Biosynthesis of an inducible glycosylated secretory enzyme (CBH I) of Trichoderma reesei

Dissertation

Irma Salovuori

Research output: ThesisDissertationMonograph

2 Citations (Scopus)

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 languageEnglish
QualificationDoctor Degree
Awarding Institution
  • Helsinki University of Technology
Place of PublicationEspoo
Publisher
Print ISBNs951-38-2773-9
Publication statusPublished - 1987
MoE publication typeG4 Doctoral dissertation (monograph)

Fingerprint

cellulose 1,4-beta-cellobiosidase
Trichoderma reesei
polysaccharides
biosynthesis
enzymes
mannose
endo-1,4-beta-glucanase
cellulases
mycelium
secretion
chemical degradation
glycosidases
hexoses
prokaryotic cells
biochemistry
vacuoles
polyacrylamide gel electrophoresis
transmission electron microscopy
antiserum
glycoproteins

Keywords

  • biosynthesis
  • hydrolases
  • cellulase
  • Trichoderma reesei

Cite this

Salovuori, I. (1987). Biosynthesis of an inducible glycosylated secretory enzyme (CBH I) of Trichoderma reesei: Dissertation. Espoo: VTT Technical Research Centre of Finland.
Salovuori, Irma. / Biosynthesis of an inducible glycosylated secretory enzyme (CBH I) of Trichoderma reesei : Dissertation. Espoo : VTT Technical Research Centre of Finland, 1987. 72 p.
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title = "Biosynthesis of an inducible glycosylated secretory enzyme (CBH I) of Trichoderma reesei: Dissertation",
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.",
keywords = "biosynthesis, hydrolases, cellulase, Trichoderma reesei",
author = "Irma Salovuori",
year = "1987",
language = "English",
isbn = "951-38-2773-9",
series = "Publications / Technical Research Centre of Finland",
publisher = "VTT Technical Research Centre of Finland",
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address = "Finland",
school = "Helsinki University of Technology",

}

Salovuori, I 1987, 'Biosynthesis of an inducible glycosylated secretory enzyme (CBH I) of Trichoderma reesei: Dissertation', Doctor Degree, Helsinki University of Technology, Espoo.

Biosynthesis of an inducible glycosylated secretory enzyme (CBH I) of Trichoderma reesei : Dissertation. / Salovuori, Irma.

Espoo : VTT Technical Research Centre of Finland, 1987. 72 p.

Research output: ThesisDissertationMonograph

TY - THES

T1 - Biosynthesis of an inducible glycosylated secretory enzyme (CBH I) of Trichoderma reesei

T2 - Dissertation

AU - Salovuori, Irma

PY - 1987

Y1 - 1987

N2 - 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.

AB - 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.

KW - biosynthesis

KW - hydrolases

KW - cellulase

KW - Trichoderma reesei

M3 - Dissertation

SN - 951-38-2773-9

T3 - Publications / Technical Research Centre of Finland

PB - VTT Technical Research Centre of Finland

CY - Espoo

ER -

Salovuori I. Biosynthesis of an inducible glycosylated secretory enzyme (CBH I) of Trichoderma reesei: Dissertation. Espoo: VTT Technical Research Centre of Finland, 1987. 72 p.