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

对于有机垃圾的高速堆肥，加速纤维素的分解是至关重要的，纤维素是最难生物降解的物质之一。由于纤维素在堆肥中滞留时间长，固化时间长，在高温堆肥的条件下，产生纤维素酶的微生物不容易生长。本研究通过将纤维素酶基因导入从堆肥中分离到的嗜热芽孢杆菌A8菌株中，使其具有较高的纤维素酶活性并能在高温下生长，从而获得具有较高纤维素酶活力的重组菌。因此，我们决定用枯草芽孢杆菌ISW生产重组菌，并从重组菌中提取大量的质粒DNA，将其转化为A8菌株。分别以耐热纤维素酶基因(Cel A)为纤维素酶基因，pGDV1为质粒。用两种阻遏酶Pst I和Sca I切割Cel A和pGDV1，并在16℃下进行12h的连接反应。将连接后的DNA样品转化为枯草芽孢杆菌原生质体。因此，我们成功地生产了堆肥来源的嗜热芽孢杆菌A8菌株的纤维素酶基因重组菌。

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.