• KSBB Journal
• /
• 제14권3호
• /
• pp.322-327
• /
• 1999

토양에서 분리된 Streptomyces sp으로부터 cholesterol oxidase의 정제 및 효소학적 특성을 조사하였다. 본 효소의 정제는 50~80% 포화의 황산암모늄 침전 및 cholesterol affinity column chromatography를 통하여 28.3%의 수율로 정제 되었다. 정제된 효소는 SDS-PAGE에서 단일한 밴드를 보였으며 분저량은 약 60,000 dalton으로 추정되었으며, HPLC분석 결과 단일의 peak로 검출되었다. 본 효소의 특성을 검토한 결과, 금속 이온으로 Hg와 Cu의 존재시 크게 저해를 받았고, dithiothreitol 과 mercaptoethanol과 같은 저해제에 의해서 상당히 실활되었다. 본 cholesterol oxidase의 Michaelis 상수는 cholesterol을 기질로하여 Lineweaver-Burk plot분석에서 1.38mM로 추산되었다. 정제효소의 아미노산 조정은 약 416개의 잔기로 추정되며 glycine, alanine, threonine의 함량이 높은 반면, methionine, isoleucine의 함량은 극히 낮았다.

• 한국축산식품학회지
• /
• 제27권1호
• /
• pp.110-116
• /
• 2007

Lactobacillus salivarius subsp. salivarius Nam27 with a high ${\beta}$-galactosidase activity was selected for enzymatic characterization. For purification, cell pellet was disrupted by Bead Beater, by DEAE-Sepharose and Mono-Q chromatography. The specific activity of the purified enzyme was 5,312 units/mg. The molecular weight of native monomeric ${\beta}$-galactosidase was estimated to be 30,000 dalton (monomer) by the SDS-PAGE. The optimum temperature and optimum pH were $50^{\circ}C$ and 5.0, respectively. This enzyme was stable between 35 and $55^{\circ}C$. ${\beta}$-galactosidase activity was lost rapidly above pH 7.0. But ${\beta}$-galactosidase was more stable at pH 4.0 (acidic conditions). And ${\beta}$-galactosidase activity was lost rapidly above $65^{\circ}C$ after 10 min incubation. $Ca^{2+}$ and $Zn^{2+}$ metal ions enhanced ${\beta}$-galactosidase activity by 164.09% and 127.37% while $Cu^{2+}$, $Fe^{3+}$ and $Mn^{2+}$ lowered ${\beta}$-galactosidase activity by 58.29%,85.10% and 77.66% respectively. Other metal ions didn't affect ${\beta}$-galactosidase activity significantly.

• Applied Biological Chemistry
• /
• 제34권3호
• /
• pp.249-257
• /
• 1991

대두 발아 과정 중의 ${\alpha}-galactosidase$를 추출하여 염석, 이온교환 크로마토그래피 및 겔 여과 등의 방법으로 정제한 후 정제효소의 효소학적 성질을 검토하였다. 대두 ${\alpha}-galactosidase$의 활성은 $25^{\circ}C$에서 120시간 발아시켰을 때 가장 높았으며, 대두 중의 raffinose는 96시간, stachyose는 120시간 발아시켰을 때 완전히 분해되었다. 대두 ${\alpha}-galactosidase$는 황산암모늄염석, DEAE-Cellulose 및 DEAE-Sephadex A-50 이온교환 크로마토그래피, Sephadex G-150 겔 여과 등에 의하여 비활성은 825U/mg protein으로써 6.6배까지 정제되었으며 수율은 2.5%이었고 HPLC와 PAGE에 의하여 순도를 확인하였다. 정제효소의 등전점은 pH 4.8이었고, 분자량은 30,000인 monomer이었으며 정제효소의 최적작용 PH는 6.0, 최적작용온도는 $40^{\circ}C$ 이었고, $60^{\circ}C$에서 10분 처리시 25%의 잔존 활성을 나타내었다. 정제효소는 stachyose보다 raffinose를 쉽게 분해하였으며 PNPG에 대한 Km값은 5.3 mM, 활성화 에너지는 13.02 cal/mole이었다.

• 한국미생물·생명공학회지
• /
• 제19권5호
• /
• pp.477-486
• /
• 1991

Aspergillus niger가 생산하는 $\alpha$-galactosidase의 효소학적 성질을 조사하기 위하여 시험균주를 밀기울 배양한 후 생성된 $\alpha$-galactosidase를 염석, 이온교환 크로마토그래피 및 겔 여과 등의 방법으로 정제한 후 정제효소의 효소학적 성질을 검토하였다. Asp. niger를 밀기울 배지에서 $30^{\circ}C$, 4일 배양했을 때 효소활성이 가장 높았으며 $\alpha$-galactosidase는 황산암모늄 염석, DEAE-cellulose 및 DEAE-Sephadex A-50 이온교환 크로마토그래피, sephadex G-150 겔 여과 등에 의하여 23.7배까지 정제되었으며 비활성이 1,229U/mg.protein, 수율 14이었고 HPLC와 PAGE에 의해 순도가 확인되었다.

• 한국수산과학회지
• /
• 제21권2호
• /
• pp.85-96
• /
• 1988

Purification and some properties of alkaline proteinases in the pyloric caeca of skipjack, Katsuwonus vagans, were investigated. Four alkaline proteinases, temporarily designated proteinases I, II, III and IV, were identified from the tissue extract of the pyloric caeca by ammonium sulfate fractionation, DEAE-Sephadex A-50 chromatography, and Sephadex G-100 and G-200 gel filtration. Result of disc-polyacrylamide gel electrophoretic analysis showed that the purified proteinases II and III were homogenous with the yields of $1.5\%\;and\;1.2\%$, and those specific activities were increased to 33 to 37 fold over that of the crude enzyme solution, respectively. Molecular weight of the proteinases II and III determined by sephadex G-100 gel filtration were 28,500 and 24,200, respectively. The optimum conditions for the caseinolytic activity of the two enzymes were pH 9.6 and $48^{\circ}C$. The reaction rates of the two alkaline proteinases were constant to the reaction time to 80 min in the reaction mixture of $3.4{\mu}g/ml$ of enzyme concentration and $2\%$ casein solution. The Km values against casein substrate determined by the method of Lineweaver-Burk were $0.56\%$ for proteinase II and $0.30\%$ for proteinase II. The proteinases II and III were inactivated under the presence of $Ag^+,\;Hg^{2+},\;Ni{2+},\;Fe^{2+},\;and\;Cu^{2+}$, and but activated by $Mn^{2+}\;and\;Ca^{2+}$ and markedly inhibited by the soybean trypsin inhibitor and N-p-toluenesulfonyl-L-lysine chloromethyl ketone. Therefore, the proteinases II and III were found to be a group of serine proteases and assured to be trypsin-like proteinases.

• Journal of Microbiology and Biotechnology
• /
• 제30권7호
• /
• pp.1082-1091
• /
• 2020

Microbial transglutaminases (MTGs) are widely used in the food industry. In this study, the MTG gene of Streptomyces sp. TYQ1024 was cloned and expressed in a food-grade bacterial strain, Bacillus subtilis SCK6. Extracellular activity of the MTG after codon and signal peptide (SP Ync M) optimization was 20 times that of the pre-optimized enzyme. After purification, the molecular weight of the MTG was 38 kDa and the specific activity was 63.75 U/mg. The optimal temperature and pH for the recombinant MTG activity were 50℃ and 8.0, respectively. MTG activity increased 1.42-fold in the presence of β-ME and 1.6-fold in the presence of DTT. Moreover, 18% sodium chloride still resulted in 83% enzyme activity, which showed good salt tolerance. Cross-linking gelatin with the MTG increased the strength of gelatin 1.67 times and increased the thermal denaturation temperature from 61.8 to 75.8℃. The MTG also significantly increased the strength and thermal stability of gelatin. These characteristics demonstrated the huge commercial potential of MTG, such as for applications in salted protein foods.

• BMB Reports
• /
• 제38권5호
• /
• pp.624-631
• /
• 2005

Tuberculosis, caused by Mycobacterium tuberculosis, continues to be one of the main diseases to mankind. It is urgent to discover novel drug targets for appropriate antimicrobial agents against this human pathogen. The shikimate pathway is onsidered as an attractive target for the discovery of novel antibiotics for its essentiality in bacteria and absence in mammalian cells. The Mycobacterium tuberculosis aroE-encoded shikimate dehydrogenase was cloned, expressed and purified. Sequence alignment analysis shows that shikimate dehydrogenase of Mycobacterium tuberculosis exhibit the pattern of G-X-(N/S)-V-(T/S)-X-PX-K, which is highly conserved within the shikimate dehydrogenase family. The recombinant shikimate dehydrogenase spectrum determined by CD spectroscopy showed that the percentages for $\alpha$-helix, $\beta$-sheet, $\beta$-turn, and random coil were 29.2%, 9.3%, 32.7%, and 28.8%, respectively. The enzymatic characterization demonstrates that it appears to be fully active at pH from 9.0 to 12, and temperature $63^{\circ}C$. The apparent Michaelis constant for shikimic acid and $NADP^+$ were calculated to be about $29.5\;{\mu}M$ and $63\;{\mu}M$. The recombinant shikimate dehydrogenase catalyzes the substrate in the presence of $NADP^+$ with an enzyme turnover number of $399\;s^{-1}$. Zymological studies suggest that the cloned shikimate dehydrogenase from M. tuberculosis has a pretty activity, and the work should help in the discovery of enzyme inhibitors and further of possible antimicrobial agents against Mycobacterium tuberculosis.

• Journal of Microbiology and Biotechnology
• /
• 제4권2호
• /
• pp.113-118
• /
• 1994

A bacterial strain NS-83 isolated from soil was able to produce an extracellular thermostable protease. The strain was identified as Pseudomonas aeruginosa based on its morphological and physiological characteristics. A thermostable protease from this strain has been purified to homogeneity as judged by SDS-PAGE and isoelectric focusing. The purification procedures included hydrophobic interaction, ion exchange, and gel filtration chromatography. The $M_r$ and the pl of the enzyme were 32,000 and 5.9, respectively. The optimal pH at 55$^{\circ}C$ and the optimal temperature at pH 7.0 were 8.0 and 60$^{\circ}C$, respectively. The D-values of the enzyme at 60, 65, and 70$^{\circ}C$ were 22, 2.1, and 0.75 hrs, respectively. The enzyme activity was significantly inhibited in the presence of 1 mM o-phenanthroline or EDTA, suggesting that the enzyme is metalloprotease. The $K_m$, and $V_{max}$ for Hammarsten casein were found to be 3.2 mg/ml and 0.918 unit/ml, respectively. These enzymatic properties were similar to those of elastase produced from P. aeruginosa IFO 3455, but the enzyme was clearly different from the reported elastase, in respect to $Ca^{++}$ effects on enzyme-thermostability. This property, together with amino acid composition analysis, confirmed that the enzyme differs from the known P. aeruginosa elastase.

• Journal of Microbiology and Biotechnology
• /
• 제19권10호
• /
• pp.1191-1196
• /
• 2009

The production of Streptomyces griseus trypsin (SGT) by S. griseus IFO13350 transformed with the expression vector pWHM3-TR1R2, containing sprT encoding SGT and the two positive regulatory genes sgtR1 and sgtR2, was investigated in various media. Cultivation in Ferm-0 gave 1.4 times more trypsin activity than in C5/L medium. In addition, replacement of 2% glucose and 1% skim milk in Ferm-0 with 2% dextrin and 1% tryptone (designated Ferm-II) enhanced trypsin activity 4.1-fold. To simplify the purification process, the supernatant from the S. griseus transformant cultured in Ferm-II medium was fractionated with ammonium sulfate (25-55%), then subjected to Hitrap Benzamidine FF affinity column chromatography. The specific activity of SGT purified by one-step chromatography was 69,550 unit/mg protein and the overall purification yield was above 8%, indicating that this method is more effective than those previously reported. Purified SGT was most active at pH 8.0 and $50^{\circ}C$, and it maintained activity between pH 7.0 and 9.0 and at temperatures up to $70^{\circ}C$. These enzymatic properties are very similar to those of authentic eukaryotic trypsin purified from bovine pancreas.

• 생명과학회지
• /
• 제8권5호
• /
• pp.497-503
• /
• 1998

GTase와 CDase를 함께 분비$\cdot$생산하는 B. stearother-mophilus KJl6 균주의 CDase를 ammonium sulfate 침전, DBAE-cellulose, Sephadex G-100 column chromatogra-phy, 및 FPLC로 수율 7%, 비활성 12.4 units/mg, 정제도 87.6배로 정제된 CDase를 얻었으며 SDS-PAGE 상 단일 band를 확인하였다. 정제된 CDase의 분자량은 약 68,000 dalton 이었고 활성 최적 pH와 온도는 6.0와 55$^{\circ}C$였다. pH 안정성은 5.5~8.5의 범위에서 비교적 안정하였으며, 온도 안정성은 5$0^{\circ}C$에서 2시간까지는 안정하였고, 7$0^{\circ}C$에서 1시간 전처리하여도 80% 이상의 잔존활성을 나타내었다. 효소 활성은 $Cu^{+2}$와 $Hg^{+2}$와 같은 금속이온과 p-chlorome-rcuribenzoate, N-bromosuccinimide, mercaptoethanol, dithiothreitol에 의해서 효소활성이 강하게 저해되었다. 기질에 대한 반응 특이성은 $\gamma$ -CD를 가장 잘 분해하였으며, 그 외에 soluble starch나 amylose, amylopectin 등의 기질도 잘 분해하나 이들의 분해속도는 $\gamma$-CD에 비해서는 늦었다. 이들 기질의 최종 분해산물은 maltose였으며, maltose는 거의 분해되지 않았다.