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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.

本研究的研究成果归纳如下。(1)海洋小球藻溴过氧化物酶（BPO）的纯化及BPO蛋白的氨基酸序列测定：为了克隆小球藻BPO基因，我们纯化了同源性较高的BPO，并测定了纯化的BPO的16个内肽的氨基酸序列。(2)通过提取小球藻的RNA和DNA，制备BPO基因扩增用探针，成功地从小球藻中提取了RNA和DNA，然后利用根据上述结果合成的引物，获得了用于PCR扩增的探针。(3)mRNA的纯化、ds DNA的合成及其与噬菌体的包装、噬斑杂交：获得纯化的mRNA和用纯化的mRNA合成的dc DNA，将dc DNA包装到噬菌体10 p中，最后与上述探针进行噬斑杂交获得阳性噬斑。(4)阳性菌斑cDNA的提取及BPO基因的DNA序列测定：从大肠杆菌中扩增的噬菌体中提取cDNA，并成功地测定了C. pilulifera的BPO基因（bpo）的DNA序列。(5)bpo在大肠杆菌中的表达和表达的BPO的鉴定：将bpo基因插入表达pKK 223中。培养用pKK 223 [bpo]转化的大肠杆菌。我们纯化了BPO prod转化的大肠杆菌，并证实BPO具有相同的N-末端氨基酸序列，并显示activv与钒酸盐预孵育。(6)由海藻BPO合成光学活性、生理活性化合物：我们发现海藻的BPOs和真菌的氯过氧化物酶（CPO）可以催化合成具有光学生理活性的茴香硫醚亚砜（甲基苯基亚砜）作为医药和农药合成的重要起始化合物，从而为光学活性，生理活性化合物。