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KMID : 0545120010110050870
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Journal of Microbiology and Biotechnology
2001 Volume.11 No. 5 p.870 ~ p.875
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Molecular Characterization of the Genes Encoding Acetoacetyl-Coenzyme A Transferase from Serratia marcescens KCTC 2172
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Yoo, Ju Soon
Kim, Hae Sun/Lee, Young Choon/Chung, Soo Yeol/Choi, Yong Lark
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Abstract
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A DNA fragment, pCKB13, containing two genes encoding Coenzyme A transferase, was isolated from a genomic DNA library of S. marcescens KCTC 2172. The complete nucleotide sequence of the 2,081-bp BamHI fragment on pCKB13 was determined. Sequencing of the fragment led to the identification of two open reading frames showing high homology with two Coenzyme A (CoA) transferases, Acetoacetyl-CoA transferase (Acot) and Succinyl-CoA transferase (Scot), enzymes catalyzing the reversible transfer of CoA from one carboxylic acid to another. The enzyme activity of Coenzyme A transferase increased after introducing the multicopy of the cloned gene in E. coli. The recombinant protein, overexpressed by multicopy and induction with IPTG, was a polypeptide of 42 kDa, as confirmed by SDS-PAGE. The protein was purified to homogeneity through three sequential chromatographic procedures including ion-exchanged DEAE-sepharose, CM-sepharose, and Mono Q.
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