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신규 Cephalosporin-C Deacetylase (CAH)생산 규주인 Bacillus sphaericus 366M-9의선발 및 그 효소학적 특성

The Isolation of Bacillus sphaericus 366M-9Producing New Cephalosporin-C Deacetylase (CAH)and its Enzymatic Characterization.

이동희|권태종|이승훈
한국미생물·생명공학회 (The Korean Society For Microbiology And Biotechnology)|2004.09|한국미생물·생명공학회지 (Microbiology and Biotechnology Letters)|제32권 제3호|pp. 224-229

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참고문헌

주제분류생물학

초록

토양으로부터 강력한 CAH activity를 갖는 균주를 분리하여, 생화학적, 배양학적, 전자현미경 동정을 한 결과, Bacillus sphaericus로 확인되었으며, 이를 B. sphaericus 366M-9로 명명하였다. 또한 이 균으로부터 최초로 cephalosporin-C deacetylase(CAH)를 분리 정제하였다. 정제수율은 약 7.5%였으며, B. sphaericus 366M-9에서 분리한 CAH-9의 최적 활성 온도는 50oC였으며, 효소안정 온도구간은 30~50oC이다. 또한 최적 활성 pH는 7.0이었으며, 효소안정 pH구간은 pH 6.0~8.0으로 90% 이상의 잔존 활성도를 나타내었다. 효소반응속도에 미치는 기질의 영향에서는 Km 값은 0.87 mM이며, Vmax는1.22 unit/ml이었다.

Abstract

Several microorganisms (esterase-producing group) were isolated by the solid selective media containing -naphtylacetate. Among them, strain 366M-9 having a high activity of cephalosporin-C deacetylase (CAH; EC 3.1.1.41) was selected. The strain 366M-9 was identified as Bacillus sphaericus on the basis of morphological, physiological, and biochemical characteristics. The production of CAH reached at maximum value after 32 hrs, when cultivated in the optimal medium containing dextrin 2.5%, peptone 2.5%, sodium chloride 0.5%, dipotassium phosphate 0.25%, ferrous sulfate 0.02%, and 7-ACA 0.1% at 30oC with initial pH 6.0. The CAH was purified by 3 steps with ammonium sulfate precipitation, adsorption chromatography on hydroxyapatite column, and Sephadex G-200 gel chromatography. The final enzyme preparation was homogeneous as judged by the analysis of SDS-PAGE and HPLC. Optimum temperature and pH for CAH activity were 50oC and around 7.0, respectively. And the enzyme was stable at pH 6.0~8.0, up to 50oC. The Michaelis-Menten constants (Km), Vmax were 0.87 mM and 1.22 unit/ml, respectively.

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