Abstract
The biosynthesis of cellobiohydrolase (CBH I, EC 3.2.1.91) of
Trichoderma reesei was studied in order to obtain more information about the
basic biochemistry and secretion processes of filamentous fungi.The induction
and regulation of cellulase biosynthesis were studied using different
inducers.The information obtained provided a basis for choosing a method of
cloning the cellulase genes.Poly(A) mRNA was isolated from T reesei cultures
actively synthesizing cellulases and translated in vitro.In vitro synthesized
polypeptides corresponding to the cellulase proteins cellobiohydrolase I,
cellobiohydrolase II and endoglucanase I were immunoprecipitated using
antisera against the natural enzymes and characterised.When mRNA from
glucosegrown cells was added to the in vitro protein synthesis system only
negligible amounts of cellulases were immunoprecipitated, indicating that
regulation of the expression of these proteins is at the transcriptional
level.Sophorose was found to be the best inducer for cellobiohydrolase I and
it was used as inducer in in vivo labeling experiments.In order to analyze the
glycans of cellobiohydrolase I the extracellular glycoproteins of T. reesei
were metabolically labeled with ³H mannose after sophorose induction.The
labeled cellobiohydrolase I was purified by irnmunoprecipitation and SDS
polyacrylamide gel electrophoresis.The glycans of cellobiohydrolase I were
analyzed by chemical degradation procedures and by specific glycosidase
digestions.The results indicated that cellobiohydrolase I contained both O-
and N glycosidic glycans.The O-linked glycans consisted of one to four hexose
residues, which were composed mainly of mannose.The N linked glycans were of
high mannose type, with the structures (Man)5(GlcNAc)2 and (Man)9(GlcNAc)2.
Analysis of the in vivo synthesized cellobiohydrolase I revealed that the N
linked glycans are most probably added co-translationally.The first residues
of the O linked glycans may also be added co-translationally translationally,
but the chains are completed later during the secretion process, which takes
about 20 minutes.The light and transmission electron microscopy of the
morphological changes in a T. reesei mutant strain induced to produce
cellulases revealed that the induced mycelium had a higher content of vacuoles
and membraneous structures than the repressed mycelium.The glycan analysis
showed that the N linked glycans of cellobiohydrolase I were of high
mannose-type, resembling the glycans in animal cells, whereas the O-linked
glycans resembled those produced by yeast.Because many problems have been
encountered in the production of recombinant products in prokaryotes T. reesei
may have considerable potential as a host for the production of a range of
foreign glyco proteins, including those from animal sources.
Original language | English |
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Qualification | Doctor Degree |
Awarding Institution |
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Place of Publication | Espoo |
Publisher | |
Print ISBNs | 951-38-2773-9 |
Publication status | Published - 1987 |
MoE publication type | G4 Doctoral dissertation (monograph) |
Keywords
- biosynthesis
- hydrolases
- cellulase
- Trichoderma reesei