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枯草菌細胞壁結合複合リパーゼによる超高機能油脂分解菌体の創成

使用枯草芽孢杆菌细胞壁结合复合脂肪酶创建超高性能石油降解细菌

基本信息

批准号：
11132221
负责人：
関口順一
金额：
$ 1.28万
依托单位：
Shinshu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research on Priority Areas (A)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至无数据
项目状态：
已结题

项目摘要

枯草菌細胞壁溶解酵素,CwlB,の細胞壁結合ドメイン(CWB)を表層局在化のアンカーとして使い、枯草菌リパーゼ,LipB,と融合させたCWB-LipBは表層に局在し、活性を示すが、培養の後期において著しく分解されることが解った。そこでこの分解を阻止し、表層におけるCWB-LipB蛋白の蓄積量を向上させるため、表層局在化プロテアーゼWprAの欠損株、細胞表層の多くの蛋白の生産を支配するシグマD因子(SigD)の欠損株、さらにWprA SigDの2重の欠損株を構築し、これらの宿主にCWB-LipBをコードするプラスミドを導入したところ、WprA SigD2重欠損株を用いた場合に顕著に蓄積させることができた。一方3種の分泌プロテアーゼ欠損株は有意な効果を与えなかった。次にこの技法を麹菌の生産するリパーゼ(CutL)に応用した。CutL蛋白は低級脂肪酸エステルに対し特に高い加水分解活性を示し、複合リパーゼの組み合わせに好適と考えられる。そこでcutLのcDNAのクローン化を行い、次にCwlBのCWBとLipBで用いた方法で連結し、CWB-CutLを構築した。この蛋白はLiClで表層より抽出出来ること、表層蛋白のSDS-PAGEでは約50kDaのCWB-CutLの分子量に対応するバンドが見られ、そのバンドはzymographyでもリパーゼ活性を示した。このことより麹菌リパーゼの表層局在化に成功するとともに、糖の修飾が無くても活性を示すことも解った。しかし表層蓄積量はCWB-LipBと比較して少なく、WprA SigD2重変異株以外ではほとんど蓄積が認められなかった。この結果は、異種蛋白は表層に於ても同種の蛋白より不安定であることを示唆している。

The cell wall lysozyme of Bacillus subtilis, CwlB, and the cell wall of Bacillus subtilis were combined with the cell wall of E. subtilis (CWB). The cell wall of Bacillus subtilis was analyzed in the form of lysozyme, LipB, fusion, CWB-LipB, bioactivity, and after culture. The CWB-LipB protein accumulates up and down, the CWB-LipB protein accumulates up, the cell surface changes, the WprA protein is deficient, the cell surface is multiprotein, the factor D factor (SigD) is deficient, the WprA SigD 2 is overweight, the host is CWB-LipB, the host is not, the host is not, and the host is not. WprA SigD2 owes a lot of money to the plant in the first place. One side 3 times secretes the wrong plant with the intention of producing the fruit and the fruit. In the second part of the experiment, the bacteria were used in the first place. (CutL) was used in the hospital. CutL protein, low-level fatty acids, low-level fatty acids, high hydrolytic activity, complex protein, low fatty acids, high hydration activity. "cutL" cDNA "LipB", "CwlB" CWB "LipB" using the "link" method, "CWB-CutL"link". The protein LiCl was extracted, the protein SDS-PAGE was extracted, the molecular weight of 50kDa CWB-CutL was extracted, the molecular weight of the protein was extracted, the molecular weight of the protein was extracted, the zymography was extracted, the molecular weight of the protein was extracted, the molecular weight of the protein was extracted, the molecular weight of the protein was extracted, the molecular weight of the protein was extracted, the molecular weight of the protein was extracted, the molecular weight of the protein was extracted, and the molecular weight of the protein was extracted. Please tell me that the table has been successfully modified, and that the activity of the sugar repairs has been improved. In this table, the positive amount of CWB-LipB is less than that of other plants, and the weight of WprA SigD2 is higher than that of other plants. The results showed that several kinds of protein tables showed that the same protein was unstable and the protein was not stable.