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Characterization of the Intact Form of Thermotoga maritima Pectinase TmPecN Expressed in Escherichia coli

  • Kim, Chung Ho;Cheong, Jong-Joo
    • Journal of Applied Biological Chemistry
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    • v.58 no.2
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    • pp.97-100
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    • 2015
  • The thermostable pectinase gene TmPec isolated from Thermotoga maritima was introduced into the NdeI site of pRSET-B vector and expressed in its intact form in Escherichia coli BL21. The overexpressed intact form of pectinase (TmPecN protein) was partially purified by heat-denaturation procedure. TmPecN showed the highest activity between 85 and $95^{\circ}C$, and at approximately pH 6.5. Enzyme activity was stably maintained at temperatures below $85^{\circ}C$. In the presence of $Ca^{2+}$, pectinase activity of TmPecN increased to 128.4% of normal level. In contrast, $Ba^{2+}$, $Zn^{2+}$, and $Mn^{2+}$ strongly inhibited TmPecN activity. We conclude that the biochemical properties of the intact form of TmPecN are comparable to those of the recombinant protein TmPec reported previously.

Design of a Low-Power Low-Noise Clock Synthesizer PLL (저전력 저잡음 클록 합성기 PLL 설계)

  • Park, J.K.;Shim, H.C.;Park, J.T.;Yu, C.G.
    • Proceedings of the KIEE Conference
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    • 2006.10c
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    • pp.479-481
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    • 2006
  • This paper describes a 2.5V, 320MHz low-noise and low-power Phase Locked Loop(PLL) using a noise-rejected Voltage Controlled ring Oscillator(VCO) fabricated in a TSMC 0.25um CMOS technology. In order to improve the power consumption and oscillation frequency of the PLL, The VCO consist of three-stage fully differential delay cells that can obtain the characteristic of high speed, low power and low phase noise. The VCO operates at 7MHz -670MHz. The oscillator consumes l.58mA from a 320MHz frequency and 2.5V supply. When the PLL with fully-differential ring VCO is locked 320MHz, the jitter and phase noise measured 26ps (rms), 157ps (p-p) and -97.09dB at 100kHz offset. We introduce and analysis the conditions in which ring VCO can oscillate for low-power operation.

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Effect of Caffeine, cAMP and Cattle Seminal Plasma on Freezability of Buffalo Bull Semen

  • Singh, P.;Raina, V.S.
    • Asian-Australasian Journal of Animal Sciences
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    • v.13 no.7
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    • pp.901-905
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    • 2000
  • An experiment was conducted to investigate the effect of caffeine, cAMP and cattle seminal plasma on preservation of semen at ultra low temperature ($-196{^{\circ}C}$). Each semen sample was divided into four parts equal in volume and sperm concentration; three were treated with caffeine, or cAMP, or cattle seminal plasma (CSP) and the fourth was kept as control. Sperm motility, abnormal spermatozoa, live-dead count and acrosomal damage were studied at different stages of freeze preservation viz.; just after dilution, at $5{^{\circ}C}$, at glycerolisation, before freezing, just after freezing, 24 hours of storage, and one week of storage. Sperm motility (58.39, 61.33, 52.00 and 50.39 per cent), non-eosinophilic spermatozoa (72.55, 69.98, 63.31 and 67.64 per cent), abnormal spermatozoa (5.71, 4.98, 8.04 and 5.66 per cent) and acrosomal damage (13.28, 13.33, 14.80 and 14.65 per cent) were observed in cAMP, caffeine, cattle seminal plasma and control, respectively, at every stage of freeze preservation. From this study it could be concluded that freezability of buffalo semen can be improved through the addition of caffeine followed by cAMP and cattle seminal plasma.

Community Structure and Biological indicator species of Marine Benthic Algal at Intertidal zone in the Three Areas of the East Coast of Korea (동해안 조간대 3개 지역에서 해조류의 군집과 생물지표종)

  • Jeong, Hee-Dong;Hong, Sung-Eic;Kim, Sang-Woo;Han, Myung-Seog;Jang, Seong-Ho
    • Journal of the Korean Society of Marine Environment & Safety
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    • v.20 no.6
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    • pp.609-618
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    • 2014
  • We have been studied for geographic distribution, dominance and inhabitation characteristics of marine algal species at intertidal zones of Geojin in the northern part of east coast of Korea, Jukbyeon in the middle part and Gampo in the southern part in May and August 2014. For the geographic distribution, the three areas (Geojin, Jukbyeon and Gampo) were a characteristic of the temperate region. Geojin dominated with Ulva pertusa and Chaetomorpha moniligera, Jukbyeon dominated with C. moniligera and Chondria crassicaulis, Gampo dominated with Sargassum thunbergii, U. pertusa and Ectocarpus species. C/P, R/P and (R+C)/P, which can represent the marine algal flora, were 0.85, 2.10, 2.94 in Geojin, 0.58, 3.15, 3.73 in Jukbyeon and 0.80, 3.91, 4.71 in Gampo. As a result of cluster analysis, Those regions were divided in two groups, which were a group A of Geojin and group B of Jukbyeon and Gampo. This result was well matched with Jukbyeon and Gampo shared their temperate region character in the geographic distribution study.

The Processing Conditions of Extracts from Rapid Fermented Anchovy Sauce (속성멸치간장엑기스분의 가공조건)

  • Lee, Eung-Ho;Kim, Jin-Soo;Ahn, Chang-Bum;Lee, Kang-Hee;Kim, Myung-Chan;Chung, Bu-Kil;Park, Hee-Yeol
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.18 no.2
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    • pp.167-174
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    • 1989
  • AS a part of investigation to use anchovy more effectively as food source, this work was undertaken the processing conditions of extracts from rapid fermented anchovy sauce. To prepare rapid fermented products, the chopped anchovy was mixed with 50% water (w/w), and then hydrolyzed by autolysis or addition of koji under different conditions of hydrolysis. The optimal conditions for hydrolysis of chopped anchovy were revealed $50^{\circ}C$, 6 hours, pH 8.0 by autolysis, and ,$50^{\circ}C$, 6 hours, pH 7.0 by addition of 10% koji, respectively. But, pH control was not much beneficial in increasing yield. The effect of soybean protein isolote for improvement of bitter taste was also tested. The reasonable amount of added soybean protein isolate was revealed 5% on the weight basis of the chopped anchovy. The reaction mixture hydrolyzed under optimal conditions were added with 1% onion powder (w/w), 1% garlic powder(w/w) and 1% red pepper powder(w/w) for masking fishy odor, inactivated for 20 min at $100^{\circ}C$, and then centrifuged for 20 min at 4,000 rpm. The supernatant liquor was filtrated and evaporated to 50%(v/v). finally, table salt was added for bateriostatic effect and characteristic taste of rapid fermented products. the reasonable amount of added table salt was reversed 15% on the volume basis of the evaporated liquor. The hydrolysis ratio of product made by addition of water, product made by addition of koji and water, and product made by addition of soybean protein isolate, koji and water hydrolyzed under optimal conditions were 58.4%, 82.1% and 86.2%, respectively.

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Catalases in Acinetobacter sp. Strain JC1 DSM 3803 Growing on Glucose (포도당을 이용하여 성장하는 Acinetobacter sp. Strain JC1 DSM 3809에 존재하는 Catalase)

  • Shin, Kyoung-Ju;Ro, Young-Tae;Kim, Young-Min
    • Korean Journal of Microbiology
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    • v.32 no.2
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    • pp.155-162
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    • 1994
  • Cells of Acinetobacter sp. strain JC1 DSM 3803, an aerobic monoxide-oxidizing bacterium, growing on glucose exhibited high catalase activity at the mid-exponential growth phase. The enzyme activity decreased gradually after then until the early stationary phase, increased again at the mid-stationary phase, and then decreased again thereafter. Cells growing on glucose was found to contain three kinds of catalses. Cat1, Cat2 and Cat3. The activities of Cat1 and Cat3 did change significantly during growth, but that of Cat2 exhibited significant variation. Cat3 was found to present only in cells growing on glucose, but not in cells growing on carbon monoxide of methanol. The activities of call and Cat3 in cell-free extracts were stable upon treatment with ethanol and chloroform, but decreased to some extent when the enzymewere treated with 2mM $H_2O_2$ and/or 3-amino-1,2,4-triazole (AT). Cat2 was found to be extremely sensitive to the ethanol-chloroform and $H_2O_2$ treatments, but was insensitive to the AT treatment. Cat1 exhibited enzyme activity after incubation for 1 min at 80$^{\circ}C$. Cat2 and Cat3 did not show enzyme activity after incubation for 1 min at 60$^{\circ}C$ and 70$^{\circ}C$, respectively. Cat2 was found to have peroxidase activity. Cat3 was purified to homogenity in seven steps. The molecular weight of the native enzyme was estimated to be 150,000. Sodium dodecyl sulfate-gel electrophoresis revealed two identical subunits of molecular weight 65,000. The enzyme was found to show two $K_m$ values of 39 mM and 58mM. The optimal pH for the enzyme activity was 7.0, but the activities at pH 6.0, 8.0, and 9.0, were found to be comparable to that at the optimal pH. The optimal temperature for the enzyme activity was found to be 40$^{\circ}C$. The enzyme also exhibited strong activity at 20$^{\circ}C$, 30$^{\circ}C$, and 50$^{\circ}C$. The purified enzyme was not affected by the ethanol-chloroform treatment. The enzyme, howerver, showed less than 10% of the original activity when it was treated with 12 mN AT, 0.1 mM $NaN_3$ of 1mM KCN.

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Effects of Prostacyclin [PGI2] on Myocardial Protection in the Isolating Working Heart Model (적출활동심장에서 Prostacyclin [PGI2]의 심근보호효과)

  • Lee, Gil-No;Kim, Gyu-Tae
    • Journal of Chest Surgery
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    • v.20 no.4
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    • pp.643-654
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    • 1987
  • The effect of prostacyclin[PGI, ] on myocardial preservation during global ischemia was studied in the isolating working rabbit heart model. Forty hearts underwent a 15 minute period of retrograde nonworking perfusion with Krebs-Henseleit buffer solution [37*C] and were switched over to the working mode for 15 minutes. After baseline measurement of heart rate, peak aortic pressure, aortic flow, and coronary flow, all hearts were subjected to 60 minutes of ischemic arrest at 10*C induced with St. Thomas Hospital cardioplegic solution: Group I had single dose cardioplegia, Croup II double dose, Croup III oxygenated double dose, and Group IV single dose with PCI, infusion [10ng/min./gm heart weight]. Hearts were then revived with 15 minute period of nonworking reperfusion at normothermia, followed by 30 minutes of working perfusion. Repeat measurements of cardiac function were obtained and expressed as a percent of the preischemic baseline values. Oxygen content of arterial perfusate and coronary effluent was measured by designed time interval. Leakage of creatine kinase was determined during post-ischemic reperfusion period. Finally wet hearts were weighed and placed in 120*C oven for 36 hours for measurement of dry weight. In the PGI, treated group [IV], heart rate increased consistently throughout the period of reperfusion from 100*5.0% [p<0.001] to 107*6.2% [p<0.001]. The percent recovery of aortic flow showed 95*5.7% [p<0.001] at the first 3 minute and full recovery through the subsequent time. Coronary flow was augmented significantly in the 3 minute [96*6.2%, p<0.001] and then sustained above baseline values. Among the Croup I, II, and III, all hemodynamic values were significantly below preischemic levels. PGI2 relatively increased oxygen delivery [1.22*0.19ml/min, p<0.001] and myocardial oxygen consumption [0.90*0.13ml/min, p<0.001] during reperfusion period. Leakage of creatine kinase in the PGI2 group was 9.3*1.58IU/15min [p<0.001]. This was significantly lower than Group I [33.0*2.68 IU/15min]. The water content of PCI2 treated hearts [81*0.9%, p<0.001] was also lower than the other groups.

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Properties of the Proteolytic Enzymes from Mulberry Tree Barks(Morus alba Linne) (상백피에서 추출한 단백질 분해효소의 특성)

  • 권순경;박상욱;최우영
    • The Korean Journal of Food And Nutrition
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    • v.11 no.5
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    • pp.576-579
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    • 1998
  • Water extract of mulberry tree barks(Morus alba Linne) was studied for its proteolytic activity. Protein content of the extract was 1.12mg/ml and its specific activity was 5.14U/ml. The enzyme was active on various proteins : the relative acitities were 100 for casein, 63 for albumin, 58 for collagen, 45 for hemoglobin and 36 gelatin, respectively. There suggested that the ability of the enzyme to hydrolyze meat was relatively high since those are major meat proteins. Optimum pH and temperature for proteolytic activity were : pH 6.0 and 6$0^{\circ}C$. And the enzyme was stable at the pH range of 6.0 to 7.0 and temperature between 50 and 8$0^{\circ}C$. Apparent proteolytic activities could support some scientific grounds of traditional application of mulberry tree barks to home cooking for meat tenderization.

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Walnut phenolic extracts reduce telomere length and telomerase activity in a colon cancer stem cell model

  • Shin, Phil-Kyung;Zoh, Yoonchae;Choi, Jina;Kim, Myung-Sunny;Kim, Yuri;Choi, Sang-Woon
    • Nutrition Research and Practice
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    • v.13 no.1
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    • pp.58-63
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    • 2019
  • BACKGROUND/OBJECTIVES: Telomeres are located at the chromosomal ends and progressively shortened during each cell cycle. Telomerase, which is regulated by hTERT and c-MYC, maintains telomeric DNA sequences. Especially, telomerase is active in cancer and stem cells to maintain telomere length for replicative immortality. Recently we reported that walnut phenolic extract (WPE) can reduce cell viability in a colon cancer stem cell (CSC) model. We, therefore, investigated the effect of WPE on telomere maintenance in the same model. MATERIALS AND METHODS: $CD133^+CD44^+$ cells from HCT116, a human colon cancer cell line, were sorted by Fluorescence-activated cell sorting (FACS) and treated with WPE at the concentrations of 0, 10, 20, and $40{\mu}g/mL$ for 6 days. Telomere lengths were assessed by quantitative real-time PCR (qRT-PCR) using telomere specific primers and DNA extracted from the cells, which was further adjusted with single-copy gene and reference DNA ($ddC_t$). Telomerase activity was also measured by qRT-PCR after incubating the PCR mixture with cell protein extracts, which was adjusted with reference DNA ($dC_t$). Transcriptions of hTERT and c-MYC were determined using conventional RT-PCR. RESULTS: Telomere length of WPE-treated cells was significantly decreased in a dose-dependent manner ($5.16{\pm}0.13$ at $0{\mu}g/mL$, $4.79{\pm}0.12$ at $10{\mu}g/mL$, $3.24{\pm}0.08$ at $20{\mu}g/mL$ and $3.99{\pm}0.09$ at $40{\mu}g/mL$; P = 0.0276). Telomerase activities concurrently decreased with telomere length ($1.47{\pm}0.04$, $1.09{\pm}0.01$, $0.76{\pm}0.08$, and $0.88{\pm}0.06$; P = 0.0067). There was a positive correlation between telomere length and telomerase activity (r = 0.9090; P < 0.0001). Transcriptions of both hTERT and c-MYC were also significantly decreased in the same manner. CONCLUSION: In the present cell culture model, WPE reduced telomere maintenance, which may provide a mechanistic link to the effect of walnuts on the viability of colon CSCs.

Production of Bleach-Stable and Halo-Tolerant Alkaline Protease by an Alkalophilic Bacillus pumilus JB05 Isolated from Cement Industry Effluents

  • Johnvesly, B.;Naik, Gajanan R.
    • Journal of Microbiology and Biotechnology
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    • v.11 no.4
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    • pp.558-563
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    • 2001
  • A new alkalophilic strain of Bacillus pumilus JB¬05 producing bleach-stable and halo-tolerant alkaline protease was isolated from cement industry effluents in Karnataka, India. The effects of carbon and nitrogen sources on protease production by this alkalophilic strain were observed after a 30-h incubation. A high level of alkaline protease activity was obtained in the presence of starch as the carbon and peptone as the nitrogen sources. The partially purified enzyme showed an optimum temperature and pH activity at $58^{\circ}C$ and 10.5, respectively. The enzyme was completely inhibited by PMSF (95.0%) indicating it as a serine protease. It is bleach-stable as it retained 35% original activity in the presence of 10% (v/v) hydrogen peroxide at $30^{\circ}$C after 2 h and is halo-tolerant as it retained 70% original activity in the presence of 2.5 M sodium chloride at $30^{\circ}C$ after 2 h incubation.

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