Discrimination among eight modified michaelis-menten kinetics models of cellulose hydrolysis with a large range of substrate/enzyme ratios: inhibition by cellobiose.
The kinetics of exoglucanase (Cel7A) from Trichoderma reesei was investigated in the presence of cellobiose and 24 different enzyme/Avicel ratios for 47 h, in order to establish which of the eight available kinetic models best explained the factors involved. The heterogeneous catalysis was studied and the kinetic parameters were estimated employing integrated forms of Michaelis-Menten equations through the use of nonlinear least squares. It was found that cellulose hydrolysis follows a model that takes into account competitive inhibition by cellobiose (final product) with the following parameters: Km = 3.8 mM, Kic = 0.041 mM, kcat = 2 h-1 (5.6 x 10-4 s-1). Other models, such as mixed type inhibition and those incorporating improvements concerning inhibition by substrate and parabolic inhibition, increased the modulation performance very slightly. The results support the hypothesis that nonproductive enzyme substrate complexes, parabolic inhibition, and enzyme inactivation (Selwyn test) are not the principal constraints in enzymatic cellulose hydrolysis. Under our conditions, the increment in hydrolysis was not significant for substrate/enzyme ratios <6.5.
Departamento de Engenharia Biológica e Ambiental, ICETA-CETAV, Universidade de Trás-os-Montes e Alto Douro, Apartado 1013, 5001-911 Vila Real, Portugal. bezerra@utad.pt
Rui M F
RM
Bezerra
Albino A
AA
Dias
D000465:Algorithms; D002266:Carboxymethylcellulose Sodium / Q000737:chemistry / Q000378:metabolism; D002475:Cellobiose / Q000737:chemistry* / Q000378:metabolism; D002482:Cellulose / Q000737:chemistry* / Q000378:metabolism; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000737:chemistry* / Q000378:metabolism; D003198:Computer Simulation; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism; D004795:Enzyme Stability; D005960:Glucosides / Q000737:chemistry / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D016018:Least-Squares Analysis; D008956:Models, Chemical; D014242:Trichoderma / Q000201:enzymology*
D004791:Enzyme Inhibitors; D005960:Glucosides; D002475:Cellobiose; C025193:4-nitrophenyl beta-D-glucoside; D002482:Cellulose; D043366:Cellulose 1,4-beta-Cellobiosidase; D002266:Carboxymethylcellulose Sodium