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 dif¡©ferent 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 blunt¡©end 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 ex¡©tracts and permeabilized cells.
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KEYWORD
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