• 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
• /
• 제11권2호
• /
• pp.317-325
• /
• 2001

For heuristic purposes, the relative ratio of urease contents inside and outside cells was surveyed using nine ureB+ strains of Helicobacter pylori. the ratio of the enzyme specific activity appeared to vary greatly between the various H. pylori strains, ranging from 0.5 to 2.5. Besides the above compartment, urease was also richly found in the membrane fraction, especially in either peripheral or integral form. The urease distribution across the H. pylori membrane was significantly influenced by the ambient pH; the specific activity of external urease was highest at pH 5.5 with a narrow plateau, whereas the internal specific activity was highest within a pH range of 4.5 to 6.5 with a broad plateau. These finding strongly suggest that H. pylori urease is secretory and responded to the external pH. However, at pH 4.0 or below, no urease activity was detected in either the internal or external compartment, although an increase in the color development with 2,4,6-trinitrobenzene sulfonate (TNBS) was observed. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that these phenomena may be related to a specific proteolysis in certain proteins, including urease or ${\gamma}$-glutamyl transpeptidase. Interestingly, the effect of ammonium ions n alleviating the enzyme inactivation inside the H. pylori cells was remarkably similar to that of D-glucose. In addition, it would appear that the cation acted as a surrogate of not only $Na^+$ but also $K^+$ thereby increasing the H. pylori P-type ATPase activity. This is of great interest, as it implies that the urease action in H. pylori is indispensible at any locus.

• BMB Reports
• /
• 제40권3호
• /
• pp.412-418
• /
• 2007

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is an unique antioxidant enzyme that directly reduces lipid hydroperoxides in biomembranes. In the present work, the entire encoding region for Oryza sativa PHGPx was expressed in Escherichia coli M15, and the purified fusion protein showed a single band with 21.0 kD and pI = 8.5 on SDS- and IFE-PAGE, respectively. Judging from CD and fluorescence spectroscopy, this protein is considered to have a well-ordered structure with 12.2% $\alpha$-helix, 30.7%$\beta$-sheet, 18.5% $\delta$-turn, and 38.5% random coil. The optimum pH and temperature of the enzyme activity were pH 9.3 and 27$^{\circ}C$. The enzyme exhibited the highest affinity and catalytical efficiency to phospholipid hydroperoxide employing GSH or Trx as electron donor. Moreover, the protein displayed higher GSH-dependent activity towards t-Butyl-OOH and $H_2O_2$. These results show that OsPHGPx is an enzyme with broad specificity for hydroperoxide substrates and yielded significant insight into the physicochemical properties and the dynamics of OsPHGPx.

• 한국응용곤충학회지
• /
• 제32권4호
• /
• pp.426-432
• /
• 1993

경기도 일원의 양잠 농가의 먼지에서 채취한 45개의 샘플중에서 내독소 단백질 결정체를 생산하는 13개의 Bacillus thuringiensis를 분리하였다. 이 중 2개 균주는 파리목에 독성을 나타냈으며, 특히 독성검정에서 NT0423균주의 $LC_{50}$수치는 나비목의 배추좀나방이 최소 1.30$\times$$10^{6}$ CFU/ml이며, 파리목인 빨간 집모기에는 2.88$\times$$10^{5}$ CFU/ml로 나타났다. 새로 분리된 NT0423균주가 생산하는 내독소 단백질 결정체는 주사 전자현미경사진에서 전형적인 이중 피라미드모양을 보였다. 그리고 이 내독소 단백질 결정체의 SDS-PAGE 분석에서는 주요한 130kDa의 polypeptide을 나타내었다. 또한 NT0423균주의 총 플라스미드 DNA분석에서는 9개의 플라스미드를 갖고 있어 기존의 유사한 독성을 나타내는 균주들과 다른 패턴을 보였다.

• 보존과학연구
• /
• 통권19호
• /
• pp.3-22
• /
• 1998

The effect of pH on degradation of paper by some fungi, which able to degrade cellulose, was investigated. Trichoderma koningii, Aspergillus nigerand Penicillium nigulosum were cultured at $28^{\circ}C$ for 16 days in the selective medium (PH3, PH4, PH5, PH6, PH7, PH8, PH9, PH10, PHC) containing paper as substrate. Each paper was pretreated with each pH buffer (pH 3∼pH 10, D.W.)prior to addition to the selective medium. Enzyme activities in the each culture medium were measured spectroph to metrically using C.M.C., Avicel, PNPG as the substrates for endoglucanase, exoglucanase and $\beta$-glucosidase, respectively. In all experimental fungi, the enzyme activities of PH3 and PH9 medium were usually much higher than those of other experimental groups. However in the PH6medium, enzyme activity was lower than other groups. To analyze the concentration and pattern of protein in the each culture medium, the medium was concentrated by lyophilization. The protein concentration of PH3 and PH9 medium were relatively high (T.koningii; 6.31mg, 6,19mg, A.niger; 1.62mg, 1.96mg, P.nigulosum;2.50mg, 2.73mg, respectively), but that of PH6 was relatively low. The protein pattern of each medium was analyzed by using SDS-PAGE and VDS Image Master Analysis Program. The concentrations of bands in the each lane were usually high at lane2 (PH3) and lane8 (PH9) and low at lane5 (PH6). Therefore, the incresed cellulolytic activity of fungus against acidified paper could be result of structural change and deterioration of paper caused by being acidified.

• 대한미생물학회지
• /
• 제35권2호
• /
• pp.97-108
• /
• 2000

Two-dimensional gel electrophoresis (2-DE) is an essential tool of proteomics to analyse the entire set of proteins of an organism and its variation between organisms. Helicobacter pylori was tried to identify differences between strains. As the first step, whole H. pylori was lysed using high concentration urea contained lysis buffer [9.5 M Urea, 4% CHAPS, 35 mM Tris, 65 mM DTT, 0.01% SDS and 0.5% Ampholite (Bio-Rad, pH 3-10)]. The extract ($10\;{\mu}g$) was rehydrated to commercially available immobilised pH gradient (IPG) strips, then the proteins were separated according to their charges as the first dimensional separation. The IPG strips were placed on Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) to separate according to molecular mass of the proteins as the second dimension. The separated protein spots were visualised by silver staining in order to compare different expression of proteins between strains. Approximately 120 spots were identified in each mini-protein electrophoresised gel, furthermore about 65 to 75 spots were regarded as identical proteins in terms of pI value and molecular weight between strains used. In addition, distinct differences were found between strains, such as 219-1, Y7 and Y14, CH150. Two representative strains were examined using strips which had pH range from 4 to 7. This strips showed a number of isoforms which were considered large spots on pH range 3-10. Furthermore, the rest of spots on pH 4-7 IPG strips appeared very distinctive compared to broad range IPG strips. 2-DE seems to be an excellent tool for analysing and identifying variations between H. pylori strains.

• 한국잡초학회지
• /
• 제14권1호
• /
• pp.56-61
• /
• 1994

귀리의 근단(根端) 조직(組織)에 alachlor $1{\times}10^{-4}$ M 처리후(處理後) 음(陽)이온의 과산화(過酸化) 효소(酵素)를 유출(抽出)하여 에탄올과 이온 교환 수지인 DEAE-celluose 및 CM-sephadex를 이용하여 정제(精製)한 결과(結果), 약 30배의 정제율(精製率)을 얻었으며, 3개의 음(陽)이온을 띤 동위(同位) 효소(酵素)가 존재(存在)하였다. 귀리 근단(根端)에 존재(存在)하는 주(主)된 과산화(過酸化) 효소(酵素)(B fraction)는 최종(最終) 정제후(精製後) 전체(全體) 동위과산화(同位過酸化) 효소중(酵素中) 65%를 차지하였으며, 37배로 정제(精製)되었다. SDS-PAGE를 이용하여 B부분을 전개(展開)한 결과(結果), 단일(單一) band를 얻었으며, 이 때 분자량(分子量)은 약 42,500으로 추산(推算)되었다. 기내에서 동위(同位) 과산화(過酸化) 효소(酵素)의 활성(活性)은 IAA에 의하여 감소(減少)되었다. 50unit 동위(同位) 과산화(過酸化) 효소(酵素)를 귀리에 처리시(처리處理時) 무처리구(無處理區)에 비하여 근단(根端)의 세포분제(細胞分製)은 70.2%, 초엽의 세포신장(細胞伸長)은 54.2% 억제(抑制)되는 현상을 보였다.

• 한국미생물·생명공학회지
• /
• 제20권2호
• /
• pp.156-163
• /
• 1992

Cyclodextrin glucanotransferase 생산균주 선발배지를 이용하여 국내 토양으로부터 CGTase 활성이 우수한 Bacillus sp. E1균주를 분리하였다. FPLC를 이용하여 gel filtration과 anion exchange column chromatography를 한 결과 순수 정제된 단일 단백질을 얻을 수 있었으며, 정제된 효소의 최적 작용 pH 범위는 6에서 8까지 였고, 온도는 $60^{\circ}C$에서 최적을 나타냈다. 정제된 단백질의 분자량은 114,000, 등전점은 4.3이었다. 생성된 cyclodextrin은 $\beta$와 $\gamma$-cyclodextrin이 주였으며, 특이하게도 $\alpha$-cyclodextrin은 거의 생성되지 않았다. 작용 후 25시간 후 최대의 $\beta$-cyclodextrin이 생성되었으며, 이때 $\beta$-cyclodextrin과 $\gamma$-cyclodextrin의 생성비율은 7:1이었다.

• 한국미생물·생명공학회지
• /
• 제47권3호
• /
• pp.412-422
• /
• 2019

The present study reports the morphological and molecular characterization of the fungal strain, CMSS06 and evaluates its raw starch hydrolyzing ability in four different agricultural substrates (rice bran, banana peel, cassava tubers, and coconut water). The potential use of each agricultural substrate to replace the expensive fermentation media was evaluated with six different fermentation media: rice bran (RB), banana peel (BP), cassava starch (CS), cassava in coconut water (CSCW), cassava in modified coconut water (CMCW), and pure Coconut water (CW). The fungal strain CMSS06 was identified as Thielaviopsis ethacetica by the analysis of the ITS sequences. The T. ethacetica alpha amylase enzyme exhibited maximum alpha amylase activity at 72 h, pH 7.0, and $40^{\circ}C$ on soluble starch. This species resulted in the highest enzyme activity (mU/ml) of 26.06, 10.89, 58.82, 14.2, and 54.67 with the RB, BP, CS, CSCW, and CMCW fermentation media, respectively. The results indicate that CS can be used as a carbon substrate and CMCW can be used to accelerate the fermentation by T. ethacetica. The enzyme was partially purified by 40-60% ammonium sulphate fraction, and it showed total enzyme activity, total protein content, specific activity, purification fold, and a recovery of 2400 mU, 30 mg, 80 mU/mg, 2.7, and 71.1%, respectively. The molecular mass of the T. ethacetica alpha amylase was estimated on SDS-PAGE, and two bands around 50 kDa and 70 kDa were identified. The present study implies that T. ethacetica can produce alpha amylase, and it can be used to hydrolyze raw starch during the fermentation processes.

• 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.