Processive action of cellobiohydrolase Cel7A from Trichoderma reesei is revealed as 'burst' kinetics on fluorescent polymeric model substrates.
Reaction conditions for the reducing-end-specific derivatization of cellulose substrates with the fluorogenic compound, anthranilic acid, have been established. Hydrolysis of fluorescence-labelled celluloses by cellobiohydrolase Cel7A from Trichoderma reesei was consistent with the active-site titration kinetics (burst kinetics), which allowed the quantification of the processivity of the enzyme. The processivity values of 88+/-10, 42+/-10 and 34+/-2.0 cellobiose units were found for Cel7A acting on labelled bacterial cellulose, bacterial microcrystalline cellulose and endoglucanase-pretreated bacterial cellulose respectively. The anthranilic acid derivatization also provides an alternative means for estimating the average degree of polymerization of cellulose and, furthermore, allows the quantitative monitoring of the production of reducing end groups on solid cellulose on hydrolysis by cellulases. Hydrolysis of bacterial cellulose by cellulases from T. reesei revealed that, by contrast with endoglucanase Cel5A, neither cellobiohydrolases Cel7A nor Cel6A produced detectable amounts of new reducing end groups on residual cellulose.
Kipper
Kalle
K
Institute of Molecular and Cell Biology, University of Tartu, Riia 23, Tartu 51010, Estonia.
Väljamäe
Priit
P
Johansson
Gunnar
G
D001665:Binding Sites; D002240:Carbohydrate Sequence; D002482:Cellulose / Q000737:chemistry / Q000378:metabolism; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D005465:Fluorocarbon Polymers / Q000737:chemistry / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism; D008962:Models, Theoretical; D011108:Polymers / Q000737:chemistry / Q000378:metabolism; D017186:Titrimetry / Q000379:methods / Q000639:trends; D014242:Trichoderma / Q000201:enzymology*
D005456:Fluorescent Dyes; D005465:Fluorocarbon Polymers; D005656:Fungal Proteins; D011108:Polymers; D002482:Cellulose; D043366:Cellulose 1,4-beta-Cellobiosidase