Components of the cellulase systems produced by a mesophilic fungus, Trichoderma konlngii, and a thermophilic anaerobic bacterium, Clostridium thermocellum, have been purified and their actions studied alone and in mixtures. The cellulase complex to T.koningii was fractionated into eight components by gel filtration on Ultrogel AcA 45 and ion-exchange chromatography on OEAE-Sepharose and Cellex PA6 (p-aminobenzyl) cellulose. The components isolated were a major cellobiohydrolase, a major CM-cellulase, a low molecular weight (1.M.14.) CIA-cellulase, two minor CM-cellulases, p-glucosidase, a minor cellobiohydrolase and a cellobiohydrolase related component with CM-cellulase activity. None of the components alone was effective in solubilising reactive (bacterial) cellulose or cotton (native cellulose) or in forming short-fibres from cotton; but when recombined at their culture filtrate concentrations, the mixture displayed more than 90% of the culture filtrate activity in solubilising both substrates and in forming short-fibres from cotton. Cellobiohydrolases (major and minor) caused little decrease in the viscosity of reactive cellulose and released small amounts of cellobiose; this is connected with their action as exo-glucanases. All the CM-cellulase components rapidly decreased the viscosity of reactive cellulose as expected from the action of endo-glucanases. The major CM-cellulase was most effective in decreasing the viscosity of reactive cellulose and released more sugar (cellobiose) from this substrate than the other components. The L.M.W. Cli-cellulase released an appreciable amount of cellotriose as well as cellobiose with traces of higher oliuosaccharides. The other components also released cellobiose, but with some glucose in the case of the minor CM-cellulases. The major cellobiohydrolase synergised only with the major CM-cellulase and together they showed 70 and 30% of the culture filtrate activity in solubilising reactive cellulose and cotton respectively; addition of the L.M.W. CM-cellulase and/or the minor CM-cellulases markedly improved solubilisation of cotton, but not that of reactive cellulose. Thus these CM-cellulase components appeared to have a different mode of action from the synergistic major CM-cellulase. Effective fragmentation of cotton into short-fibres also required a concerted action of cellulolytic components and cellobiohydrolase (exo-glucanase) as well as the major CM-cellulase was essential. a-Glucosidase made little contribution to cellulolysis. Cell-free culture broth of C.thermocellum contained cellulase activity capable of degrading, in the presence of CaLl and a thiol reducing agent, native cellulose (cotton) and derived forms of cellulose (Avicel and filter paper) at a rate, and to an extent, comparable with that achieved by cellulase from Trichoderma reesei. The cellulase was most active at 70°C and at pH 5.7-6.1. Fractionation of cell-free culture broth of C.thermocellum on Ultrogel AcA 45 provided a low molecular weight CM-cellulase (containing most of the B-glucosidase of the broth) and a high molecular weight CM-cellulase/cellulase fraction. The latter was further resolved into two, CM-cellulase fractions; one of which was free of cellulase and a-glucosidaso activity; The other was active on Avicel (microcrystallinecellulose). This isolated cellulase fraction failed to hydrolyse Avicel extensively and did not synergise with other fractions. Considerable cellulase activity, but not Cli-cellulase activity, was lost during fractionation, particularly on lyophilisation of partially purified preparations. The results provide evidence for the presence of different cellulolytic components in the C.thermocellum cellulase system. One of these may be an exo-glucanase but there is no direct evidence, as yet, for its presence.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:638742 |
| Date | January 1983 |
| Creators | Sakajoh, M. |
| Publisher | Swansea University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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