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KMID : 0380019950100030264
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Korean Journal of Biotechnology and Bioengineering
1995 Volume.10 No. 3 p.264 ~ p.269
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Cloning and Expression of D-amino Acid Oxidase from Trigonopsis variabilis for Cephalosporin C Biotransformation
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Abstract
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Trigonopsis variabilis is a strong prdfducer o-amino acid oxidase that can transform cephalosporin C (ceph C) to a keto-adipyl-7-aminocephalosporanic acid (AKA-7ACA). Polymerase chain reaction (PCR) was applied to isolate the D-AAO gene from T. variabilis. To clone the PCR fragment, four different methods were examined using enzymatic reactions of Taq DNA Polymerase, Klenow, T4 DNA
polymerase I, Alkaline phosphatase Calf Intestinal, and T4 kinase. Ligation of phosphorylated bluntend PCR fragment and dephosphorylated blunt-end of pUC18 plasmid yielded the best cloning efficien
cy. One of recombinant E. coil transformants showed D-AAO activity against ceph C in both cell extracts and permeabilized cells.
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KEYWORD
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