• Korean Journal of Microbiology
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• v.28 no.1
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• pp.65-70
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• 1990

The objective of the current study is to elucidate the biological roles of proteinase inhibitor in microorganisms. As the first step, a strain of Streptomyces fradiae was selected as a producer of extracellular proteinase inhibitor. The proteinase inhibitor was purified from culture broth through ultrafiltration, gel-filtration and ion-exchange chromatography. Molecular weight of the proteinase inhibitor was estimated to be 16, 800 by SDS polyacrylamide gel electrophoresis. It was found that the proteinase inhibitor inhibited only alkaline serine proteinases such as subtilisin, $\alpha$-chymotrypsin and Promase E but not trypsin and other proteinases. The mode of inhibition against Pronase E with succinyl-phenylalanine-p-nitroanilide as a substrate was competitive.

• The Microorganisms and Industry
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• v.14 no.1
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• pp.22-28
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• 1988

DNA 클로닝과 조작기술의 발전은 어떤 유전자의 특정한 위치에 선택적으로 돌연변이를 도입할 수 있는 site-specific mutagenesis 기술을 창출해 내었다. 이 기술로 DAN 염기의 치환, 결실, 삽입등을 클론된 유전자에 직접 도입할 수가 있게 되어 생체의 유전자 조작이나 유전자의 산물인 단백질의 구조와 기능을 의도적으로 변화시키는 protein engineering에 광범위하게 이용되고 있다. Protein engineering은 주로 단백질의 촉매 및 생리활성의 증가, 효소의 특성및 기질 특이성의 변화, 단백질 구조의 안정화 및 내염성 증가, 분자량의 감소, 효소및 생리활성 단백질의 구조의 안정화및 내열성 증가 등에 활용되고 있으며 산업적 유용성이 큰새로운 단백질의 창조에도 기여할 것으로 기대를 모으고 있다. Site-specific mutagenesis 기술로 현재 가장 널리 이용되는 것이 in vitro상에서 수행하는 oligonucleotide-directed site specific mutagenesis이다. 이 방법은 생화학적으로 합성한 특정한 염기서열을 가진 oligonucleotide들을 일종의 mutagen으로 사용하거나 효소적 DNA 합성을 위한 primer로 사용하여 클론된 DNA의 염기서열을 선택적으로 개조하거나 혹은 다른 조작을 하는 것이다. 여기서는 돌연변이율을 높이는 여러가지 개량된 방법들이 나왔으며 그중의 몇가지를 소개하였다.

• Korean Journal of Microbiology
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• v.34 no.3
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• pp.82-86
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• 1998

An alkaline proteinase secreted from Yarrowia lipolytica 504D was purified by salting-out and column chromatography. The molecular weight of the purified enzyme was about 32,000 Da estimated by SDS-PAGE. The optimal condition for the activity of the enzyme was at pH 9.5 and $42^{\circ}C$ The enzyme was stable up to $45^{\circ}C$ and at the range of pH 4-10. Because the enzyme was inhibited by PMSF as well as EDTA, EGTA, and phenan-throlin, it is uncertain whether the enzyme is serine proteinase or metalloproteinase. However, almost all metal salts tested did not increase the enzyme activity, and Ca salt restored the activity of the enzyme inactivated by EDTA. Therefore, the purified enzyme seems to be an serine proteinase (E.C. 3.4.21.14).

• Microbiology and Biotechnology Letters
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• v.31 no.1
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• pp.57-62
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• 2003

A bacteria strain producing extracellular $\beta$-mannanase was isolated from soil and was identified as Bacillus subtilis by 16S rRNA sequence comparison and biochemical determinations. The optimum pH and temperature for the $\beta$-mannanase activity were 5.0 and 5.5$^{\circ}C$, respectively. The zymogram technique revealed a single protein band exhibiting $\beta$-mannanase activity from the culture supernatant. The molecular mass of the enzyme was estimated at approximately 130 kDa. The addition of 0.5% lactose or 0.5% locust bean gum to the LB medium caused to Increase significantly the $\beta$-mannanase productivity from Bacillus subtilis JS-1. The cells grown on LB medium supplemented with lactose produced maximal enzyme activity at the stationary phase. In contrast to this, the $\beta$-mannanase was induced at the logarithmic phase from the cells grown on LB medium supplemented with locust bean gum. The discrepancy in induction times suggests that $\beta$-mannanase was induced by different induction mechanisms depending on the carbon sources in Bacillus subtilis JS-1 .

• Korean Journal of Ecology and Environment
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• v.35 no.4 s.100
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• pp.326-330
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• 2002

A wetland soil was sterilised by two methods and changes in microbial enzyme activities were assessed. The short-term effects were determined by toluene addition, while the longer-term effects of elimination was monitored by ${\gamma}$-radiation. The changes in ${\beta}$- glucosidase, ${\beta}$-xylosidase, cellobiohydrolase, phosphatase, arylsulphatase, and N-acetylglucosaminidase activities were determined by using methylumbelliferyl model substrates and comparing with the activities of control samples. Toluene addition induced different responses of enzymes. For example, phosphatase activity increased by the treatment while ${\beta}$-glucosidase and arylsulphatase activities decreased. In contrast, ${\gamma}$-radiation decreased all enzyme activities compared to control by 40-80%. The overall results of the toluene and ${\gamma}$-radiation experiments indicate that the large amounts of enzymes are stabilised outside of living cells, at least in the short term, but that the persistence of enzymes is maintained by de-novo synthesis of microbes.

• Microbiology and Biotechnology Letters
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• v.16 no.2
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• pp.79-84
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• 1988

Dye materials and cross linking agents were used for the determination of $\beta$-glucanase activities. The objective of this study was to prepare the blue coloured substrates which are sensitive, specific and simple for the determination of $\beta$-glucanase in malt and Bacillus subtilis K-4-3 enzymes. This method is based on the principle of measuring colorimetrically the split product of coloured and cross linked substrate. The best coupling of dye stuff of $\beta$-glucan was cibacron blue 3G-A and the colour released can suitably be measured at 623nm. Optimal concentration of dye and cross linking agents was 1.5g and 1.25$m\ell$ under 0.1N NaOH. The sensitivity comparison proved that the stained $\beta$-glucan method is much more sensitive than the DNS method to determine reducing sugar released by the enzyme.

• Korean Journal of Food Science and Technology
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• v.54 no.3
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• pp.351-361
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• 2022

This study aimed to investigate the differences in the isomaltooligosaccharides present during Makgeolli production according to the type of Nuruk used, fermentation period, and presence of microorganisms. Makgeolli was fermented for 15 days using three kinds of Nuruk (Soyul, Sansung, and Jinju) with and without microbial growth inhibitors. Isomaltooligosaccharide contents were analyzed using high performance anion exchange chromatography. The most abundant isomaltooligosaccharide was panose, which was highest in Makgeolli produced using Soyul Nuruk (SH) on day 6 (24.7 mM), followed by Makgeolli prepared using Sansung Nuruk (SS) on day 2 (18.2 mM) and Makgeolli prepared using Jinju Nuruk (JJ) on day 3 (10.8 mM). Isomaltotriose and isomaltotetraose, which were generated in the control, were not detected when microbial growth was suppressed. Based on these results, isomaltooligosaccharide production is affected more by the enzymes produced by microorganisms during Makgeolli fermentation than by Nuruk itself.

• Korean Journal of Microbiology
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• v.42 no.1
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• pp.34-39
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• 2006

Wood-rot fungi produce extracellular lignin-degrading enzymes, the best known of which are lignin peroxidase, Mn-peroxidase and laccase. In this experiment, some of them produced all of three enzymes. Many other wood-rot fungi produced one or two of those enzymes with various combinations. In this experiment, we tried to clarify the relationship between the pattern of enzyme production and degradative activity of several dye compounds. From the 36 strains of 23 species of wood-rot fungi, Mn-peroxidase activity was found in 30 strains of the fungi tested, whereas the activity of lignin peroxidase and laccase was detected in 11 strains and 12 strains of species, repectively, in Kirks low nitrogen media. In relation to the activity of lignin degrading enzymes and degradation of dye compounds, the white-rot fungi with three kinds of enzymes tested showed the best dye decolorizers. The fungi with Mn-peroxidase activity only decolorized poly R-478 and remazol brilliant blue R dye in proportion to the enzyme activity, while methylene blue, bromophenol blue and congo red dye were degraded in regardless of enzyme activity. Those dyes were degraded in relation to the growth rate of mycelium. Brown-rot fungi did not degrade all the dye compounds except bromophenol blue, in spite of moderate growth rate.

• Microbiology and Biotechnology Letters
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• v.26 no.5
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• pp.427-434
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• 1998

An alkaline protease was 4-fold purified, yielding 2.3% of recovery by ammonium sulfate precipitation, CM-cellulose column chromatography and Sephadex G-100 column chromatography. The purified enzyme was estimated to be monomeric with molecular weight of about 62,000 from polyacrylamide gel eletrophoresis (PAGE) and sodiumdodecylsulfate polyacrylamide gel electrophoresis (SDS-FAGE). The optimal pH and temperature of the alkaline pretense activity were 11.0 and 50$^{\circ}C$, respectively, exhibiting high stability at pH value from 6.0 to 11.0 at 50$^{\circ}C$ for 30 minute. The alkaline pretense was activated by MnSO$_4$, CaCl$_2$, and was inhibited by CuSO$_4$, ZnSO$_4$, HgCl$_2$, EDTA and EGTA. Also, the enzyme was found to be a metaloenzyme requiring Mn$\^$2+/ as cofactor. The NH$_2$-terminal amino acid of alkaline protease was alanine. The Km and Vmax values of this enzyme for casein was 4.0 mg/$m\ell$ and 5,500 unit/$m\ell$, respectively.

• Microbiology and Biotechnology Letters
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• v.31 no.4
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• pp.311-321
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• 2003

Bacteria produce lipases, which can catalyze both the hydrolysis and the synthesis of long chain triglycerides. These reactions usually proceed with high regioselectivity and enantioselectivity, and, therefore, lipases have become very important biocatalysts used in organic chemistry. 3D lipase structures were solved from several bacterial lipases. They have an $\alpha/\beta$ hydrolase fold and a catalytic triad consisting of a nucleophilic serine, and an aspartate or glutamate residue that is hydrogen bonded to a histindine. Active sites are covered with $\alpha$-helical lid structure, of which movement is involved in the enzyme's activation at oil/water interface. Four substrate binding pockets were identified for triglycerides: an oxyanion hole and three pockets accommodating the fatty acids bound at positions sn-1, sn-2, and sn-3. These pockets determine the enantiopreference of a lipase. The understanding of structure-function relationships as well as the development of molecular evolution techniques will enable researchers to tailor new lipases for biotechnological applications.