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Applied Biochemistry and Biotechnology

, Volume 134, Issue 1, pp 27–38 | Cite as

Simulaaneous ethanol and cellobiose inhibition of cellulose hydrolysis studied with integrated equations assuming constant or variable substrate concentration

  • Rui M. F. BezerraEmail author
  • Albino A. Dias
  • Irene Fraga
  • António Nazaré Pereira
Original Research Articles

Abstract

The integrated forms of the Michaelis-Menten equation assuming variable substrate (depletion) or constant substrate concentration were used to study the effect of the simultaneous presence of two exoglucanase Cel7A inhibitors (cellobiose and ethanol) on the kinetics of cellulose hydrolysis. The kinetic parameters obtained, assuming constant substrate (K m =21 mM, K ic =0.035 mM; K icl =1.5×1015mM; kcat=12 h−1) or assuming variable substrate (K m =16 mM, K ic =0.037 mM; K icl =5.8×1014 mM; kcat=9 h−1), showed a good similarity between these two alternative methodologies and pointed out that bothethanol and cellobiose are competitive inhibitors. Nevertheless, ethanol is a very weak inhibitor, as shown by the large value estimated for the kinetic constant K icl . In addition, assuming different concentrations of initial accessible substrate present in the reaction, both inhibition and velocity constants are at the same order of magnitude, which is consistent with the obtained values. The possibility of using this kind of methodology to determine kinetic constants in general kinetic studies is discussed, and several integrated equations of different Michaelis-Menten kinetic models are presented. Also examined is the possibility of determining inhibition constants without knowledge of the true accessible substrate concentration.

Index Entries

Cellulase kinetics ethanol inhibition exoglucanase Cel7A integrated Michaelis-Menten equations 

Nomenclature

A

ethanol

E

free enzyme

f0.95

point of Fpa, pb (F distribution) curve with area 0.95 (to its right)

I

all inhibitors

kcat

catalytic constant (h−1)

kic

competitive inhibition constant (mM) to cellobiose

Kicl

competitive inhibition constant (mM) to ethanol

Kiu

uncompetitive inhibition constant (mM) to cellobiose

Kiul

uncompetitive inhibition constant (mM) to ethanol

Km

Michaelis constant (mM)

n

experimental points

P

reaction product (cellobiose)

pA, pB

parameters

P0

initial product

Pt

product at time t (min)

S

substrate

t

time (min)

Vmax

maximum velocity

ω

quotient used to test significance of improvement of different models interconvertible by addition or elimination of parameters by comparison of F-value

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Copyright information

© Humana Press Inc 2006

Authors and Affiliations

  • Rui M. F. Bezerra
    • 1
    Email author
  • Albino A. Dias
    • 1
  • Irene Fraga
    • 1
  • António Nazaré Pereira
    • 2
  1. 1.CETAV, Departmento de Engenharia Biológica e AmbientalUniversidade de Trás-os-Montes e Alto DuoroVila RealPortugal
  2. 2.Departmento de Indústrias AlimentaresUniversidade de Trás-os-Montes e Alto DouroVila RealPortugal

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