• Journal of Microbiology and Biotechnology
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• v.31 no.6
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• pp.882-889
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• 2021

In order to use an enzyme industrially, it is necessary to increase the activity of the enzyme and optimize the reaction characteristics through molecular evolution techniques. We used the error-prone PCR method to improve the reaction characteristics of LipCA lipase discovered in Antarctic Croceibacter atlanticus. Recombinant Escherichia coli colonies showing large halo zones were selected in tributyrin-containing medium. The lipase activity of one mutant strain (M3-1) was significantly increased, compared to the wild-type (WT) strain. M3-1 strain produced about three times more lipase enzyme than did WT strain. After confirming the nucleotide sequence of the M3-1 gene to be different from that of the WT gene by four bases (73, 381, 756, and 822), the secondary structures of WT and M3-1 mRNA were predicted and compared by RNAfold web program. Compared to the mean free energy (MFE) of WT mRNA, that of M3-1 mRNA was lowered by 4.4 kcal/mol, and the MFE value was significantly lowered by mutations of bases 73 and 756. Site-directed mutagenesis was performed to find out which of the four base mutations actually affected the enzyme expression level. Among them, one mutant enzyme production decreased as WT enzyme production when the base 73 was changed (T→ C). These results show that one base change at position 73 can significantly affect protein expression level, and demonstrate that changing the mRNA sequence can increase the stability of mRNA, and can increase the production of foreign protein in E. coli.

• Journal of Microbiology and Biotechnology
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• v.26 no.6
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• pp.1087-1097
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• 2016

An intracellular lipase from Anoxybacillus flavithermus HBB 134 was purified to 7.4-fold. The molecular mass of the enzyme was found to be about 64 kDa. The maximum activity of the enzyme was at pH 9.0 and 50℃. The enzyme was stable between pH 6.0 and 11.0 at 25℃, 40℃, and 50℃ for 24 h. The K_m and V_max of the enzyme for pNPL substrate were determined as 0.084 mM and 500 U/mg, respectively. Glycerol, sorbitol, and mannitol enhanced the enzyme thermostability. The enzyme was found to be highly stable against acetone, ethyl acetate, and diethyl ether. The presence of PMSF, NBS, DTT and β-mercaptoethanol inhibited the enzyme activity. Hg²⁺, Fe³⁺, Pb²⁺, Al³⁺, and Zn²⁺ strongly inhibited the enzyme whereas Li⁺, Na⁺, K⁺, and NH₄⁺ slightly activated it. At least 60% of the enzyme activity and stability were retained against sodium deoxycholate, sodium taurocholate, n-octyl-β-D-glucopyranoside, and CHAPS. The presence of 1% Triton X-100 caused about 34% increase in the enzyme activity. The enzyme is thought to be a true lipase since it has preferred the long-chain triacylglycerols. The lipase of HBB 134 cleaved triolein at the 1- or 3-position.

• Journal of Microbiology and Biotechnology
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• v.22 no.11
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• pp.1501-1509
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• 2012

A novel bacterial strain, MG7, with high cellulase activity was isolated and identified by morphological characteristics and molecular phylogeny analysis as Paenibacillus barcinonensis. Maximum production of cellulase by MG7 was observed at pH 7.0 and $35^{\circ}C$. The enzyme was purified with a specific activity of 16.88 U/mg, the cellulase activity was observed in a zymogram, and its molecular mass (58.6 kDa) was confirmed by SDS-PAGE. The purified enzyme showed maximum activity at pH 6.0 and $65^{\circ}C$ and degraded cellulosic substrates such as carboxy methyl cellulose (CMC), Avicel, filter paper, and ${\beta}$-glucan. The enzyme showed stability with 0.5% concentration of various surfactants. The $K_m$ and $V_{max}$ of cellulase for CMC and Avicel were found to be 0.459mg/ml and 10.46mg/ml/h, and 1.01 mg/ml and 10.0 mg/ml/h, respectively. The high catalytic activity and its stability to temperature, pH, surfactants, and metal ions indicated that the cellulase enzyme by MG7 is a good candidate for biotechnological applications.

• KSBB Journal
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• v.27 no.1
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• pp.61-66
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• 2012

In the screening process of organic solvent tolerant bacteria showing good growth in media containing several kinds of organic solvents, one strain was isolated and identified as Bacillus sp. BCNU 5006. The strain was able to tolerate many organic solvents including benzene, toluene, xylene, octane, dodecane, butanol and ethylbenzene. Likewise, it could also utilize these solvents as the sole source of carbon with significant enzyme production. The lipolytic enzyme stability of Bacillus sp. BCNU 5006 was studied in the presence of several kinds of solvents at a 25% (v/v) concentration. The highest enzyme stability was observed in the presence of octane (107%), followed by ethylbenzene (88%), decane (86%), and chloroform (85%). Especially, BCNU 5006 lipase was determined to be more stable than immobilized enzyme (Novozyme 435) in the presence of octane, chloroform and xylene. This organic solvent tolerant Bacillus sp. BCNU 5006 could be expected as a potential bioremediation agent and biocatalyst for biodegradation and provide on organic-solvent-based enzymatic synthetic method in industrial chemical processes.

• Journal of Ginseng Research
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• v.7 no.1
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• pp.88-93
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• 1983

Studies were carried out to elucidate the protein stabilizing effect of ginseng. Malate dehydrogenase (EC 1.1.1.37) was used as a protein and the rate constant of the enzyme inactivation was determined under the heat denaturation condition. There was an optimum pH for the enzyme stability, the rate constant of the enzyme inactivation was minimum at BH 8.8. The rate constant was increased at lower and higher pH regions than the optimum pH. The inactivation reaction followed the Arrehnius law and the activation energy was measured as 36.8kcal/mole. The reaction rate was not affected by the enzyme concentration and thus it was assumed to be unimolecular first order reaction. The water extract of red ginseng decreased the rate constant of Malate dehydrogenate under heat inactivation condition to stabilize the enzyme activity. Purified ginseng saponin also stabilized the enzyme against heat inactivation.

• Analytical Science and Technology
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• v.29 no.3
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• pp.105-113
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• 2016

The present study investigated the immobilization of lipases on silica nanoparticles and silica-coated magnetite nanoparticles as supports with a functional group to enhance the stability of lipase. The influence of functional groups, such as the epoxy group and the amine group, on the activity and stability of immobilized lipase was also studied. The epoxy group and the amino group were introduced onto the surface of nanoparticles by glycidyl methacrylate and aminopropyl triethoxysilane, respectively. Immobilized Candida rugosa lipase on silica nanoparticles and silica-coated magnetite nanoparticles with a functional group showed slightly lower initial enzyme activities than free enzyme; however, the immobilized Candida rugosa lipase retained over 92 % of the initial activity, even after 3 times reuse. Lipase was also immobilized on the silica-coated magnetite nanoparticles by cross-linked enzyme aggregate (CLEA) using glutaraldehyde and covalent binding, respectively, were also studied. Immobilized Candida rugosa lipase on silica nanoparticles and silica-coated magnetite nanoparticles by CLEA and covalent binding showed higher enzyme activities than free enzyme, while immobilized Candida rugosa lipase retained over 73 % of the initial activity after 5 times reuse.

• Korean Journal of Microbiology
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• v.52 no.2
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• pp.230-235
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• 2016

This study was carried out to analyze the differences in enzyme activity and stability between the native Fe superoxide dismutase (FeSOD) and the 6xHis-tagged superoxide dismutase (6xHis-FeSOD) of Streptomyces subrutilus P5. The optimum pHs for both native FeSOD and 6xHis-FeSOD were 7, while the pH range of the activity was narrower for the 6xHis-FeSOD. The native FeSOD was stable at pH 4-9, but the 6xHis-FeSOD lost its stability at pH > 9. The temperatures of the optimum activities were same for both types of enzymes. However, the heat stability of the 6xHis-FeSOD was clearly decreased; even at $20^{\circ}C$ the enzyme lost the activity after 360 min. In contrast, the native FeSOD was stable after 720 min at below $40^{\circ}C$. $H_2O_2$ inhibition was occurred already at 0.5 mM for the 6xHis-tagged enzyme. Therefore, from the results that the 6xHis-FeSOD retained the enzyme activity at pH 6-7 and $20-40^{\circ}C$, it can be assumed that the protein structure became destabilized under different storage conditions and sensitive to the enzyme inhibitor.

• Journal of the Korean Society of Food Science and Nutrition
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• v.23 no.5
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• pp.856-862
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• 1994

In salt-preserved foods of every kinds, it was examined the growth condition of halophilic bacteria that induced a change of colour, taste, nutritive substance, a production condition of enzyme and a character of crude enzyme. Used bacteria is H. halobium a kind of extremely halophilic bacteria, and the required of optimum culture needed a quite long time of crude enzyme production is 168 hours. Optimum pH is about 7-7.5, so the traditional food of such neutrality pH as soybean paste and soy sauce particularly come into trouble because the growth can flourish in neutrality or alkaliescence, and the crude enzyme also appeared that best activation between pH 6 and pH 8. The optimum temperature is about 37$^{\circ}C$, the optimum temperature of enzyme is about 40 $^{\circ}C$ and the temperature stability is settled for 15 minutes and it is completely inactivated at 10 minutes. In the influence of each metal ion, Fe++ and Mn++ a stimulated the growth of H.halobium and the activation of enzyme, Cu++ and Zn++ were identified that made the growth and the activation of enzyme inhibit.

• Journal of the Korean Society of Food Science and Nutrition
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• v.23 no.6
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• pp.964-972
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• 1994

The 4.3-kb gene coding for L-lactate dehydrogenase of Bacillus stearothermophilus has been subcloned and expressed in E. coli cells. The enzyme was purified 200-fold with 25% yield by heat treatment , DEAE-Sephadex, and NAD++ -Sepharose CL-4B affinity chromatography followed by gel filtration through Sephadex G-200 . The molecular weight of the purfied enzyme was estimated to be about 35, 000 and 140, 000 on SDS-polyacrylamide gel electrophoresis and gel filtration, respectively. indicating that the enzyme is composed of four identical subunits. THe enzyme for pyruvate reduction and lactate oxdiation was stable at 60 and 75$^{\circ}C$ for 30 min, and the optimal temperatures for both reactions were 60 and 7$0^{\circ}C$, respectively. The enzyme had an optimal pH at 5.5 and 8.5 in pyruvate reduction and lactate oxidation, respectively. The pH stability of enzyme of pyruvate reduction was table between pH 5 and 7. more than 90% of enzyme activity was lost at 1mM FeSO4 and p-chloromercuribonzoate. The maximal activation of the enzyme was obtained with 0.8mM fructose 1, 6-bisphosphate.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.32 no.1 s.55
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• pp.29-33
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• 2006

This article describes an enzyme stabilization method that allows the use of enzymes irrespective of environmental factors, especially heat, while maintaining their activity for a long time. We have designed enzyme microcapsules that consist of papain enzyme cores, poly(propylene glycol) interlayers, and poly(${\epsilon}-caprolactone$) walls. By confocal laser scanning microscopy measurements and the thermal stability of papain-loaded microcapsules, it is demonstrated that the papain is surrounded by a hydrophobic polyol layer and stabilized by the exclusive volume effect. In our study, improved thermal stability can be obtained by using more hydrophobic long-chained polyols, which is understood to be attributed to the effective formation of a hydrophobic polyol layer between the papain and the polymer wall by means of conformational anchoring in the interface.