Characterization of endoglucanase cela from the rumen bacterium Clostridium longisporum
Doctoral Thesis
1994
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University of Cape Town
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Abstract
Cellulose is the most abundant organic compound on earth and offers great potential as a source of renewable energy and other chemicals. Cellulases are being studied to elucidate the enzymatic degradation of cellulose. We are interested in the molecular mechanisms of microbial degradation of cellulose in the rumen. The long-term aim is the potential genetic modification of lignocellulolytic activities in the rumen. ยท Clostridium longisporum was obtained from Varel (1989) because oxygen-resistant endospores might be suitable "vectors" for the introduction of genetically modified enzyme systems into the rumen via animal feeds. It is a sporadically occurring rumen bacterium and its role in ruminal cellulolysis is unclear. The aim of this project was the initial characterization of cellulases produced by C. longisporum. The celA gene was obtained by screening a library of C. longisporum genomic DNA in Escherichia colifor clones expressing CMCase activity. Approximately 38 CMCase-positive clones were obtained and the plasmid pCM4 was isolated from the clone expressing the highest activity. Southern analysis indicated that another plasmid, pCM64, contained a larger insert including the insert of pCM4. A total of 3620 bp were sequenced and a 1548-bp open reading frame, termed celA, was found. This gene showed homology with other endo-B-1,4-glucanases from family 5 (Henrissat & Bairoch, 1993). Plasmid pCM64 was found to contain the whole celA gene encoding endoglucanase CelA, while pCM4 has a 5'-truncated gene, termed celM5', which encodes a fusion protein, CelMN', that was initiated from an ATG codon in the vector. Sequence analysis of celA revealed the presence of a type I cellulose-binding domain (Beguin & Aubert, 1994) at the COOH-terminus of CelA.
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Bibliography: pages 84-101.
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Mittendorf, V. 1994. Characterization of endoglucanase cela from the rumen bacterium Clostridium longisporum. University of Cape Town.