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Journal of Chemical Engineering of Japan, Vol. 33, No. 6, pp. 886890, 2000
Introduction
Heterogeneous enzymatic catalysis includes:
a. mass transfer of substrate to the external surface of
catalyst pellet,
b. diffusion of substrate from the external surface to
domain of enzyme,
c. adsorption of substrate,
d. enzymatic reaction,
e. desorption of product,
f. diffusion of product back to the surface of catalyst,
and
g. mass transfer of product to the bulk of fluid stream.
Steps (a) and (g) are external, while (b) and (f) are
internal mass transfer effects. These are essentially
different from homogeneous kinetics. The overall rate
of an immobilized enzymatic process is always influenced by these mass transfer effects. These effects may
become even rate limiting steps.
The rate of external mass transfer of component
A, is given by
NA = KLa(Cb Cs)
(1)
886
(2 )
(3)
Fig. 1
erature. Effective diffusivity measurements of a solute within a porous particle, are made using a chromatographic method involving the response to a pulse
input of solute to a column packed with the porous
particles of interest.
The effect of mass transfer on reaction rate, is indicated by the effectiveness factor, , which is a function of Thiele modulus, . in turn is a function of
characteristic length of the catalyst pellet, L. Thiele
modulus is given by Wadiak and Carbonell (1975), as:
= L[r(Cs)/Deff Cs]1/2
(4)
Experimental
Fig. 2
80C and held there for one hour. The pH of the solution is checked to ensure that it is at 7.0, otherwise
adjusted using dilute acetic acid.
1.2 Preparation of enzyme in buffer solution
A solution of glucoamylase is prepared by taking
a weighed quantity in the sodium acetate buffer. The
solid insoluble material is filtered. The filtrate contains
the protein that catalyses the reaction, starch to glucose. The protein content in the enzyme is determined
by Lowrys method as reported by Anderson and
Morrison (1925). It is found that 1000 mg of enzyme
contains 63.7 mg of protein. All calculations in this
work are based on the amount of pure glucoamylase
taken for conversion.
1.3 Method of immobilization and optimization of
parameters
Cyanuric chloride reacts rapidly with the free hydroxyl groups present in cellulose. The dichloro-striazyl cellulose may then be reacted with the enzyme
and finally the third chloro group substituted with
amine or other chemical group. A schematic representation is given in Fig. 1. 0.5 g of 2,4,6-trichloro-s-triazine is dissolved in 50 cc of 1:1 acetone water mixture at a temperature of 40C. To this is added 10 g of
cellulose pellets and reacted under stirring for 2 minutes. A mixture of 12 cc of 1.0 mol/liter hydrochloric
acd and 8 cc of sodium carbonate solution is added
and stirred for further 5 minutes. The reaction mixture
is filtered and the cellulose-di-chloro-s-triazyl obtained
is washed first with distilled water and then with acetate buffer. The activated pellets are suspended in
glucoamylase solution. The mixture is incubated at 4C
for 24 hours. The enzyme-cellulose complex is then
washed with acetate buffer until no protein is detectable in the washings. The effects of time, temperature
and pH of activation are studied to get the optimum
values as, 4 minutes, 40C and pH of 5.6. The results
indicate that the enzyme requires about 24 hours of
incubation time to completely saturate all the active
sites on the activated cellulose complex.
887
Fig. 3
Modeling
Fig. 4
(5)
( )) = pC1, r / t
(6 )
C1,r/r = 0 at r = 0
(7)
(8)
C1,r = 0 at t = 0, 0 r R
(9)
By introducing the fractional conversion, X and suitable dimensionless groups, equations can be written
in the dimensionless form, for steady state.
(1/Pe)(2X1/Y2 X1/ Y) + U(X1, 1 X 1) = 0
(10)
(11)
Fig. 5
Fig. 6
Fig. 7
netic constants and assuming no external mass transfer resistance, theoretical effectiveness charts are prepared for various kinetic models.
Comparison of effectiveness factors for different
kinetic models is given in Fig. 5. The substrate inhibition model always experiences higher effectiveness
factors than other models. Under certain circumstances,
the effectiveness factor may even exceed unity. Because the enzyme molecules are protected by a diffusion barrier, substrate plus product inhibition offsets
the effect of diffusion resistance on substrate inhibition and in turn gives a lower effectiveness factor. The
product inhibition model depletes the effectiveness
factor further.
The effect of initial substrate concentration on
effectiveness factor and Thiele modulus curves for
substrate inhibition model are plotted. A comparison
for various initial substrate concentrations, is given in
Fig. 6. Higher substrate concentrations give higher
effectiveness factors. The curves approach first order
limit at low substrate concentration and zero order limit
at high substrate concentration.
Figure 7 simulates the real reaction environment
of a fixed bed reactor. For a given substrate concentra889
tion and Thiele modulus, and enzyme catalyst with inhibition kinetics, the highest effectiveness factor is at
the entrance of the reactor. The effectiveness factor
drops along the reactor due to decrease of substrate
concentration and increase of product concentration.
Conclusion
The reaction velocity is proportional to the enzyme
concentration in both the cases of free and immobilized enzyme.
The homogeneous kinetics of glucoamylase is best
represented by a modified substrate inhibition model
incorporated with an adjustable parameter. The equation is, V = Vmax S(A + BS)/(KM + S + 2/KI). The kinetic
constants are V max = 6.54 mol/min/mg.protein, KM =
6.57 mg/ml, KI = 310.34 mg/ml, A = 5.18 and B =
0.19 ml/mg. The temperature stability of immobilized
glucoamylase is superior to that of soluble enzyme.
Free glucoamylase lost all its activity at 70C upon
incubation for one hour whereas immobilized enzyme
retained 83% of initial activity.
Immobilized enzyme is stable in lower pH ranges.
Enzyme progressively gets denatured as pH is raised.
The diffusion and reaction model which includes
external and internal mass transfer effects and axial
dispersion predicts quite well the performance of the
fixed bed reactor. By solving the simultaneous diffusion and reaction model numerically, best kinetic constants are obtained as Vmax = 5.3 mol/min/mg.protein,
KM = 4.47 mg/ml, KI = 211.03 mg/ml, A = 5.18 and B
= 0.19 ml/mg.
The theoretical analysis of effectiveness factor in
this study proved useful for judging the significance
of diffusion resistance in the overall rate process of
biological systems.
Nomenclature
A
= cross-sectional area of fixed bed reactor
[cm 2]
a
= external area of catalyst pellet per unit weigh of
mass
[cm 2/g]
B1
= Biot number K LR/D eff
Cb
= concentration of diffusing component in bulk solution
[mmol]
C0
= initial substrate concentration at entrance of fixed
bed reactor
[mmol]
C1
= substrate concentration in liquid phase in fixed bed
reactor
[mmol]
890
C 1,r
D AB
D eff
Dp
EL
J A*
KI
KL
KP
KS
L
NA
Pe
R
r
T
t
V0
X1
X1,
Y
Z
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
D S/D
bed void fraction
void fraction of pellet
r/R
pellet density
tortuosity factor
Thiele modulus
Literature Cited
Anderson, T. W. and K. D. Morrison; Estimation of Protein by
Lowrys Method, J. Biol. Chem ., LXV, 393398 (1925)
Bollmeier, J. P. and S. Middleman; Inactive Immobilized Enzyme
Carrier for Measurement of Ef fective Diffusivity, Biotech.
Bioeng., 21, 23032307 (1979)
Satterfield, C. N.; Mass Transfer in Heterogeneous Catalysis, MIT
Press, Cambridge, USA (1970)
Sirotti, D. A. and A. Emery; Diffusivity of Glucose and Cellobiose into an Immobilized Enzyme Carrier , Biotech. Bioeng.,
25, 17731777 (1983)
Smith, J. M.; Chemical Engineering Kinetics, 3rd ed., McGraw Hill,
New York, USA (1981)
Toda, K. and M. Shoda; Measurement of Ef fective Diffusivity of
Substrate in the Absence of Immobilized Enzyme, Biotech.
Bioeng., 17, 481485 (1975)
Wadiak, D. T. and R. C. Carbonell; Ef fect of Mass Transfer on
Reaction Rate, Biotech. Bioeng., 17, 176182 (1975)
Wen, C. Y. and L. T. Fan; Models for Flow Systems in Chemical
Reactors, Mercel Dekker, New York, USA (1975)
Wilson, E. J. and C. J. Geankopolis; J D Factor for External Mass
Transfer in Heterogeneous Chemical Reactors, Ind. Eng.
Chem., 5, 916 (1966)