Mannan-hydrolysis by hemicellulases: enzyme-polysaccharide interaction of a modular beta-mannanase

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Mannan-hydrolysis by hemicellulases: enzyme-polysaccharide interaction of a modular beta-mannanase. / Hägglund, Per.

Dr Henrik Stålbrand, Getingevägen 60, Box 124, 221 00 LUND, Sweden, 2002. 174 s.

Forskningsoutput: AvhandlingDoktorsavhandling (sammanläggning)

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APA

Hägglund, P. (2002). Mannan-hydrolysis by hemicellulases: enzyme-polysaccharide interaction of a modular beta-mannanase. Dr Henrik Stålbrand, Getingevägen 60, Box 124, 221 00 LUND, Sweden,.

CBE

Hägglund P. 2002. Mannan-hydrolysis by hemicellulases: enzyme-polysaccharide interaction of a modular beta-mannanase. Dr Henrik Stålbrand, Getingevägen 60, Box 124, 221 00 LUND, Sweden,. 174 s.

MLA

Hägglund, Per Mannan-hydrolysis by hemicellulases: enzyme-polysaccharide interaction of a modular beta-mannanase Dr Henrik Stålbrand, Getingevägen 60, Box 124, 221 00 LUND, Sweden,. 2002.

Vancouver

Hägglund P. Mannan-hydrolysis by hemicellulases: enzyme-polysaccharide interaction of a modular beta-mannanase. Dr Henrik Stålbrand, Getingevägen 60, Box 124, 221 00 LUND, Sweden, 2002. 174 s.

Author

Hägglund, Per. / Mannan-hydrolysis by hemicellulases: enzyme-polysaccharide interaction of a modular beta-mannanase. Dr Henrik Stålbrand, Getingevägen 60, Box 124, 221 00 LUND, Sweden, 2002. 174 s.

RIS

TY - THES

T1 - Mannan-hydrolysis by hemicellulases: enzyme-polysaccharide interaction of a modular beta-mannanase

AU - Hägglund, Per

N1 - Defence details Date: 2002-05-31 Time: 10:15 Place: Center of Chemistry and Chemical engineering, Hörsal C External reviewer(s) Name: Warren, Anthony Title: Professor Affiliation: Dep of Microbiology and Immunology, University of British Columbia, 300-6174 University Blvd, Vancouver BC --- Article: I. Ademark, P., Lundqvist, J., Hägglund, P., Tenkanen, M., Torto, N., Tjerneld, F. and Stålbrand, H. (1999) Hydrolytic properties of a b-mannosidase purified from Aspergillus niger. J. Biotechnol. 75, 281-289 Article: II. Ademark, P., de Vries, R. P., Hägglund, P., Stålbrand, H. and Visser, J. (2001) Cloning and characterization of Aspergillus niger genes encoding an alpha-galactosidase and a beta-mannosidase involved in galactomannan degradation. Eur. J. Biochem. 268, 2982-90. Article: III. Hägglund, P., Sabini, E., Boisset, C., Wilson, K., Chanzy, H. and Stålbrand, H. (2001) Degradation of mannan I and II crystals by fungal endo-b-1,4-mannanases and a b-1,4-mannosidase studied with transmission electron microscopy. Biomacromolecules 2, 694-699 Article: IV. Hägglund, P., Eriksson, T., Collén, A., Nerinckx, W., Claeyssens, M. and Stålbrand, H. (2002) A cellulose-binding module of the Trichoderma reesei b-mannanase Man5A increases the mannan-hydrolysis of complex substrates. Submitted for publication in J. Biotechnol. Article: V. Xu, B., Hägglund, P., Stålbrand, H. and Janson, J. C. (2002) endo-beta-1,4-Mannanases from blue mussel, Mytilus edulis: purification, characterization, and mode of action. J. Biotechnol. 92, 267-77. Article: VI. Lundqvist, J., Hägglund, P., Eriksson, T., Persson, P., Stoll, D, Siika-aho, M., Gorton, L. and Stålbrand, H. Degradation of glucomannan and O-acetyl-galactoglucomannan by mannoside- and glucoside-hydrolases. Manuscript Article: VII. Hägglund, P., Anderson, L. and Stålbrand, H. The active site cleft of Trichoderma reesei b-mannanase Man5A: analysis of mutants of the catalytic glutamates and arginine 171 positioned in the +2 subsite. Manuscript

PY - 2002

Y1 - 2002

N2 - The enzymatic degradation of plant polysaccharides is a process of fundamental importance in nature which involves a wide range of enzymes. In this work, the structure and function of hemicellulose-degrading enzymes was investigated. The focus was on a beta-mannanase (TrMan5A) produced by the filamentous fungus Trichoderma reesei. This enzyme is composed of a catalytic module and a carbohydrate-binding module (CBM). In this thesis, the enzyme-polysaccharide interaction in both these modules was investigated. The results demonstrate that the CBM of TrMan5A is important for hydrolysis of complex mannan substrates containing cellulose. Furthermore, the increase in activity could be linked to binding of the CBM to the complex substrate. Binding studies revealed that the CBM binds to cellulose, but not to mannan. Studies of the enzyme/polysaccharide interaction in the active site cleft of the catalytic module of TrMan5A showed that a mutant of Arg 171 displayed activity in the same range as the wild-type enzyme toward polymeric substrates. However, the Arg171 mutant was impaired in hydrolysis of small substrates. Interestingly, this mutant also appears to have a more alkaline activity pH-optimum than the wild-type. The low or abolished activity observed with mutants of the predicted catalytic glutamates (Glu169 and Glu276) support their importance in hydrolysis. In addition to TrMan5A, the properties of a beta-mannanase (MeMan5A) from blue mussel and a beta-mannosidase (AnMan2A) from Aspergillus niger, were studied in this work. Investigations on the catalytic properties of the enzymes showed that all three enzymes are capable of degrading polymeric mannan. Furthermore, analysis by transmission electron microscopy revealed that TrMan5A and AnMan2A degrades highly crystalline mannan. Degradation of glucomannan and galactoglucomannan by several polysaccharide-degrading enzymes shows that these substrates can be hydrolysed by both mannoside- and glucoside-hydrolases. Furthermore, the results presented show that cellulases potentially are able to hydrolyse other components in the plant cell wall. Altogether, the results presented demonstrates the need to use complex substrates in order to reveal the mechanisms of plant polysaccharide degradation. In conclusion, this work has shown that the enzyme/polysaccharide interaction in the two modules of TrMan5A is important in determining the overall enzymatic activity and specificity.

AB - The enzymatic degradation of plant polysaccharides is a process of fundamental importance in nature which involves a wide range of enzymes. In this work, the structure and function of hemicellulose-degrading enzymes was investigated. The focus was on a beta-mannanase (TrMan5A) produced by the filamentous fungus Trichoderma reesei. This enzyme is composed of a catalytic module and a carbohydrate-binding module (CBM). In this thesis, the enzyme-polysaccharide interaction in both these modules was investigated. The results demonstrate that the CBM of TrMan5A is important for hydrolysis of complex mannan substrates containing cellulose. Furthermore, the increase in activity could be linked to binding of the CBM to the complex substrate. Binding studies revealed that the CBM binds to cellulose, but not to mannan. Studies of the enzyme/polysaccharide interaction in the active site cleft of the catalytic module of TrMan5A showed that a mutant of Arg 171 displayed activity in the same range as the wild-type enzyme toward polymeric substrates. However, the Arg171 mutant was impaired in hydrolysis of small substrates. Interestingly, this mutant also appears to have a more alkaline activity pH-optimum than the wild-type. The low or abolished activity observed with mutants of the predicted catalytic glutamates (Glu169 and Glu276) support their importance in hydrolysis. In addition to TrMan5A, the properties of a beta-mannanase (MeMan5A) from blue mussel and a beta-mannosidase (AnMan2A) from Aspergillus niger, were studied in this work. Investigations on the catalytic properties of the enzymes showed that all three enzymes are capable of degrading polymeric mannan. Furthermore, analysis by transmission electron microscopy revealed that TrMan5A and AnMan2A degrades highly crystalline mannan. Degradation of glucomannan and galactoglucomannan by several polysaccharide-degrading enzymes shows that these substrates can be hydrolysed by both mannoside- and glucoside-hydrolases. Furthermore, the results presented show that cellulases potentially are able to hydrolyse other components in the plant cell wall. Altogether, the results presented demonstrates the need to use complex substrates in order to reveal the mechanisms of plant polysaccharide degradation. In conclusion, this work has shown that the enzyme/polysaccharide interaction in the two modules of TrMan5A is important in determining the overall enzymatic activity and specificity.

KW - Metabolism

KW - Biochemistry

KW - hemicellulase

KW - beta-mannanase

KW - carbohydrate-binding modules

KW - Biokemi

KW - metabolism

M3 - Doctoral Thesis (compilation)

SN - 91-628-5203-5

PB - Dr Henrik Stålbrand, Getingevägen 60, Box 124, 221 00 LUND, Sweden,

ER -