Development of production process for physiologically active substanses using haenzymes from marine alga and microoganism

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The research results of this study are summarized as follows.(1) Purification of bromoperoxidase (BPO) from a marine alga Corallina pilulifera andamino acid sequencing peptides of the purified BPO protein : Aiming at cloning of C.pilulifera BPO gene, we purified BPO of homogeneity and sequenced amino acids of 16 internal peptides of the purified BPO.(2) Extraction of RNA and DNA from C.pilulifera and preparation of probe for amplification of BPO gene by l succeeded in extraction of RNA and DNA from C.pilulifera, and then we obtained probe for amplification o by PCR using primers synthesized based on the above result.(3) Purification of mRNA of, synthesis of ds DNA and its packaging to lambdagt10 phage, and plaque hybridization : obtain purified mRNA and synthesized dc DNA using the purified mRNA,packaged dc DNA into lambdagt10 p finally obtained positive plaques by plaque hybridization with the above probe.(4) Extraction of cDNA from positive plaques and DNA sequencing of BPO gene : We extracted cDNA from posit proliferated in E.coli and succeeded in DNA sequencing of BPO gene (bpo) of C.pilulifera.(5) Expression of bpo in E.coli and identification of expressed BPO : The bpo gene was inserted in an express pKK223. E.coli which was transformed with the pKK223 [bpo], was cultivated. We purified BPO prod transformed E.coli and confirmed that the BPO had the same N-terminal amino acid sequence and showed activ was preincubated with vanadate.(6) Synthesis of optically active, physiologically active compound by marine algal BPO : We found that dependent BPOs of marine algae and fungal chloroperoxidase (CPO) catalyzed the synthesis of optical physiologically active thioanisole sulfoxide (methyl phenyl sulfoxide), an important compound as a starting the synthesis of medicines and pesticides, and as a result, we could show a possiblity of application of the synthesis of optically active, physiologically active compounds.