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High-rate composting by using a recombinant of thermophilic bacterium producing cellulose binding cellulase

使用产生纤维素结合纤维素酶的嗜热细菌重组体进行高效率堆肥

基本信息

批准号：
10680541
负责人：
NAKASAKI Kiyohiko
金额：
$ 2.18万
依托单位：
Shizuoka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10680541/
关键词：
compost high-rate composting microorganism organic matter degradation cellulose thermophile recombinant cellulase

项目摘要

For high rate composting of organic waste, it is essential to accelerate the decomposition of cellulose, one of the mostly hard to be biodegradable substances. The reason why cellulose remains in compost for a long period and it takes time for compost to be cured, microorganism that produces cellulase is not easy to grow under the condition of a high temperature of thermophilic composting. In this study, we tried to make recombinant bacterium of having high cellulase activity and of being able to grow at a high temperature, by introducing cellulase genes into thermophilic Bacillus strain, A8 isolated from compost.We ascertained that the introduction of cellulase genes into plasmid and the direct transformation of them into the strain A8 have a very low value of transformation efficiency, making it difficult to produce recombinant bacteria. Therefore, we decided to produce recombinant bacterium using Bacillus subtilis ISW, and extract a large quantity of plasmid DNAs from the recombinant bacteria, and transform them into A8 strain. Thermostable cellulase genes (Cel A) derived from Clostridium thermocellum were used for cellulase genes, and pGDV1 for plasmid, respectively. Cel A and pGDV1 were cut off using two types of repressible enzymes Pst I and Sca I and were subject to ligation reaction at 16 C for 12 hours. DNA samples after ligation were transformed into protoplast of B. subtilis ISW. As a result, we succeeded in producing cellulase genetic recombinant bacteria of thermophilic Bacillus strain A8 originated from compost.

为了实现有机垃圾的高效堆肥，必须加速纤维素的分解，纤维素是最难生物降解的物质之一。纤维素会在堆肥中长时间保留，堆肥需要时间固化，这是因为产生纤维素酶的微生物在高温堆肥的高温条件下不易生长。在本研究中，我们试图通过将纤维素酶基因导入从堆肥中分离的嗜热芽孢杆菌菌株A8中来获得具有高纤维素酶活性且能够在高温下生长的重组细菌，我们确定将纤维素酶基因导入质粒并将其直接转化到菌株A8中具有很低的转化效率值，使得难以产生重组细菌。因此，我们决定利用枯草芽孢杆菌ISW生产重组菌，并从重组菌中提取大量质粒DNA，转化到A8菌株中。纤维素酶基因以热纤梭菌耐热纤维素酶基因（CelA）为载体，质粒以pGDV 1为载体。用两种类型的阻遏酶Pst I和Sca I切断CelA和pGDV 1，并在16 ℃下进行连接反应12小时。将连接后的DNA样品转化到B的原生质体中。枯草杆菌ISW。结果表明，我们成功地从堆肥中获得了嗜热芽孢杆菌A8菌株的纤维素酶基因重组菌。