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

利用海藻和微生物的酶生产生理活性物质的工艺开发

基本信息

批准号：
07556092
负责人：
IZUMI Yoshikazu
金额：
$ 3.01万
依托单位：
Tottori University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1997
项目状态：
已结题

项目摘要

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.

这项研究的研究结果总结如下。（1）纯化BPO蛋白的海洋藻氧化酶（BPO）的纯化纯化溴氧化酶（BPO）：旨在克隆纯化的BPO基因的克隆的纯化BPO基因和均具有阶级氨基酸酸性氨基酸性氨基酸的肽的肽的肽： bpo。（2）从C. pilulifera中提取RNA和DNA以及L l从C. pilulifera中提取RNA和DNA来扩增BPO基因的探针，然后我们根据上述结果通过PRCR合成的PRCR获得了pCR的探针，并根据上述结果与dna unabl anbs and。噬菌体和斑块杂交：使用纯化的mRNA获得纯化的mRNA并合成DC DNA，将DC DNA包装到lambdagt10 p中，最终获得了通过斑块杂交与上述探针的阳性斑块。 C. pilulifera的BPO基因（BPO）的DNA测序。（5）BPO在e.coli中的表达和表达的BPO的鉴定：BPO基因插入了Express PKK223中。培养了用PKK223 [BPO]转化的大肠杆菌。 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生理活性的硫烷硫化硫氧化物（甲基亚磺氧化物），一种重要的化合物，作为开始合成药物和农药，因此，我们可以显示出合成光学活性，生理活性化合物的合成的可能性。