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Discrimination among eight modified michaelis-menten kinetics models of cellulose hydrolysis with a large range of substrate/enzyme ratios

Inhibition by cellobiose

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Abstract

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: K m=3.8 mM, K ic=0.041 mM, k cat=2 h−1 (5.6×10−4 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.

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Abbreviations

E :

free enzyme

EI :

enzyme inhibitor complex

EII :

enzyme inhibitor complex for second inhibitor molecule

EIS :

enzyme substrate inhibitor complex

ES :

enzyme substrate complex (nonproductive)

Et :

total enzyme

f 0.95 :

point of Fp ApB (F distribution) curve with area 0.95 (to its left)

k cat :

catalytic constant (h−1)

K ic :

competitive inhibition constant (mM)

K ip :

parabolic inhibition constant (mM)

K is :

substrate inhibition constant (mM)

K iu :

uncompetitive inhibition constant (mM)

K m :

Michaelis constant (mM)

n :

experimental points

p A′ pB :

parameters

P :

product (µg/mL) (cellobiose)

P 0 :

initial product

P t :

product at time t (min)

R 2 :

determination coefficient

S :

substrate (%) (cellulose)

SSE :

sum of squares error

t :

time (min)

V max :

maximum velocity

w :

quotient used to test significance of improvement of different models that are interconvertible by addition or elimination of parameters in comparison with F value

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Authors and Affiliations

  1. Departamento de Engenharia Biológica e Ambiental, ICETA-CETAV, Universidade de Trás-os-Montes e Alto Douro, Apartado 1013, 5001-911, Vila Real, Portugal

    Rui M. F. Bezerra & Albino A. Dias

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  1. Rui M. F. Bezerra
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  2. Albino A. Dias
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Correspondence to Rui M. F. Bezerra.

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Bezerra, R.M.F., Dias, A.A. Discrimination among eight modified michaelis-menten kinetics models of cellulose hydrolysis with a large range of substrate/enzyme ratios. Appl Biochem Biotechnol 112, 173–184 (2004). https://doi.org/10.1385/ABAB:112:3:173

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  • DOI: https://doi.org/10.1385/ABAB:112:3:173

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