Cellulolytic enzymes capable of efficiently degrading crystalline cellulose are a
complex mixture of endo- (endoglucanases) and exo-acting (cellobiohydrolases)
enzymes. One approach to separating these enzymes is affinity chromatography. A new
ligand, p-aminophenyl l-thio-β-D-cellobioside (APTC), is introduced for this purpose.
The property of APTC in affinity chromatography is demonstrated using Trichoderma
reesei cellulases. The behavior of these enzymes on APTC-affinity column was
essentially equivalent to that reported for the same enzymes on p-aminobenzyl 1-thio-β-
D-cellobioside (ABTC)-columns; ABTC being the traditional ligand for affinity
chromatography of exocellulases. The primary advantage of the APTC ligand is its ease
of preparation.
The affinity between CBHs and APTC may be considerably affected by
nonspecific interactions. In this study, the significance of nonspecific protein/matrix
interactions in affinity chromatography of cellulolytic enzymes is evaluated. The role of pH, NaCl, coupling conditions and stationary phase functional groups (N-hydroxysuccinimide
ester and cyanogen bromide) on the affinity purification of
Trichoderma reesei CBHs has been systematically determined. The results suggest that
the apparent discrepancies in existing methods for the affinity purification of CBHs are
due to nonspecific interactions, i.e. ionic interactions, between the enzymes and the
stationary phase matrix.
Exocellulases can be classified into two classes, based on their hydrolytic
specificities. Class I enzymes preferentially hydrolyze cellulose from the reducing end,
while Class 11 enzymes preferentially hydrolyze cellulose from the nonreducing end.
Trichoderma reesei CBH I is a class I enzyme and CBH 11 is a class II enzyme. CBH I
and CBH II are both retained on the APTC-affinity column; showing that both CBH
classes bind to immobilized APTC. To further understand the differences in the two
CBH classes, the behavior of CBH I and CBH II on the APTC-affinity column was
compared. The affinity of CBH I for immobilized APTC was found to decrease when
glucose was present in the system. In contrast, glucose was found to increase the affinity
of CBH 11 for immobilized APTC. An outcome of this difference is that in the presence
of glucose CBH I can be selectively eluted from the column. Equilibrium binding studies
with each enzyme clearly reflect that CBH II has a higher affinity for immobilized APTC
than CBH I. / Graduation date: 1998
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/27239 |
| Date | 25 June 1997 |
| Creators | Piyachomkwan, Kuakoon |
| Contributors | Penner, Michael H. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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