국내 토양에서 분리한 Thermoactinomyces CS707이 분비하는 단백분해효소의 분리정제 및 특성분석

다국어 초록 (Multilingual Abstract)

Thermophilic actinomyces producing extracellular protease was isolated from soil sample in Jeju province, Korea, and was named as Thermoactinomyces CS707. Culture broth of Thermoactinomyces CS707 showed the highest protease activity at late exponential growth phase when grown in OSYM medium(Oatmeal 2.0%, Dried yeast 1.0%, Soybeen meal 1.0%, Mannitol 1.0%, pH 8.3) at 48℃, 180rpm. Three forms of protease, TA-1, TA-2 and TA-3, were isolated through ammonium sulfate fractionation, Ultrogel AcA 34 column chromatography and DEAE-Trisacryl column chromatography. The optimum temperature for the activities of pretense TA-1, TA-2 and TA-3 were 55℃ , 65℃ and 65℃, respectively. About 50% of the original pretense activities(TA-1, TA-2 and TA-3) remained after being treated at 60℃ for 30min. The optimum pH values of protease TA-1, TA-2 and TA-3 were 7.5, 6.5 and 10.0, respectively. Protease TA-1, TA-2 and TA-3 were stable in the pH range of 6~ll.5, 4~9 and 5~11, respectively. The activity of protease TA-1 was Inhibited by Cu^(2+), SDS, metal chelators EDTA and EGTA, and phenylmethylsulfonylfluride(PMSF). The activity of protease TA-2 was inhibited by Cu^(2+), Ni^(2+) and PMSF, Whereas the metal chelators EDTA and EGTA did not affect the enzyme activity of TA-2. The activity of protease TA-3 was inhibited by SDS, EDTA, EGTA and PMSF.

목차 (Table of Contents)

• 목차
• 도목차 = ⅰ
• 표목차 = ⅱ
• ABSTRACT = ⅲ
• Ⅰ. 서론 = 1
• Ⅱ. 재료 및 방법 = 4
• 1. 균 분리 및 보관 = 4
• 2. DNA추출 = 4
• 3. 16S rDNA의 염기서열 = 5
• 4. Phylogenic analysis = 5
• 5. 균주의 액체 배양 = 6
• 6. 효소활성도 측정 = 6
• 7. 단백질 정량 = 6
• 8. 효소의 정제 = 6
• 1) 조효소 조제 = 7
• 2) Ammonium sulfate분획 = 7
• 3) Gel permeation chromatography = 7
• 4) DEAE-Trisacryl column chromatography = 7
• 9. 효소활성도와 안정성에 대한 온도의 영향 = 7
• 10. 효소활성도와 안정성에 대한 pH의 영향 = 8
• 11. 효소활성도에 대한 금속이온과 효소활성저해제의 영향 = 8
• Ⅲ. 결과 및 고찰 = 10
• 1. Thermoactinomyces CS707의 동정 = 10
• 1) 형태학적 특성 분석 = 10
• 2) 분자생물학적 특성 분석 = 10
• 2. Protease의 생산 = 11
• 3. 효소의 정제 = 11
• 4, 효소활성도와 안정성에 대한 온도의 영향 = 12
• 5. 효소활성도와 안정성에 대한 pH의 영향 = 12
• 6. 효소활성도에 대한 금속이온의 영향 = 12
• 7. 효소활성도에 대한 효소활성저해제의 영향 = 13
• Ⅳ. 결론 = 26
• Ⅴ. REFERENCES = 27