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Purification and Characterization of Two Alkaline Protease Produced by Pseudomonas sp. BK7
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  • Purification and Characterization of Two Alkaline Protease Produced by Pseudomonas sp. BK7
저자명
Lee. Eun-Goo,Park. Eun-Hee,Hyun. Hyung-Hwan
간행물명
Journal of microbiology and biotechnology
권/호정보
2000년|10권 5호|pp.677-684 (8 pages)
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한국미생물생명공학회
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이 논문은 한국과학기술정보연구원과 논문 연계를 통해 무료로 제공되는 원문입니다.
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Pseudomonas sp. BK7, an alkalophile, displayed the highest growth and protease activity when grown in a fermenter which was controlled at a pH level of 9.0, and the enzyme production was significantly enhanced by the increase of agitation speed. Two forms of alkaline proteases (BK7-1 and BK7-2) were fractionated and purified to near homogeneity. Protease BK7-1 was purified through CM-Sepharose CL-6B and Sephadex G-75 column chromatographies, and Protease BK7-2 was purified through CM-Sepharose CL-6B, DEAE-Sepharose, and Sephadex G-75 column chromatographies. The molecular weights of proteases BK7-1 and BK7-2 determined by gel filtration chromatography were 20,700 and 40,800, respectively. The $K_m$ value, isoelectric point, and optimum pH of protease BK7-1 were 2.55 mg/ml, 11.0, and 11.0, respectively, whereas those of protease BK7-2 were 1.57 mg/ml, 7.2, and 10.0, respectively. Both proteases were practically stable in the pH range of 5-11. The optimum temperatures for the activities of both protease BK7-1 and BK7-2 were $50^{circ}C$ and $45^{circ}C$, respectively. About 56% of the original protease BK7-2 activity remained after being treated at $50^{circ}C$ for 30 min but protease BK7-1 was rapidly inactivated at above $25^{circ}C$. Both proteases were completely inhibited by phenylmethane sulfonyl fluoride, a serine protease inhibitor. Protease BK7-2 was stable against EDTA, EGTA, STP, and detergents such as SDS and LAS, whereas protease BK7-1 was found to be unstable.