• 한국식품과학회지
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• 제31권1호
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• pp.219-223
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• 1999

우리나라의 전통 발효식품인 김치에서 혈전용해 효소를 생산하는 미생물을 분리하고 그 중 Bacillus amyloliquefaciens, Bacillus brevis, Micrococcus luteus의 세 종을 Bergey's manual of systematic bacteriology등에 의하여 동정하였다. 분리된 미생물을 효소 유도 배지에서 배양한 결과 B. amyloliquefaciens는 2.58 plasmin unit/mL, B. brevis는 1.48 plasmin unit/mL, 그리고 M. luteus는 2.03 plasmin unit/mL의 혈전용해효소 생산능을 보여 주였다. 각 균주에서 생산된 세포의 단백질을 SDS-PAGE와 fibrin zymography assay에 의해 분석한 결과 B. brevis와 M. luteus에서는 서로 다른 분자량을 가진 $3{\sim}4$개의 혈전용해 효소가 존재하였으며 B. amyloliquefaciens에서는 분자량이 약 29 kDa인 단일 band의 혈전용해 효소가 생산되었음을 확인하였다.

• 미생물학회지
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• 제30권4호
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• pp.252-259
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• 1992

Lentinus edodes 에서 분비된 laccase 를 DEAE Sephadex A-50, Con A-Sepharose, Sephadex G-150 크로마토그래피를 통해서 순수분리하였다. 효소는 분자량이 87 KDa 정도인 하나의 소단위체로 되어 있고, 12.0% 의 당을 함유하고 있었다. 그리고 N-말단 아미노산 서열은 Pleurotus oxtreatus 와 Cloriolus hirsutus 의 laccase 와 유사하였다. 효소의 최적 pH 는 4.8 이고 최적 온도는 $40^{\circ}C$ 이었다. 그리고 본 효소는 pH 7-9 와 $30^{\circ}C$이하에서 비교적 안정하였다. Syringaldazine 에 대한 $K_{M}$ 은 $0.4\mu$ M 이었고 $k_{cat}$ 은$ 77 sec^{-1}$ 이었다. This layer chromatography 에 의한 본 효소의 반응 산물들의 분리양상이 Pleurotus ostreatus 의 laccase 의 경우와 유사하였다.

• 미생물학회지
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• 제38권4호
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• pp.218-218
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• 2002

Using two drugs, tunicamycin and brefeldin A, which affect protein processing, we investigated the intracellular processing mechanism of secreted aspartyl proteinase 1 (SAPl) of Candide albicans. Three intracellular forms of SAPI were detected by immunoblotting using menoclonal antibody (MAb) CAPl. Their molecular weights were approximately 40, 41 and 45 kDa, respectively. The 41 kDa protein is a glycoprotein and may be the same as the extracellular form judging by its molecular mass. The 40 kDa protein was the unglycosylated form and its molecular mass coincided with deglycosylated SAPl and the 45 kDa protein was also the unglycosylated form. Neither the 40 and 45 kDa proteins were detected in the culture supernatant of C. albicans. These suggested that the 40 and 45 kDa proteins might be intracellular precursor forms of SAPI. These results show that SAPI is translated as a 45 kDa precusor form in the endoplasmic reticulum and the 45 kDa precursor farm undergoes proteolytic cleavage after translocation into the Golgi apparatus, generating the 40 kDa precursor form. This 40 kDa precursor is converted into a 41 kDa mature form through glycosylation in the Golgi apparatus. The mature form of the 41 kDa protein is sorted into secretary vesicles and finally released into the extracellular space through membrane fusion. When the glycan region of SAPl was digested with N-glycosidase F, both stability and activity of the enzyme decreased. These results indicate that the glycan attached to the enzyme may, at least in parti be related to enzyme stability and activity.

• 한국미생물·생명공학회지
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• 제20권2호
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• pp.150-155
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• 1992

토양 분리 균주로부터 얻은 배양액을 20-80 황산암모늄으로 분획한 후 세포의 cytosine deaminase의 성질을 검토하였다. 이 효소는 cytosine과 5-fluorocytosine(5-FC)에 기질 특이성을 가짐으로서 각각 uracil과 5-fluorouracil(5-FU)로 전환을 촉매하였다. 효소안정성에 대한 온도와 보존기간은 tris-HC1 완충용액에서 검토한 결과 최적온도는 $50^{\circ}C$ 부근이하에서 90 이상의 잔존활성을, 보존시간은 4일정도에서 80 이상의 잔존활성을 유지하였다. 최대 pH 8.0과 $37^{\circ}C$의 온도에서 나타났다. 활성에 적당한 pH는 8.0~8.5였고, 온도는 37~$45^{\circ}C$의 범위였다.

• Applied Biological Chemistry
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• 제44권4호
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• pp.246-250
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• 2001

토양시료로부터 높은 활성의 pretense를 생산하는 세균을 수십종 분리하였다. 분리된 세균중에서 protease활성과 성장속도면에서 가장 우수한 균주를 선별하여 WRD-2로 명명하였으며, 형태학적, 생화학적 및 생리학적 특성을 조사한 후 Bacillus sp로 동정되었다. WRD-2조효소액의 protease 최적 활성은 pH 6.0, 온도는 $40^{\circ}C$로 중성 protease임을 알 수 있었고, 온도 $20{\sim}40^{\circ}C$에서 높은 protease활성을 나타내었다. 또한, 초기 pH에 따른 protease 활성에서는 pH 6에서 가장 높은 활성을 나타내었고, 배양배지의 영향은 탄소원으로 maltose 3%, 질소원으로 yeast extract 4%에서 가장 높은 pretense활성을 나타내었다.

• Bulletin of the Korean Chemical Society
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• 제30권6호
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• pp.1360-1364
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• 2009

The bacterium Sphingomonas chungbukensis DJ77 produces the extracellular polysaccharide gellan in high yield. Gellan produced by this bacterium is widely used as a gelling agent, and the enzyme UDP-glucose pyrophosphorylase (UGP) is thought to play a key role in the gellan biosynthetic pathway. The UGP gene has been successfully cloned and over-expressed in E. coli. The expressed enzyme was purified with a molecular weight of approximately 32 kDa, as determined by a SDS-polyacrylamide gel, but the enzyme appears as ca. 63 kDa on a native gel, suggesting that the enzyme is present in a homodimer. Kinetic analysis of UDP-glucose for UGP indicates $K_m$ = 1.14 mM and $V_{max}$ = 10.09 mM/min/mg at pH 8.0, which was determined to be the optimal pH for UGP catalytic activity. Amino acid sequence alignment against other bacteria suggests that the UGP contains two conserved domains: An activator binding site and a glucose-1-phosphate binding site. Site-directed mutagenesis of Lys194, located within the glucose-1-phosphate binding site, indicates that substitution of the charge-reversible residue Asp for Lys194 dramatically impairs the UGP activity, supporting the hypothesis that Lys194 plays a critical role in the catalysis.

• Journal of Microbiology
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• 제38권4호
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• pp.218-223
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• 2000

Using two drugs, tunicamycin and brefeldin A, which affect protein processing, we investigated the intracellular processing mechanism of secreted aspartyl proteinase 1 (SAPl) of Candide albicans. Three intracellular forms of SAPI were detected by immunoblotting using menoclonal antibody (MAb) CAPl. Their molecular weights were approximately 40, 41 and 45 kDa, respectively. The 41 kDa protein is a glycoprotein and may be the same as the extracellular form judging by its molecular mass. The 40 kDa protein was the unglycosylated form and its molecular mass coincided with deglycosylated SAPl and the 45 kDa protein was also the unglycosylated form. Neither the 40 and 45 kDa proteins were detected in the culture supernatant of C. albicans. These suggested that the 40 and 45 kDa proteins might be intracellular precursor forms of SAPI. These results show that SAPI is translated as a 45 kDa precusor form in the endoplasmic reticulum and the 45 kDa precursor farm undergoes proteolytic cleavage after translocation into the Golgi apparatus, generating the 40 kDa precursor form. This 40 kDa precursor is converted into a 41 kDa mature form through glycosylation in the Golgi apparatus. The mature form of the 41 kDa protein is sorted into secretary vesicles and finally released into the extracellular space through membrane fusion. When the glycan region of SAPl was digested with N-glycosidase F, both stability and activity of the enzyme decreased. These results indicate that the glycan attached to the enzyme may, at least in parti be related to enzyme stability and activity.

• 한국식품영양과학회지
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• 제23권6호
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• pp.973-979
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• 1994

Microorganisms, including the strain IJ-3 isolated from soil, were found to secrete an extracellular soybean protein coagulating enzyme and the strain, IJ-3 was identified as Genus Bacillus according to the Bergey's manual . The enzyme coagulated protein in soymilk , thus forming a curd at pHs 5.8-6.4 and at 55-75℃. The optimum temperature for soybean protein coagulating activity was 65-75℃ and the enzyme was stable at temperature below 50℃ and was found to be stable with about 60-100% of the original activity at a with pH ranges(pH6-7). The molecular weight of enzyme was estimated to be 28,000 by SDS-PAGE. The curd formed with the enzyme from Bacilus sp. IJ-3 has a smooth texture, and a mild taste without any bitterness or a beany flavor.

• Journal of Microbiology and Biotechnology
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• 제19권10호
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• pp.1184-1190
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• 2009

A thermostable extracellular $\beta$-mannanase from the culture supernatant of a fungus Aspergillus niger gr was purified to homogeneity. SDS-PAGE of the purified enzyme showed a single protein band of molecular mass 66 kDa. The $\beta$-mannanase exhibited optimum catalytic activity at pH 5.5 and $55^{\circ}C$. It was thermostable at $55^{\circ}C$, and retained 50% activity after 6 h at $55^{\circ}C$. The enzyme was stable at a pH range of 3.0 to 7.0. The metal ions $Hg^{2+}$, $Cu^{2+}$, and $Ag^{2+}$ inhibited complete enzyme activity. The inhibitors tested, EDTA, PMSF, and 1,10-phenanthroline, did not inhibit the enzyme activity. N-Bromosuccinimide completely inhibited enzyme activity. The relative substrate specificity of enzyme towards the various mannans is in the order of locust bean gum>guar gum>copra mannan, with $K_m$ of 0.11, 0.28, and 0.33 mg/ml, respectively. Since the enzyme is active over a wide range of pH and temperature, it could find potential use in the food-processing industry.

• Journal of Microbiology and Biotechnology
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• 제11권6호
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• pp.957-963
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• 2001

Bacillus subtilis YL-1004 was isolated from soil for the development of agents to control dental caries. This strain produced an extracellular lytic enzyme that hydrolyzed the Streptococcus mutans cell wall. The lytic enzyme was purified to homogeneity by affinity chromatography and gel permeation chromatography to give a single band on SDS-PAGE and non-denaturing polyacrylamide gel electrophoresis. The molecular weight of the enzyme was deduced from SDS-PAGE and gel chromatography to be 38 kDa and the PI to be 4.3 from isoelectric focusing. Sirty $\%$ of its lytic activity remained after incubation at $50^{\circ}C$ for 30 min, and its optimal temperature was $37^{\circ}C$ . The enzyme showed its highest activity at pH 8.0 and was stable at pHs ranging from 4.0 to 9.0. Treatment with several modifiers showed that a cysteine residue was involved in the active site of the enzyme. This lytic enzyme from Bacillus subtilis YL-1004 exhibited specificity towards Streptococci and also showed autolytic activity on Bacillus subtilis YL-1004.