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. / Graduation date: 1999
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/27212 |
| Date | 07 May 1999 |
| Creators | Sangseethong, Kunruedee |
| Contributors | Penner, Michael H. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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