• BMB Reports
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• 제29권6호
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• pp.500-506
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• 1996

We have isolated a water-extracted novel regulator for blood coagulation from an earthworm, Lumbricus rubellus. As a folk remedy, the earthworm has been known to facilitate blood circulation. After complete heat inactivation of endogenous proteases in the earthworm, an anticoagulant(s) was purified through ammonium sulfate fractionation and three consecutive gel permeation chromatography of Sephacryl S-300, Sephadex G-75, and G-150 by measuring activated partial thromboplastin time (APTT) The anticoagulant was further purified to 2,800 fold with a C4 reversed-phase HPLC This activity was stable under heat ($100^{\circ}C$ for 30 min) and acidic conditions (0.4 N HCl). The effects of this partially purified anticoagulant on thrombin were observed with various substrates such as N${\alpha}$-benzoyl-DL-arginine-p-nitroanilide (BApNA), H-D-phenylalanyl-L-pipecoyl-L-arginine-p-nitroanilide (S-2238), N${\alpha}$-p-tosyl-L-arginine methyl ester (TAME), and fibrinogen as a natural substrate. Only TAME hydrolysis, due to an esterase activity of the enzyme, was inhibited among the chromogenic substrates. In addition, the anticoagulant not only inhibited the conversion of fibrinogen to fibrin but also prolonged the fibrin clot formation monitored with the in vitro coagulation test. Based on these observations, we suggest the significance of measuring the ability of antithrombotic drugs to inhibit the esterase activity of thrombin. In this report, it was also shown that the earthworm indeed contained a water-extractable, heat- and acid-stable anticoagulant which could be used as a novel antithrombotic agent.

• 한국응용곤충학회지
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• 제20권1호
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• pp.6-14
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• 1981

모자익 Virus병에 감염된 마늘인편에 열처리, 화학치료제의 효과를 본 시험결과는 다음과 같다. 수중 또는 기중 열처리에 있어서 공히 $37^{\circ}\~57^{\circ}C$에서 35일간-1시간의 처리로서는 마늘 Virus의 불활성 화효과가 없었고, $62^{\circ}\~72^{\circ}C$에 $90\~5$분간의 처리로서 마늘엽에 나타난 병징이 감소되기는 하였으나 식물체의 생장력이 매우 약화되었으므로 열처리로서는 마늘 Virus를 완전히 불활성화 시킬 수 없었다. 마늘 인편을 $10\~50ppm$의 Malachite Green, 동농도의 2,4-Dichlorophenoxy Acetic Acid 또는 $20\~100ppm$의 Quinhydrone 수용액에 24시간 침윤처리한 후에 식재생장한 마늘엽에 모자익 병징은 감소되었으나 고농도에서 마늘의 생육이 억제되었다. $10\~50ppm$의 Naphthyl Activ A챵에 침윤한 마늘 인편을 식재 하였을 때에도 생장한 마늘엽에 모자익 병징이 감소되기는 하였으나 완전히 없어지지는 않았다. 수정한 Murashigh-Skoog 배지에 $0.5\~1.5ppm$의 Naphthyl Acetic Acid를 가용하여 마늘의 조직을 배양하였을 때에는 신생한 마늘식생체내에 Virus가 불활성화되어 엽에는 모자익 병징이 나타나지 않았으며 조직세포내에는 봉입체가 형성되지 않고, 정상적인 핵을 가진 건전한 식물체를 얻을 수 있었다.

• 한국식품과학회지
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• 제12권3호
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• pp.209-215
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• 1980

파파이야 라텍스를 $(NH_4)_2SO_4$,분획 및 CM-cellulose column을 통과시켜 두개의 획분으로 분리하고 각 획분 별로 pH 7.0, $60{\sim}90^{\circ}C$에서 열 불활성 실험을 행하고 다음과 같은 열역학적인 자료를 얻었다. 1. fraction I 의 z-value는 $25^{\circ}C$이었고 $70^{\circ}C$에서 활성화 엔탈피 (enthalpy of activation) 89.5 kJ/mol, 활성화엔트로피 (entropy of activation) -44.0 kJ/mol K, 활성화 자유에너지(free energy of activation) 104.6 kJ/mol이었다. 2. fraction II 의 z-value는 $23^{\circ}C$이고 $70^{\circ}C$에서 활성화 엔트로피 -22.0 kJ/mol K, 활성화 자유에너지 104.2 kJ/mol이었다. 3. fraction I과 fraction II를 혼합한 경우의 z-value는 $24.6^{\circ}C$, $70^{\circ}C$에서 활성화 엔탈피 90.9 kJ/mol, 활성화 앤트로피 -38.8 J/mol K, 활성화 자유에너지 104.2 kJ/mol이었다. 4. 조 추출액의 z-value는 $23.2^{\circ}C$이고 $70^{\circ}C$에서 활성화 엔트로피 113.8 kJ/mol, 활성화 엔트로피 22.0 kJ/mol K, 활성화자유에너지 106.2 kJ/mol이었다. 이상의 결과에서 fraction I 이 fraction II 보다 열에 안정하다고 볼 수 있었고 파파이야 라텍스에 있는 단백질 분해 효소의 열 안정성은 주로 fraction I 에 기인하는 것 을 알 수 있었다.

• 한국식품영양과학회지
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• 제45권8호
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• pp.1233-1238
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• 2016

감귤의 저장 중 미생물학적 안전성 확보 및 품질 향상을 위해 0.5% 푸마르산과 $50^{\circ}C$ 열수 세척처리 후 15, 30 ppmv 이산화염소 가스 훈증 처리하여 $4{\pm}1^{\circ}C$에서 30일간 저장하였다. 푸마르산, 열수 세척처리, 15와 30 ppmv 병합처리 시 접종된 Listeria monocytogenes를 3.5~3.7 log CFU/g 감소시켰다. 또한, 감귤의 병합처리 후 30 ppmv 병합처리구에서 저장 30일 후에 효모 및 곰팡이 수에 있어서 2.54 log CFU/g의 감소 효과를 나타냈고, 대조구와 비교하여 부패율을 48% 낮추는 효과를 보였다. 저장 중 당도, 산도는 대조구와 비교하여 유의적인 차이를 나타내지 않았고, 병합처리는 감귤 시료의 표면 색도에 부정적인 영향을 끼치지 않았다. 따라서 본 연구 결과 이산화염소 가스, 중온 열수 세척, 푸마르산 병합처리는 감귤 시료의 품질에 영향을 끼치지 않으면서 미생물학적 안전성 및 품질을 향상할 수 있는 효과적인 허들기술이라고 판단된다.

• 생약학회지
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• 제30권3호
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• pp.306-313
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• 1999

Polyphenol Oxidase(PPO) was purified from an extract of Ginkgo biloba leaves by ammonium sulfate fractionation followed by sephadex G-150 column chromatography, which resulted in a 18-fold increase in specific activity. The enzyme was most active at pH 8.5 and the temperature optimum for the PPO catechol oxidation reaction was $45^{\circ}C$. Heat inactivation studies showed that heating for 7, 9 and 48 min, at 80, 70 and $60^{\circ}C$ respectively caused a 50% loss in enzymatic activity and that the enzyme was completely inactivated after heat treatment at $90^{\circ}C$ for 60 min. Km values of the PPO for catechol, hydroquinone and 4-methylcatechol derived from Lineweaver-Burk plots were $6.06\;{\times}\;10^{-4}M,\;1.02\;{\times}\;10^{-3}M,\;1.41\;{\times}\;10^{-3}M$ respectively. Of the substrates tested, 4-methylcatechol was oxidized most readily and the enzyme did not oxidize monophenols. The enzyme datalyzed browning reaction was completely inhibited in the presence of reducing reagents, namely ascorbic acid, cysteine, glutathione, 2-mercaptoethanol, potassium metabisulfite at 0.5 mM level. Sodium chloride showed very little inhibition effect on Ginkgo biloba leaves PPO. Lineweaver-Burk analysis of inhibition data revealed that the inhibition by cysteine, 2-mercaptoethanol, potassium cyanide was competitive with ki values of $1.1\;{\times}\;10^{-5}M,\;2.4\;{\times}\;10^{-5}M,\;8\;{\times}\;10^{-5}M$, respectively. Among the divalent cations, $Cu^{2+}ion$ was a strong activator on PPO and $Mn^{2+}ion$ was little or no effect on PPO activity $Ni^{2+}ion$ was an inhibitor on PPO.

• Journal of Microbiology and Biotechnology
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• 제27권2호
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• pp.219-225
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• 2017

An aqueous chlorine dioxide ($ClO_2$) treatment combined with highly activated calcium oxide (CaO) and mild heat was tested for inactivating naturally existing bacteria and Escherichia coli O157:H7 inoculated on fresh-cut kale. Kale samples were treated with different concentrations of $ClO_2$ (10, 30, and 50 ppm), CaO (0.01%, 0.05%, 0.1%, and 0.2%), and mild heat ($25^{\circ}C$, $45^{\circ}C$, $55^{\circ}C$, and $65^{\circ}C$) as well with combinations of 30 or 50 ppm $ClO_2$ and 0.2% CaO at $55^{\circ}C$ for 3 min. An increasing concentration of $ClO_2$ and CaO significantly reduced the microbial population compared with the control. In addition, mild heating at $55^{\circ}C$ elicited greater microbial reduction than the other temperatures. A combined treatment of 50 ppm $ClO_2$ and 0.2% CaO at $55^{\circ}C$ reduced the population of naturally existing bacteria on kale by 3.10 log colony forming units (CFU)/g, and the counts of E. coli O157:H7 were below the detection limit (1 log CFU/g). In addition, no significant differences in the Hunter color values were evident in any treatment during storage. Therefore, a combined treatment of $ClO_2$ and active CaO at $55^{\circ}C$ can be an effective sanitizing method to improve the microbiological safety of fresh-cut kale without affecting its quality.

• 한국응용곤충학회지
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• 제27권4호
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• pp.211-218
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• 1988

담배거세미나방(Spodoptera litura: SI), 누에(Bombyx mori: Bm) 및 흰불나방(Hyphantria cunea : Hc)으로부터 분리된 핵다각체병 바이러스(nuclear polyhedrosis virus : NPV)의 다각체의 단백질의 특성과 그에 대한 alkaline protease의 영향을 SDS-PAGE 및 혈정학적 방법으로 분석했다. Alkaline protease를 부활화시킨 후 다가체 단백질을 SDS-PAGE한 결과, BmNPV의 경우 30kD, SINPV와 HcNPV는 31kD의 단일 major band 및 이것들의 종합체(polymer)인 57kD와 66kD의 minor band들이 관찰되었다. Alkaline protease의 부활화를 성략한 SI NPV 다각체를 알칼리 용액으로 시간 차이를 두고 처리한 후 SDS-PAGE한 결고, 알칼리 처리시간이 경과함에 따라서 alkaline protease활성에 의해 다각체 단백질이 일정한 pattern으로 저분자화됨이 뚜렷하였다. SINPV 와 BmNPV 다각체 단백질의 항체를 제조하여 SINPV, BmNPV및 HcNPV 다각체 단백질간의 혈정학적 상동성을 이중형역광산법과 Western blot으로 비교한 결과는 3종 모두에서 공통 antigenic determinants의 존재가 인정되었으며 major 다각체 단백질의 종합체 형성과 alkaline protease에 의한 분해를 확인했다.

• Journal of Applied Biological Chemistry
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• 제56권3호
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• pp.165-170
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• 2013

The SCO3388 gene from Streptomyces coelicolor is homologous to tmrB, the tunicamycin resistance gene of Bacillus subtilis. The SCO3388-inactivation strain (SY-tbl-1) was generated by replacing SCO3388 with thiostrepton resistance gene. Spores of S. coelicolor derivatives were prepared on mannitol-soy flour (MS) agar on which SY-tbl-1 displayed no significant defect in growth and development. When plated on R4 agar, spores of SYtbl-1 displayed retardation in growth and sporulation, whereas its mycelium gave rise to normal growth. Thus, SCO3388 is suggested to be involved in the dormant spore germination. Expression of SCO3388 under the ermE1 promoter restored but only partially the ability to sporulate in SY-tbl-1. Neither SY-tbl-1 nor SY-tbl-1/ermE1p-SCO3388 showed a difference in tunicamycin resistance to the wild type whereas, interestingly, the introduction of ermE1p-SCO3388 dramatically enhanced spore survival to heat and detergent treatments, suggesting that SCO3388 might play a role in the maintenance of spore cell wall integrity.

• Journal of Microbiology and Biotechnology
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• 제28권9호
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• pp.1457-1466
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• 2018

In the present study, the stabilizing effect of four different biological osmolytes on Bacillus licheniformis ${\gamma}$-glutamyl transpeptidase (BlGGT) was investigated. BlGGT appeared to be stable under temperatures below $40^{\circ}C$, but the enzyme retained less than 10% of its activity at $60^{\circ}C$. The tested osmolytes exhibited different degrees of effectiveness against temperature inactivation of BlGGT, and sucrose was found to be the most effective among these. The use of circular dichroism spectroscopy for studying the secondary structure of BlGGT revealed that the temperature-induced conformational change of the protein molecule could be prevented by the osmolytes. Consistently, the molecular structure of the enzyme was essentially conserved by the osmolytes at elevated temperatures as monitored by fluorescence spectroscopy. Sucrose was further observed to counteract guanidine hydrochloride (GdnHCl)-and urea-induced denaturation of BlGGT. Taken together, we observed evidently that some well-known biological osmolytes, especially sucrose, make a dominant contribution to the structural stabilization of BlGTT.

• Journal of Microbiology and Biotechnology
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• 제5권1호
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• pp.14-17
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• 1995

Endoglucanase gene of Pseudomonas sp. LBC505 was previously cloned in pUC19 to yield plasmid pLCl. The Pseudomonas sp. LBC505 endoglucanase gene was subcloned in a temperature-regulated Es-cherichia coli expression vector, pAS1, containing the leftward promoter $P_L$ of bacteriophage lambda. The level of gene expression was controlled by the thermal inactivation of the heat-sensitive lambda cI857 repressor. Best yield of endoglucanase was obtained by lowering the incubation temperature to $37^{\circ}C$ after induction at $42^{\circ}C$ for 1h. Under these conditions enzyme production continued for about 5h at a gradually decreasing rate. Ecoli harboring recombinant plasmid pASC10 expressed 4.3 times as much CMCase activity as E.coli containing pLCl. To enhance the expression level of endogl, ucanase gene, we have also changed the presumptive Shine-Dalgamo sequence (AGAGGT) of the gene to consensus sequence (AGGAGGT) by site-directed mutagenesis. The genes mutated were subcloned in pASl resulting in the formation of recombinant plasmid pASS50. E.coli harboring the plasmid pASS50 expressed 6.2-fold higher levels of CMCase activity than that of E.coli harboring pLC1.