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Yarrowia lipolytica TH65가 생산하는 Alkaline Proteinase의 정제 및 특성
유춘발,김창화,진영호,진익렬 한국미생물생명공학회 ( 구 한국산업미생물학회 ) 1996 한국미생물·생명공학회지 Vol.24 No.3
최적조건에서 생산한 효소를 40~65% ammonium sulfate 분획, DEAE-cellulose chromatography, Sephadex G-100과 Sephadex G-75 gel filtration을 이용하여 수율 14%, 15배 정제하였다. 정제효소의 분자량은 SDS-PAGE로 측정한 결과 31,500 정도였고, 최적온도와 최적 pH는 각각 40℃와 8.5~9.0이었다. 대부분의 2가 금속이온은 효소활성을 저해하였으며, 특히 Hg^2+, Zn^2+, Co^2+ 등은 강하게 저해하였다. PMSF는 효소활성을 완전히 저해시켰으며, EDTA, EGTA 및 phenanthroline은 부분적으로 저해시켰으나 Ca^2+을 첨가하면 모두 효소활성이 복원되는 것으로 나타나 metalloprotease는 아닌 것으로 생각되었다. 이상의 결과로 보아 본 효모 Yarrowia lipolytica TH65가 생산하는 효소는 alkaline serine proteinase로 생각된다. An alkaline proteinase produced by Yarrowia lipolytica TH65 was purified by 40~65% ammonium sulfate fractionation, DEAE-cellulose chromatography, and gel filtration with Sephadex G-100 and Sephadex G-75. The purified enzyme was shown as a single ban on SDS-PAGE, and its molecular weight 31,500. Optimum temperature and pH were 40℃ and 8.5~9.0, respectively, and the enzyme was stable below 40℃ and in the pH range of 6~8. The enzyme was strongly inhibited by divalent ions, completely by PMSF, and partially by EDTA, EGTA, and phenanthroline. But the inhibitory effect in the presence of EDTA, EGTA and phenanthroline could be reversed by addition of Ca^2+. Thus, these results indicated that the purified enzyme was an alkaline serine proteinase (E.C. 3.4.21.14).
유춘발 대구대학교 (한사대학) 산업기술연구소 1986 産業技術硏究 Vol.5 No.-
Thermophilic Streptomyces sp. TS 20-1 which produced an antibiotic substance against Bacillus thuringiensis was isolated from soil. Escherichia coli was less sensitive to the antibiotic than B. thuringiensis. The antibiotic was purified from culture filtrate with charcoal adsorption, and extracted with chloroform and acetone. Purified antibiotic was emitted single blue fluorescent spot under UV irradiation on paper chromatography in carbontetrachloride: chloroform (8:1) solvent system. The substance was stable in acid, but it was unstable by heat.
Extracellular Proteinase를 생산하는 효모의 분리동정과 효소의 생산
김창화,이태형,유춘발,진익렬 한국미생물생명공학회 ( 구 한국산업미생물학회 ) 1996 한국미생물·생명공학회지 Vol.24 No.4
토양 및 농업용수와 하수 등으로부터 분리한 237주의 효모를 대상으로 단백질 분해력이 있는 효모 13주를 분리한 후, 알카리 조건에서 활성이 우수한 효모 TH65의 형태학적 및 생리생화학적 특성을 조사하고, 그 결과를 Saccharomycopsis lipolytica KCCM 12495 및 S. lipolytica KCCM 35426와 비교한 바, 발효성이 없고 대부분의 당류와 nitrate를 이용하지 못하는 등 대조 균주와 거의 동일한 특성을 보임에 따라 분리균주를 Yarrowia lipolytica TH65로 동정하였다. 분리효모의 protease 생산성을 조사한 결과, 탄소원으로는 glycerol과 glucose가 좋았고, glucose 농도는 0.5%가 적당하였다. 질소원으로는 0.6% skim milk에서 가장 많은 효소가 유도되었고, 아미노산에 의한 영향을 조사한 결과는 cysteine, cystine 및 tryptophane에 의해 효소 생산성이 감소되는 것으로 나타났다. Yeast extract 농도의 영향을 조사한 결과는 0.1~0.5%에서 양호하였으나, 첨가된 대부분의 무기염과 인산염은 효소생산성을 증가시키지 못하는 것으로 나타났다. 배양초기 pH는 9~11에서 가장 좋았고, 배양온도는 18℃에서 가장 좋았으며, 배양시간에 따른 효소생산성은 36시간에서 최고치를 나타내었다. A yeast strain TH65 producing a high level of proteinase under alkaline condition was isolated, and identified as Yarrowia lipolytica by morphological, physiological, and biochemical characteristics. In proteinase productivity, glycerol and glucose among tested carbon sources were very effective, and optimum concentration of glucose was 0.5%. Skim milk was found to be most effective nitrogen source in productivity, and its optimum concentration was 0.6%. But, cysteine, cystine and tryptophane decreased the proteinase productivity. Yeast extract was relatively effective at the range of 0.1~0.5%. The yeast showed maximum production of proteinase at 18℃, pH 9~11, and cultivation time of 36 hours.
소재현,김원찬,신재호,Choon-Bal Yu,이인구 한국식품과학회 2010 Food Science and Biotechnology Vol.19 No.5
A β-glucosidase, efficiently hydrolyzing isoflavone glycoside to isoflavone aglycone, was purified from Pichia guilliermondii K123-1, isolated from Korean soybean paste by ammonium sulfate precipitation, ion exchange column chromatography, gel filtration, and fast protein liquid chromatogram (FPLC). The molecular mass of purified enzyme was estimated to be 45 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE). The optimum temperature for enzyme activity was 45℃ and it decreased dramatically above 50℃. The maximal activity was at pH 4.5 and more than 80% of the activity was retained for 24 hr in the pH range from 4.0 to 8.0 at 4℃. The N-terminal amino acid sequence of the enzyme was determined to be GLNWDYDNDK. Based on its substrate specificity and catalytic properties, the activity of the purified β-glucosidase was more effective when the sugar moiety of the glycoside was glucose and the size of the aglycone similar to that of the isoflavones. The purified β-glucosidase efficiently converts genistin and daidzin to genistein and daidzein 1.96 and 1.75 times more than almond meal β-glucosidase.
Cloning and Characterization of a Cyclohexanone Monooxygenase Gene from Arthrobacter sp. L661
김영목,정용현,Soon-Hyun Jung,Choon-Bal Yu,이인구 한국생물공학회 2008 Biotechnology and Bioprocess Engineering Vol.13 No.1
Cyclohexanone monooxygenase (CHMO), a type of Baeyer-Villiger oxidation, catalyzes the oxidation of cyclohexanone into ε-caprolactone, which has been utilized as a building block in organic synthesis. A bacterium that is capable of growth on cyclohexanone as a sole carbon source was recently isolated and was identified as Arthrobacter sp. L661. The strain is believed to harbor a CHMO gene (chnB), considering the high degradablity of cyclohexanone. In order to characterize the CHMO, a chnB gene was cloned from Arthrobacter sp. L661. The deduced amino acids of the chnB gene evidenced the highest degree of homology (90% identity) with the CHMO of Arthrobacter sp. BP2 (accession no. AY123972). The CHMO of L661 was shown to be functionally expressed in Escherichia coli cells, purified via affinity chromatography, and characterized. The specific activity of the purified enzyme was 24.75 μmol/min/mg protein. The optimum pH was 7.0 and the enzyme maintained over 70% of its activity for up to 24 h in a pH range of 6.0 to 8.0 at 4℃. The CHMO of L661 readily oxidized cyclobutanone and cyclopentanone whereas less activity was detected with those of Arthrobacter sp. BP2, Rhodococcus sp. Phi1, and Rhodococcus sp. Phi2, thereby suggesting that the CHMO of L661 evidenced the different substrate specificities compared with other CHMOs. These results can provide us with useful information for the development of biocatalysts applicable to commercial organic syntheses, especially because only a few CHMO genes have been identified thus far.