The work presented studies the cellulolytic system of <I>Trichoderma koningii</I> with particular reference to its ability to produce "short fibres" in the early stages of cellulose degradation. The culture filtrate of this organism was shown to produce short fibres from both filter paper (Whatman No.1) and cotton (Texas, non-dewaxed). The optimum conditions for production were identified and an assay system developed to measure this activity. Assay using filter paper was rapid and sensitive in determining short fibre producing activity, all results were subsequently confirmed on the more resistant substrate (cotton). The cellulase system was separated using an ion exchanger with a non-carbohydrate matrix and affinity chromatography on cellulose. Initial separation on ion exchange yielded the main cellobiohydrolase (CBH 1). Another fraction from this column separated on cellulose columns gave purified fractions of β-glucosidase, CM-cellulase and the short fibres forming activity (D2Cc). Only this latter fraction produced short fibres and synergised with CM-cellulase and β-glucosidase to increase short fibre production. Short fibres produced by D2Cc were more susceptible to subsequent hydrolysis by culture filtrate or CBH 1, degraded (bacterial) cellulose showed no physical changes on action of D2Cc but subsequent hydrolysis by CBH 1 or culture filtrate was increased. The main product of D2Cc was cellobiose but some cellotriose was detected from filter paper. D2Cc was inactive against cellobiose and cellotriose, both were potent inhibitors of D2Cc activity. Cellobiose was also an inhibitor of CBH 1 but cellotriose was not. D2Cc was shown to reduce the DP of bacterial cellulose. D2Cc and CBH 1 synergised in hydrolysing degraded cellulose, filter paper and cotton. The suggested role for this enzyme component is that it produces short fibres in concert with CM-cellulase which are then attacked by CBH 1 to produce cellobiose which is utilized by the organism.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:639202 |
| Date | January 1997 |
| Creators | Thomas, D. J. |
| Publisher | Swansea University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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