protein of 315 amino acid residues with a molecular weight of 35 kDa and a pI of 6.06. The deduced protein showed 74% and 68% amino acid sequence identities with the putative esterases from Photobacterium profundum SS9 and Photobacterium damselae, respectively. Absence of a signal peptide indicated that it was a cell-bound protein. Sequence analysis showed that the protein contained the signature G-X-S-X-G included in most serine-esterases and lipases. The MA1-3 esterase was produced in both soluble and insoluble forms when E. coli cells harboring the gene were cultured at $18^{\circ}C$. The enzyme was a serine-esterase and was active against $C_2$, $C_4$, $C_8$ and $C_{10}$ p-nitrophenyl esters. The optimum pH and temperature for enzyme activity were pH 8.0 and $30^{\circ}C$, respectively. Relative activity remained up to 45% even at $5^{\circ}C$ with an activation energy of 7.69 kcal/mol, which indicated that it was a cold-adapted enzyme. Enzyme activity was inhibited by $Cd^{2+}$, $Cu^{2+}$, $Zn^{2+}$, and $Hg^{2+}$ ions.

• 미생물학회지
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• 제46권1호
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• pp.73-79
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• 2010

극지연구소(KOPRI)는 국내외적으로 유일하게 남북극 지역에서 분리한 저온적응성 박테리아 균주를 대상으로 culture collection(약 6,300균주)을 구축하여 운영하고 있다. 보유 중인 프로테아제(protease) 생산 균주들(총 874균주) 중에서 활성이 높은 프로테아제를 생산하는 78개의 균주들을 1차 선발한 후, 1% skim milk가 포함된 0.1${\times}$ ZoBell 고체배지에 접종하고 다양한 온도($5-30^{\circ}C$)에서 배양하면서 세포외분비성 프로테아제의 활성을 비교하였다. 위의 신속하고 직접적인 균주 스크리닝 방법을 통해서, 최종적으로 저온활성 프로테아제를 생산하는 15개의 저온적응성 균주들을 선발하였다. 최종 선발된 균주들은 16S rRNA 유전자의 분석결과 Pseudoalteromonas (13균주)와 Flavobacterium (2균주) 속(genus)으로 분류되었고, $5-15^{\circ}C$ 저온에서도 활성을 나타내는 저온성 프로테아제를 생산하였다. 15개 균주들이 생산하는 각각의 프로테아제는 특이적 화합물에 의한 효소활성 억제 정도에 따라 5개의 그룹(serine protease, aspartic protease, cysteine protease, metalloprotease, 그리고 미분류 프로테아제)으로 분류되었다. 본 실험을 통해서 선발한 남북극 유래 박테리아 균주들은 새로운 저온활성 프로테아제를 발굴하기 위한 유용한 생물자원으로서의 가치를 가지고 있다.

• Journal of Microbiology and Biotechnology
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• 제21권3호
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• pp.236-242
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• 2011

A psychrotrophic bacterium, Arthrobacter sp. ON14, isolated from Antarctica, was shown to exhibit a high ${\beta}$-galactosidase activity at a low temperature. A genomic library of ON14 was constructed and screened for ${\beta}$-galactosidase genes on functional plates containing 5-bromo-4-chloro-3-indolyl-${\beta}$-D-galactopyranoside (X-gal) as the substrate. Two different ${\beta}$-galactosidase genes, named as galA, galB, were found in ON14. Computational analyses of the genes revealed that the encoded protein GalA belongs to family 2 of glycosyl hydrolysases and is a cold-active protein, whereas GalB belongs to family 42 of glycosyl hydrolysases and is a mesophilic protein. Reverse transcription analyses revealed that the expression of galA is highly induced at a low temperature ($4^{\circ}C$ ) and repressed at a high temperature ($28^{\circ}C$ ) when lactose is used as the sole carbon source. Conversely, the expression of galB is inhibited at a low temperature and induced at a high temperature. The purified GalA showed its peak activity at $15^{\circ}C$ and pH 8. The mineral ions $Na^+$, $K^+$, $Mg^{2+}$, and $Mn^{2+}$ were identified as enzyme activators, whereas $Ca^{2+}$ had no influence on the enzyme activity. An enzyme stability assay revealed that the activity of GalA is significantly decreased when it is incubated at $45^{\circ}C$ for 2 h, and all its activity is lost when it is incubated at $50^{\circ}C$.

• Journal of Microbiology and Biotechnology
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• 제26권12호
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• pp.2087-2097
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• 2016

Most cold-adapted enzymes possess higher $K_m$ and $k_{cat}$ values than those of their mesophilic counterparts to maximize the reaction rate. This characteristic is often ascribed to a high structural flexibility and improved dynamics in the active site. However, this may be less convincing to cold-adapted metabolic enzymes, which work at substrate concentrations near $K_m$. In this respect, cold adaptation of a shikimate kinase (SK) in the shikimate pathway from psychrophilic Colwellia psychrerythraea (CpSK) was characterized by comparing it with a mesophilic Escherichia coli homolog (EcSK). The optimum temperatures for CpSK and EcSK activity were approximately $30^{\circ}C$ and $40^{\circ}C$, respectively. The melting points were $33^{\circ}C$ and $45^{\circ}C$ for CpSK and EcSK, respectively. The ${\Delta}G_{H_2O}$ (denaturation in the absence of denaturing agent) values were 3.94 and 5.74 kcal/mol for CpSK and EcSK, respectively. These results indicated that CpSK was a cold-adapted enzyme. However, contrary to typical kinetic data, CpSK had a lower $K_m$ for its substrate shikimate than most mesophilic SKs, and the $k_{cat}$ was not increased. This observation suggested that CpSK may have evolved to exhibit increased substrate affinity at low intracellular concentrations of shikimate in the cold environment. Sequence analysis and homology modeling also showed that some important salt bridges were lost in CpSK, and higher Arg residues around critical Arg 140 seemed to increase flexibility for catalysis. Taken together, these data demonstrate that CpSK exhibits characteristics of cold adaptation with unusual kinetic parameters, which may provide important insights into the cold adaptation of metabolic enzymes.