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

本研究的研究结果总结如下：(1)从海洋藻类珊瑚中纯化溴过氧化物酶（BPO）并对纯化后的BPO蛋白进行氨基酸肽测序：针对珊瑚BPO基因的克隆，对纯化后的BPO进行了同源性纯化，并对纯化后的BPO的16个内部肽进行了氨基酸测序。(2)从毛蚕中提取RNA和DNA并制备BPO基因扩增探针，成功提取毛蚕的RNA和DNA，然后利用上述结果合成的引物进行PCR得到扩增探针o。(3)纯化mRNA，合成ds DNA并将其包装到lambdagt10噬菌体中，并进行斑块杂交：获得纯化的mRNA并使用纯化的mRNA合成dc DNA，将dc DNA包装到lambdagt10 p中，最后用上述探针进行斑块杂交获得阳性斑块。(4)阳性斑块cDNA的提取和BPO基因的DNA测序：从大肠杆菌中增殖的阳性斑块中提取cDNA，成功测序了毛利菌BPO基因（BPO）。(5) bpo在大肠杆菌中的表达及表达bpo的鉴定：将bpo基因插入表达的pKK223中。培养用pKK223 [bpo]转化的大肠杆菌。我们纯化了转化大肠杆菌的BPO prod，证实该BPO具有相同的n端氨基酸序列，并与钒酸盐预孵育后显示出活性。(6)利用海藻BPO合成光生理活性化合物：我们发现海藻BPO的依赖性和真菌氯过氧化物酶（CPO）催化合成了光生理活性的巯基亚砜（甲基苯基亚砜），这是药物和农药合成的重要起始化合物，因此我们可以显示出在合成光生理活性化合物方面的应用可能性。

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.