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

纤维素是最难生物降解的物质之一，要实现有机废物的高效堆肥，必须加快纤维素的分解。纤维素在堆肥中停留时间长、堆肥固化需要时间的原因是，产生纤维素酶的微生物在嗜热堆肥的高温条件下不易生长。本研究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，试图获得具有高纤维素酶活性且能在高温下生长的重组菌。我们确定将纤维素酶基因导入质粒并直接转化到菌株A8中，其转化效率值非常低，难以产生重组菌。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，转化为A8菌株。纤维素酶基因分别来源于热胞梭菌（Clostridium thermocellum）的耐热纤维素酶基因（Cel A）和质粒pGDV1。用两种抑制酶Pst I和Sca I切断细胞A和pGDV1，在16℃下进行结扎反应12小时。将结扎后的DNA样本转化为枯草芽孢杆菌的原生质体。结果，我们成功地从堆肥中获得了嗜热芽孢杆菌菌株A8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.