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KMID : 0545120010110030452
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Journal of Microbiology and Biotechnology
2001 Volume.11 No. 3 p.452 ~ p.457
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Characterization of Glutaryl 7-ACA Acylase from Pseudomonas diminuta KAC-1
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KIM, DAE-WEON
KANG, SANG-MO/YOON, KI-HONG
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Abstract
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The glutaryl 7-aminocephalosporanic acid (glutaryl 7-ACA) acylase was purified from Pseudomonas diminuta KAC-1 cells isolated from soil, and characterized. The acylase was purified by procedures including ammonium sulfate fractionation and column chromatographies on DEAE-Sepharose, Phenyl-Sepharose, Q-Sepharose, and Superose 12H/R. The native acylase was found to be composed of two subunits with molecular masses of approximately 55kDa and 17kDa, respectively. The isoelectric point of the enzyme was 4.0. The specific activities of the purified acylase were 8.0 and 7.0U/㎎ on glutaryl 7-ACA and glutaryl 7-aminodesacetoxy cephalosporanic acid (glutaryl 7-ADCA ), respectively, and K_m values were 0.45mM for glutaryl 7-ACA and 0.67mM for glutaryl 7-ADCA. The enzyme had a pH optimum at 8.0 and a temperature optimum at 40℃. The acylase catalyzed the synthesis of glutaryl 7-ACA from glutaric acid and 7-ACA as well as the hydrolysis of glutaryl 7-ACA, although the reaction rate of the synthesis was slower than that of the hydrolysis. In addition, it was found that the enzyme had a glutaryl transferase activity, thereby transferring the glutaryl group from one cephalosporin nucleus to another.
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