Cloning and heterologous expression of the endoglucanase gene of Bacillus licheniformis CFFD2

• Main Authors: Chih-Hsiang Wang, 王芝翔
• Other Authors: Fu-Shyan Wen
• Format: Others
• Language: zh-TW
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/47850192587419309687

碩士 === 國立中興大學 === 生命科學系所 === 99 === In response to the global issues of energy crisis and environmental pollution caused by overuse of fossil fuels, researchers in various countries have devoted all efforts to develop clean, renewable alternative energy, biomass energy has been on research focus during the past decade, even biomass-energy technology has gone through four generations of evolution, among them, however, the second generation of biomass energy technology which uses lignocellulose as raw material still attracts more attention today. Lignocellulose is particularly well-suited for biomass energy production because of its high carbohydrate content, large-scale availability, low cost, and environmentally friendly production. Lignocellulose is composed of cellulose, hemicellulose ( both are carbohydrate polymers ) and lignin, and its whole structure is very compact, so its complete degradation needs the cooperative action of cellulase, hemicellulase and ligninase. These degrading enzymes are almost produced by microorganisms, but the degrading enzymes produced by one microorganism always differ from others in function and activity, different decomposing and producing microorganisms must be cultivated together in order to utilize lignocellulose to produce biomass energy. This growing mode would poses problem of competition in nutrients and growing space among microorganisms, the best solution is genetically to make a good producing microorganism also a good decomposer. Clostridium xylanolyticum Ter3 is an early bacterial isolate of our lab, it has good xylanolytic ability and can produce hydrogen, ethanol and methanol, but it has only a little cellulolytic ability. In this study, an attempt was made to strengthen the cellulolytic ability of C. xylanolyticum Ter3. At first, three Bacillus licheniformis isolates, CFFD1, CFFD2 and CFFD4 were tested on their cellulolytic ability, their endoglucanase ( CMCase ) genes were also sequenced, then the complete CMCase gene ( bglC ) of CFFD2 which expressed higher CMCase activity was inserted into a shuttle vector of E. coli and Clostridium spp., pIMP1. When the constructed plasmid pBglC was transfered into E. coli DH5a by transformation and the transformant E. coli DH5a ( pBglC ) was grown in PY medium containing carboxylmethylcellulose (CMC), filter paper or natural lignocellulose (such as bagasse, rice straw and forage), it displayed stronger CMCase activity than B. licheniformis CFFD2 grown in the same media. However, the transformation of C. xylanolyticum Ter3 was unsuccessful, it seems that C. xylanolyticum Ter3 contained unknown restriction enzyme system to digest pBglC. Alternatively, pBglC is tried to transfer into C. acetobutylicum which has known restriction-methylation system, experimental work is proceeding now.