Cellulolytic and xylanolytic enzymes of Trichoderma reesei: Dissertation

Arja Lappalainen

Research output: ThesisDissertationCollection of Articles


Enzymes hydrolyzing cellulose and xylan are important in the utilization of biomass in biotechnical processes.The work presented here concerns the cellulolytic and xylanolytic enzymes of Trichoderma reesei.Purification procedures, detection methods for the different components of the complex enzyme mixtures and some properties of the purified enzymes are described.T. reesei secretes into the culture liquid several enzymes which have very similar molecular and ionic properties. ß-Glucosidase (1,4 ß-D-glucoside glucohydrolase, EC, ß-xylosidase (1,4-ß-D-xylan xylohydrolase, EC and xylanases I and II (1,4-ß-D-xylan xylanohydrolase, EC 3.2.1.- and EC could be separated from the culture filtrate on the basis of their failure to adsorb on amorphous cellulose and using gel- and ion-exchange chromatography.Cellobiohydrolases I and II (1,4-ß-D-glucan cellobiohydrolase, EC and EC 3.2.1.-) and endoglucanase I (1,4-(1.3;1,4)-D-glucan 4-glucanohydrolase EC were purified by means of immunoaffinity chromatography.Special attention was given to the hydrolytic properties of purified enzymes and the synergistic effects of cellulolytic enzymes in the hydrolysis of cellulosic materials.In order to investigate the role of ß-glucosidase in the hydrolysis of cellulose, Aspergil lus niger ß-glucosidase was also purified.Some kinetic parameters were determined for purified T. reesei and A. niger ß-glucosidases.End product inhibition by glucose was much stronger for the T. reesei enzyme (Kj = 0.7 mM) than for A. niger enzyme (Kj = 2.1 mM).On the other hand, T. reesei ß-glucosidase had the higher affinity (lower Kmvalues) for both the substrates studied, cellobiose and p nitrophenyl ßglucoside.This means that at low substrate concentrations the use of T. reesei ß-glucosidase may be more advantageous.Cellobiohydrolase I hydrolyzes amorphous cellulose and produces short insoluble fibres and small amounts of cellobiose from cotton.It does not attack soluble substituted celluloses. Endoglucanase I hydrolyzes soluble ß-1,4-glucans and has no activity towards insoluble substrates.Cellobiohydrolase II hydrolyzes both soluble ß-1,4-glucans and insoluble amorphous cellulose, liberating cellobiose.It modifies native cotton by increasing its ability to take up alkali, without however releasing any detectable soluble material.Cellobiohydrolase I and II together hydrolyze native cotton as extensively as the culture filtrate alone. Endoglucanase I did not cooperate with cellobiohydrolases in the hydrolysis of insoluble native cellulose.Xylanase I has only a low capacity to liquify insoluble birch xylan (ß-1,4-linked poly xylose) but it is more active in hydrolyzing different branched xylan preparations (arabinoxylan, O-acetyl-4-0 methylglucuronoxylan, water extract of steamed birchwood).It seems to be rather non-specific and its main activity is clearly the hydrolysis of lamin arin (ß-1,3-glucan).Xylanase II has high activity both in liquifying insoluble birch xylan and in hydrolyzing branched xylan preparations.The addition of partially purified xylosidase considerably enhanced xylose production in hydrolyses with both xylanases but did not affect the overall liquefaction of insoluble xylan.Procedures are described for the preparation of pure components from the mixture of cellulolytic and xylanolytic enzymes produced by T. reesei and new data about the properties of the individual proteins are presented.In particular the hydrolytic properties of purified cellobiohydrolases and endoglucanase indicate a need for reclassification of these enzymes and re-evaluation of the mechanism of cellulose hydrolysis.
Original languageEnglish
QualificationDoctor Degree
Awarding Institution
  • University of Helsinki
  • Niku-Paavola, Marja-Leena, Supervisor, External person
Award date25 Nov 1988
Place of PublicationEspoo
Print ISBNs951-38-3271-6
Publication statusPublished - 1988
MoE publication typeG5 Doctoral dissertation (article)


  • glycoside hydrolases
  • Trichoderma reesei
  • Aspergillus niger


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