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Title: Structure and properties of two galactomannan-degrading enzymes
Author: Halstead, J. R.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 1998
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The capacity of microorganisms to hydrolyse the plant cell wall polysaccharides, cellulose and xylan, has been investigated extensively, but to date there is little information concerning the degradation of the hemicellulose, galactomannan. This research focusses on the structures and properties of two bacterial galactomannan-degrading enzymes; an endo-β-1,4-mannanase and an α-galactosidase. One of the most active cellulase/hemicellulase systems known is the multienzyme cellulosome complex of the thermophilic anaerobe, Clostridium thermocellum. Previous work has shown that even though C. thermocellum does not utilise xylose, the cellulosome includes highly active xylan-degrading enzymes, which facilitate complete dissolution of the plant cell wall matrix enabling C. thermocellum to maximise production of potentially fermentable sugars. Similarly, although C. thermocellum does not utilise mannose for growth, its culture supernatant contains enzymes that hydrolyse mannan. As a first step in investigating the molecular basis for this activity and establishing whether the mannan-degrading activity is part of the cellulosome, a recombinant phage with the ability to hydrolyse azo-carob galactomannan had been previously isolated from a random library of C. thermocellum genomic DNA constructed in λ ZAPII. The cloned fragment of DNA containing a putative mannanase gene (manA) was excised into pBluescript SK- and sequenced in both strands revealing an open-reading frame (ORF) of 1767 nucleotides, encoding a protein (Mannanase A, ManA) of 589 amino acids, with a predicted molecular weight of 66816 Daltons. The putative catalytic domain of ManA was located between residues 165 and 507 and displayed 32.4 % sequence identity with other mannanases belonging to glycosyl hydrolase Family 26. Immediately downstream of the catalytic domain and separated from it by a short proline/threonine linker was a duplicated motif highly conserved in other catalytic subunits of the C. thermocellum cellulosome.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available