|Budget Amount *help
¥3,400,000 (Direct Cost : ¥3,400,000)
Fiscal Year 1999 : ¥1,100,000 (Direct Cost : ¥1,100,000)
Fiscal Year 1998 : ¥2,300,000 (Direct Cost : ¥2,300,000)
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.