Title page for ETD etd-07282010-020204
|Type of Document
||Biosynthesis and identification of enzymes of the cellulase system of Trichoderma reesei.
||Master of Science
||Biochemistry and Nutrition
|Brown, Ron D. Jr.
|Gregory, Eugene M.
|Hess, John L.
|Linkins, A. E. III
|Date of Defense
The biosynthesis of cellulolytic enzymes by resting cells of
T. reesei QM 9414 incubated in 17 mM potassium phosphate buffer,
pH 6.0, and Q-β-D-glucopyranosy1-(172)α-D-glucopyranose was investigated.
A maximum of 200 mi1liunits aryl-β-D-g1ucosidase, 9000 mi1liunits
endo-l,4-β-D-glucanaseand 200 milliunits Avicelase per milliliter
of culture supernate was produced after 24 hours of incubation;
at that time, extracellular protein production reached a maximum of
0.5 mg/ml. Optimum enzyme yields were obtained with 3-4 mg dry weight
cells/ml, and 1 nIDi 0-β-D-glucopyranosyl-(1->2)α-D-glucopyranose.
Inclusion of metals, e.g., zinc, cobalt, manganous and ferrous ions
enhanced enzyme production when glutamic acid was present, in which
case aryl-β-D-glucosidase, endoglucanase and Avicelase activity were
enhanced by 20, 100 and 40 percent, respectively. Under the same
conditions asparagine enhanced only ary1-β-D-glucosidase activity.
Some of the enzymic components of the cellulase system produced
by T. reesei under these conditions were purified by ion exchange
chromatography on DEAE-Sephadex A-50. Two cellobiohydrolases were
isolated in pure form. One of these was identical to the previously
isolated ce1lobiohydrolase D on the basis of amino acid composition
carbohydrate content, electrophoretic mobility and ultraviolet spectrum.
Similar data for the other cellobiohydrolase suggest that it
represents an enzyme not previously identified. An endoglucanase was
also isolated which, on the basis of its amino acid composition and
electrophoretic mobility, appears similar to the previously identified
Endoglucanase IV. More precise characterization of these enzymes
is currently under investigation.
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