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KMID : 0545120000100050677
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Journal of Microbiology and Biotechnology
2000 Volume.10 No. 5 p.677 ~ p.684
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Purification and Characterization of Two Alkaline Proteases Produced by Pseudomonas sp.BK7
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LEE, EUN GOO
PARK, EUN-HEE/HYUN, HYUNG-HWAN
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Abstract
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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, DEAESepharose, 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㎎/㎖, 11.0, and 11.0, respectively, whereas those of protease BK7-2 were 1.57㎎/㎖, 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℃ and 45℃, respectively. About 56% of the original protease BK7-2 activity remained after being treated at 50℃ for 30min but protease BK7-1 was rapidly inactivated at above 25℃. 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.
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KEYWORD
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