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

Hägglund, Per LU (2002)
Abstract
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... (More)
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. (Less)
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author
opponent
  • Professor Warren, Anthony, Dep of Microbiology and Immunology, University of British Columbia, 300-6174 University Blvd, Vancouver BC
organization
publishing date
type
Thesis
publication status
published
subject
keywords
Metabolism, Biochemistry, hemicellulase, beta-mannanase, carbohydrate-binding modules, Biokemi, metabolism
pages
174 pages
publisher
Dr Henrik Stålbrand, Getingevägen 60, Box 124, 221 00 LUND, Sweden,
defense location
Center of Chemistry and Chemical engineering, Hörsal C
defense date
2002-05-31 10:15
external identifiers
  • other:ISRN: LUNKDL/(NKBK-1074)/1-174/2002
ISBN
91-628-5203-5
language
English
LU publication?
yes
id
f48e132a-9725-45e9-9eb7-28d585e87014 (old id 464686)
date added to LUP
2007-10-14 15:16:26
date last changed
2016-09-19 08:45:07
@phdthesis{f48e132a-9725-45e9-9eb7-28d585e87014,
  abstract     = {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.<br/><br>
<br/><br>
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.<br/><br>
<br/><br>
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.},
  author       = {Hägglund, Per},
  isbn         = {91-628-5203-5},
  keyword      = {Metabolism,Biochemistry,hemicellulase,beta-mannanase,carbohydrate-binding modules,Biokemi,metabolism},
  language     = {eng},
  pages        = {174},
  publisher    = {Dr Henrik Stålbrand, Getingevägen 60, Box 124, 221 00 LUND, Sweden,},
  school       = {Lund University},
  title        = {Mannan-hydrolysis by hemicellulases: enzyme-polysaccharide interaction of a modular beta-mannanase},
  year         = {2002},
}