Graduate Thesis Or Dissertation
 

Immobilized cellooligosaccharides in the study of trichoderma reesei cellobiohydrolases

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  • A novel type of model substrates, i.e. immobilized p-aminophenyl-β-D-cellooligosaccharides, was developed and used in the study of exocellulases. The two major cellobiohydrolases from Trichoderma reesei, CBH I and CBH II were used as representative enzymes. p-Aminophenyl derivatives of cellobiose (PAPG₂), cellotriose (PAPG₃), and cellotetraose (PAPG₄) were synthesized from the reaction of p-nitrophenol and peracetylated glycosyl bromide of the corresponding cellooligosaccharides under the phase-transfer catalyzed conditions, followed by deacetylation and catalytic hydrogenation. p-Aminophenyl cellooligosaccharides were then tethered via their amino functional groups to N-hydroxy succinimide-activated agarose. The ability of CBH I and CBH II to associate with and catalyze the hydrolysis of reducing end tethered cellooligosaccharides was tested. CBH I catalyzed the hydrolysis of free PAPG₂ but CBH II did not. Both CBH I and CBH II reversibly bound, but did not hydrolyze, immobilized PAPG₂. Hence, the immobilized PAPG₂ was tested for the affinity chromatographic application. PAPG₂ is shown to be an effective ligand for the chromato graphic fractionation of cellobiohydrolases (CBHs). The PAPG₂-derivatized agarose specifically retained the CBH component of relatively complex cellulase mixtures. The purity of the resulting CBH preparation was comparable to that of corresponding enzyme preparations obtained using more traditional thioglycoside-based affinity ligands. The application of PAPG₂ as an affinity ligand suggests that the immobilized reducing end-blocked ligand associate with the T. reesei CBHs in a catalytically nonproductive mode. The catalytic activity for the hydrolysis of free and immobilized arylcellodextrins by the CBH I and CBH II were determined. CBH II attacked free and immobilized PAPG₃ and PAPG₄ in a typical exo manner in which cellobiose is a major hydrolytic product released from the nonreducing end. The rate of hydrolysis increases with increasing chain length suggesting the extended binding sites (at least 4 binding sites). Like CBH II, CBH I preferentially cleaved immobilized PAPG₃ and PAPG₄ at a second glycosidic linkage from the nonreducing end; the rate of hydrolysis increases as a function of chain length. However, it attacked free aryl-cellodextrins in a random manner. The rate of hydrolysis increases only from PAPG₂ to PAPG₃ and significantly drops in PAPG₄. This suggests that CBH I interacts with free and immobilized substrates in different modes.
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