Mass-Transfer Limitations for
Immobilized Enzyme-Catalyzed Kinetic
Resolution of Racemate in a
Fixed-Bed Reactor
Guo-Hua Xiu,1,* Lei Jiang,1 Ping Li2
1
Institute of Chemistry, Chinese Academy of Sciences,
Beijing 100080, P.R. China
2
Department of Chemical Engineering, Shenyang Institute of Chemical
Technology, Shenyang, P.R. China
Received 4 August 2000; accepted 28 December 2000
Abstract: A mathematical model has been developed for
immobilized enzyme-catalyzed kinetic resolution of
racemate in a fixed-bed reactor in which the enzyme-
catalyzed reaction (the irreversible uni–uni competitive
Michaelis–Menten kinetics is chosen as an example) was
coupled with intraparticle diffusion, external mass trans-
fer, and axial dispersion. The effects of mass-transfer
limitations, competitive inhibition of substrates, deacti-
vation on the enzyme effective enantioselectivity, and
the optical purity and yield of the desired product are
examined quantitatively over a wide range of parameters
using the orthogonal collocation method. For a first-
order reaction, an analytical solution is derived from the
mathematical model for slab-, cylindrical-, and spherical-
enzyme supports. Based on the analytical solution for the
steady-state resolution process, a new concise formula-
tion is presented to predict quantitatively the mass-
transfer limitations on enzyme effective enantioselectiv-
ity and optical purity and yield of the desired product for
a continuous steady-state kinetic resolution process in a
fixed-bed reactor. © 2001 John Wiley & Sons, Inc. Biotechnol
Bioeng 74: 29–39, 2001.
Keywords: immobilized enzyme; kinetic resolution;
mass-transfer limitations; fixed-bed reactor; analytical
solution; orthogonal collocation method
INTRODUCTION
Enzyme-catalyzed resolution of racemate has stimulated
much interest with regard to production of drugs and agro-
chemicals in the last decade. Lipase, a relatively inexpen-
sive enzyme, has been widely employed as a biocatalyst for
such reactions. This enzyme is usually immobilized onto
porous support material to improve its activity and thermo-
dynamic stability and to separate easily from the mixture for
reuse. For the immobilized enzyme-catalyzed kinetic reso-
Correspondence to: G. H. Xiu
* Present address: LSRE Laboratory of Separation and Reaction Engi-
neering, Faculty of Engineering, University of Porto, Rua Dr. Roberto
Frias s/n, 4200-465 Porto, Portugal. Telephone: 351-22-5081618; fax: 351-
22-5081674; e-mail: ghxiu@hotmail.com.
Contract grant sponsor: China Postdoctoral Science Foundation.
© 2001 John Wiley & Sons, Inc.
lution process, mass-transfer limitations may exist, espe-
cially for a more reactive system. If diffusion transfer limi-
tation occurs in the resolution process, the enzyme effective
enantioselectivity will be lowered to a certain extent, and
will result in a decrease in the optical purity and yield of the
desired product. Matson and Lopez (1990) first described
this phenomenon theoretically for a membrane bioreactor.
Fixed-bed reactors have traditionally been used for most
large-scale catalytic reactors because of their efficiency, low
cost, and ease of construction, operation, and maintenance.
Efforts have been made to study the reaction kinetics, mass-
transfer mechanism, and mathematical model of fixed-bed
reactors. Most researchers have focused on steady-state op-
erations (Bailey and Ollis, 1986; Froment and Bischoff,
1990, Marrazzo et al., 1975; Smith, 1981). Recently, inter-
est in the mathematical modeling of unsteady-state opera-
tion has increased significantly because of its importance in
fixed-bed reactor design and process control (Zheng and
Gu, 1996). Therefore, kinetic resolution of racemate by im-
mobilized enzyme in a fixed-bed reactor is a topic for in-
vestigation.
In terms of design of immobilized enzyme fixed-bed re-
actors, three major processes should be considered in order
to achieve maximal efficiency and high-quality production
(Indlekofer et al., 1996): (1) transport of the reactant (prod-
uct) molecules form (to) the bulk phase to (from) the vicin-
ity of the immobilized enzyme, in which axial dispersion,
external mass transfer, and intraparticle diffusion should be
involved; (2) stereoselective transformation of the reactant
molecules by chemical reaction catalyzed by immobilized
enzyme; and (3) enzyme deactivation. For the second and
third processes, many fundamental experiments have been
carried out to determine the reaction mechanism, deactiva-
tion rate of enzyme, and enzyme enantioselectivity in batch
and fixed-bed reactors (Garcia et al., 2000; Indlekofer et al.,
1993; Rakels et al., 1994; Straathof et al., 1992; Tsai et al.,
1997). However, quantitative investigation about mass-
transfer limitations and the characteristics of the resolution
process in a fixed-bed reactor has been rare.
 For an intrinsic reaction catalyzed by an enzyme, Chen et
al. (1982) was the first to find a quantitative relationship
between enantiomeric excess, racemate conversion, and in-
trinsic enantiomeric ratio. Although the enzyme effective
enantioselectivity for an immobilized enzyme depends on
both the intrinsic enantiomeric ratio and effectiveness fac-
activity. Consider a system with zero initial racemic con-
centrations in which a racemic mixture step is introduced to
the inlet of the reactor at time zero. For the reaction/mass-
transfer controlling process, the material balance equation
of an individual enantiomer, j (j ⳱ R-, S-), in the porous
support matrix is then given by:

冉 冊 冉 冊
tors (Indlekofer et al., 1996; Matson and Lopez, 1990), it
⭸cj 1 ⭸ ⭸cj t
must also evaluate the impact of mass-transfer restrictions ␧p = Deff p rp − ␳p XE exp − vj (1)
on enzyme effective enantioselectivity for the immobilized ⭸t r ⭸r ⭸r t 1
enzyme-catalyzed kinetic resolution process.
where exp(−t/t1) is the rate of enzyme deactivation, t1 is the
Matson and Lopez (1990) reported a quantitative analysis
time constant of enzyme deactivation, vj is the special re-
taking into account the simultaneous reaction/intraparticle
action rate of enantiomers, and p is the shape factor of the
diffusion processes pertinent to competitively reacting en-
porous support (p ⳱ 2 for sphere, p ⳱ 1 for cylinder, and
antiomers in an immobilized enzyme system for first-order
p ⳱ 0 for slab).
reaction. Indlekofer et al. (1996) numerically investigated
The pore diffusion equation [Eq. (1)] is coupled with the
the effectiveness factors in single catalyst pellets at steady
external bulk concentrations in the stream flowing through
state for competitive Michaelis–Menten kinetics and mass-
the fixed-bed reactor through the following boundary con-
transfer limitations on the optical purity and yield of the
dition:
desired product for a fixed-bed reactor at specified operat-
ing conditions.
In a previous study (Xiu et al., 2000), the mass-transfer Deff 冉 冊
⭸cj
⭸r r =R
= kf 关Cj − 共cj兲r =R兴 (2)
limitations for an immobilized enzyme-catalyzed kinetic
resolution process in a batch reactor was studied. In this The second boundary condition is given by the symmetry
work, however, the objectives are: condition at the center of the porous support:
1. To analyze quantitatively the mass-transfer limitations
(taking into account the axial dispersion, external mass- 冉 冊⭸cj
⭸r r =0
=0 (3)
transfer resistance, and intraparticle diffusion resistance)
on the enzyme effective enantioselectivity and the opti- The mass balance equation for the substrates in the fluid
cal purity and yield of the desired product for the im- phase is:
mobilized enzyme-catalyzed kinetic resolution process
⭸Cj ⭸2Cj u0 ⭸Cj 共1 − ␧b兲 共1 + p兲
in a fixed-bed reactor using a numerical method (for = Dax 2 − − kf 关Cj − 共cj兲r =R兴
Michaelis–Menten kinetics) and analytical solution (for ⭸t ⭸Z ␧b ⭸Z ␧b R
first-order reaction) over a wide range of model param- (4)
eters.
with the Danckwerts boundary conditions at the reactor inlet
2. Based on an analytical solution in an extreme situation,
and outlet:
to present a concise formula for predicting the effects of
mass-transfer limitations, inhibition of substrates, and en-
zyme deactivation on enzyme effective enantioselectiv- Z = 0, Dax 冉 冊 ⭸Cj
⭸Z Z=0
=−
u0
关C − 共Cj兲Z=0兴
␧b j0
(5)

冉 冊
ity and optical purity and yield of the desired product for
irreversible uni–uni competitive Michaelis–Menten and ⭸Cj
Z = L, =0 (6)
other complex reaction kinetics. ⭸Z Z=L

The initial conditions for Eqs. (1)–(6) are:

MATHEMATICAL FORMULATION
t = 0, cj = 0, Cj = 0 (7)
Consider that AS and AR are fast and slow-reacting enantio- It is further assumed that the special reaction rate, vj,
mers that compete for the same site on the enzyme (Chen et derives from the irreversible uni–uni competitive Michae-
al., 1982). For the following reaction, we assume this to be lis–Menten kinetics (Scheme 1). For an individual enantio-
virtually irreversible and that there is no product inhibition: mer, j (j ⳱ R-, S-), this can be expressed as:
Vmj Ⲑ Kmj
Enz + Aj → Enz + Bj
Scheme 1.
共j = R, S兲 vj = 冉 冊
Vmj
Kmj
1+
cj
cR
+
cS
(8)

The other assumptions employed in this work for the
heterogeneous kinetic resolution process are isothermal,
constant effective diffusivity of the substrate species inside
the porous support, and homogeneously distributed enzyme
KmR KmS
The enantioselectivity or enantiomeric ratio, E, of the
enzyme is defined as the ratio of the specificity constants
(Vm/Km) of the enzyme for the R- and S-enantiomer

30 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 74, NO. 1, JULY 5, 2001

(Straathof and Jongejan, 1997). The enantiomeric ratio for
the S-enantiomer preferentially selective process can be de- 冉 冊
⭸xj
⭸␰ ␰=1
= Bi关yj − 共xj兲␰=1兴 (11)
fined as:

E=
VmS Ⲑ KmS
(9)
冉 冊
⭸xj
⭸␰ ␰=0
=0 (12)
VmR Ⲑ KmR
⭸yj 1 ⭸2yj ⭸yj
The number of parameters used in this mathematical = − − 共1 + p兲St关yj − 共xj兲␰=1兴 (13)
⭸␶ Pe ⭸␨2 ⭸␨
model can be reduced by nondimensionalization. For our
work, the following dimensionless variables and groups are
introduced: ␨ = 0, 冉 冊
⭸yj
⭸␨ ␨=0
= −Pe关1 − 共yj兲␨=0兴 (14)

xj =
cj
,y =
Cj
Cj0 j Cj0
dimensionless concentrations ␨ = 1, 冉 冊
⭸yj
⭸␨ ␨=1
=0 (15)

Z xj = 0, yj = 0, ␶ = 0 (16)
␨= dimensionless axial distance
L
where:
r
␰= dimensionless radial distance xj
R Rj = (17)
xR xS
1+ +
u0t ␤R ␤S
␶= dimensionless contact time
L␧b
Owing to the nonlinearity of Rj, an analytical solution
kf R could not be obtained from Eqs. (10)–(17). The orthogonal
Bi = Biot number collocation method is used to transform the actual system of
Deff
Eqs. (10)–(17) into a system of ordinary differential equa-
u0 L Peclet number based on reactor tions of initial-value type. These equations could then be
Pe = integrated into the time domain using Gear’s stiff variable
Dax␧b length
step integration routine to calculate the changes of exit con-

冉 冊 ␶ centrations of substrates with time (Li et al., 2000).

␾S = ␾S共0兲exp − Thiele modulus for S-enantiomer In the extreme situation (for both the boundary conditions
2␶1
and the expression of Rj), there exists an analytical solution

冑
for Eqs. (10)–(17). In the following sections, we first derive
VmS␳pXE an analytical solution, and present a more general expres-
␾S共0兲 = R ␾S in the case of ␶1 → ⬁ sion for the exit concentration at steady state from Danck-
KmSDeff
werts boundary conditions and a first-order reaction as-
␾S sumption based on the work of Marrazzo et al. (1975). We
␾R = Thiele modulus for R-enantiomer then propose a formula. The general formula and its derived
公E equations are helpful in calculating the enzyme effective
␾S共0兲 enantioselectivity and for predicting the optical purity and
␾R共0兲 = ␾R in the case of ␶1 → ⬁ yield of the desired product for an immobilized enzyme-
公E catalyzed kinetic resolution process in a fixed-bed reactor.
Kmj dimensionless Michaelis–Menten
␤j =
Cj0 constant Analytical Solution for First-Order Reaction in a
Fixed-Bed Reactor
␧bDeff L
Dr = bed-length parameter For first-order reaction kinetics, Eq. (17) becomes:
␧PR2u0
Rj = xj (18)
BiDr␧P共1 − ␧b兲
St = Stanton number This situation is valid only for the enzymatic kinetics with
␧b
xj Ⰶ ␤j (or, in dimension form, cj Ⰶ Kmj), or for dilute
substrate concentration leading to xj Ⰶ ␤j.
Then, by writing in dimensionless form, Eqs. (1)–(8) be-
For the purpose of deriving an analytical solution from
come:
the equations just given, the following assumptions should
1 ⭸xj 1 ⭸ p ⭸xj
=
Dr ⭸␶ ␰p ⭸␰
␰
⭸␰ 冉 冊
− ␾2j Rj (10)
be made:
1. ␶1 → ⬁; that is, the enzyme activity is time-independent.

XIU, JIANG, AND LI: MASS TRANSFER LIMITATIONS IN A FIXED-BED REACTOR 31

2. The Danckwerts boundary conditions are replaced by the BiEj
following equations: hj = (27)
D2j + E2j

冑公
yj共0,␶兲 = 1 (19)
关␾j共0兲兴4 + ␤2 Ⲑ Dr2 + 关␾j共0兲兴2
yj共␨ → ⬁,␶兲 = 0 (20) dj = (28)
2

冑公
which were used by Rosen (1952).
Raghavan and Ruthven (1983) pointed out that the dif- 关␾j共0兲兴4 + ␤2 Ⲑ Dr2 − 关␾j共0兲兴2
ference in the Danckwerts and Rosen boundary conditions ej = (29)
2
leads to a significant difference in the solutions only when
both the axial Peclet number, Pe, and the bed-length pa- For expression of ␭j , Dj , and Ej , see Table I.
rameter, Dr, are small. From a practical point of view, it is For high values of Pe (DL → 0), we can disregard all
not possible to make both of them small. Therefore, the terms of the order 1/Pe, and then Eq. (21) becomes:
solution derived based on Eqs. (19) and (20) can represent
兰 exp关− 共1 + p兲Stq ␨兴sin兵␤␶ − ␨关␤ +
yj⬁ 1 ⬁
this based on Danckwerts boundary conditions. yj = +
Because both enantiomers are converted according to 2 ␲ 0
j

first-order kinetics, the conversion of one enantiomer is in- d␤

共1 + p兲Sthj兴其 共Pe → ⬁兲 (30)
dependent from the conversion of the other. Similar to the ␤
work of Xiu and Li (1999), the following time-domain so-
lution of yj is obtained based on Rosen’s boundary condi- For the particular case:

冋 冉 冊册
tions: Bi

冉 冑公 冊
yj⬁ = exp − 共1 + p兲St␨ 1 − 共Pe → ⬁兲
Bi + ␭j
a2j + b2j + aj
兰 exp
yj⬁ 1 ⬁ Pe␨
yj = + −␨ sin (31)
␲

冉 冑公 冊
2 0 2 2
The analytical solution of Eqs. (21)–(29) has a general
a2j + b2j − aj d␤ form that takes into account the first-order reaction from the
␤␶ − ␨ (21)
2 ␤ work of Rasmuson and Neretnieks (1980).

冋冉 冊册
The exit concentrations of substrates at steady state
Pe (yj⬁)␨⳱1 are equal to the zeroth moment of the unreacted
yj⬁ = exp − ␦j ␨ (22)
2 fractions of substrates (Suzuki and Smith, 1971). Fortu-

冑
nately, we can derive a more general expression of (yj⬁)␨⳱1
␦j =
Pe2
4
+ 共1 + p兲PeSt 1 − 冉Bi
␭j + Bi 冊 (23)
from the zeroth moment based on the Danckwerts boundary
conditions. The final result is given by:

冋 Pe
册 1 Pe
冉 冊
冉 冊
aj = Pe + 共1 + p兲Stqj (24) 共yj⬁ 兲␨=1 = exp
4 Pe ␦j 2
+ sinh ␦j + cosh ␦j
4␦j Pe
bj = Pe关␤ + 共1 + p兲Sthj兴 (25) (32)
BiDj It should be noted that Marrazzo et al. (1975) derived Eq.
qj = 1 − (26)
D2j + E2j (32) for p ⳱ 2 with a dimension form.

Table I. Expressions of ␭j, Dj, and Ej (j ⳱ R-, S-).

p ␭j Dj Ej

2 ␾j共0兲coth␾j共0兲 − 1 dj sinh 2dj + ej sin 2ej ej sinh 2dj − dj sin 2ej

− 1 + Bi
cosh 2dj − cos 2ej cosh 2dj − cos 2ej

1a I1关␾j共0兲兴 ⬁
共d2j − ej2兲共␥n + dj2 − e2j 兲 + 4dj2e2j
⬁

兺 兺 共␥
4␥ndjej
I0关␾j共0兲兴 2 + Bi
n=1 共 ␥n + dj2 − ej2兲2 + 4d2j ej2 n=1 n + dj − ej 兲 + 4dj ej
2 2 2 2 2

0 ␾j共0兲tanh␾j共0兲 dj sinh 2dj − ej sin 2ej ej sinh 2dj + dj sin 2ej

+ Bi
cosh 2dj + cos 2ej cosh 2dj + cos 2ej

a
I1[␾j(0)]/I0[␾j(0)] ⳱ ⌺⬁n⳱1 2␾j(0)/(␥n + [␾j(0)]2) (Xiu and Li, 1999), in which I0 and I1 are the
modified Bessel functions of zero and first orders, and √␥n are the roots of Bessel functions of zero
order.

32 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 74, NO. 1, JULY 5, 2001

RESULTS AND DISCUSSION
Typical Time Courses of Exit Concentrations and
Relationship Among Dr, the Racemate
Conversion, ␩, and Excess of Remaining
Substrate, eeS (or Product Excess eeP)
Figure 1 presents typical courses of exit concentrations, yj,
for the immobilized enzyme-catalyzed kinetic resolution
process in a fixed-bed reactor for the irreversible uni–uni
competitive Michaelis–Menten kinetics (first-order reaction
is included), with and without enzyme deactivation at E ⳱
20, Bi ⳱ 10, Pe ⳱ 50, Dr ⳱ 1.4, and [␾S(0)]2 ⳱ 10, under
the following conditions: p ⳱ 2, ␨ ⳱ 1, ␧b ⳱ 0.5, and ␧p
⳱ 0.5. For the first-order reaction without enzyme deacti-
vation (␶1 ⳱ ⬁), the curves are also calculated by the ana-
lytical solutions [Eqs. (21)–(29)], as shown in Figure 1
(open circles). The integrand of Eq. (21) is the product of an
exponential decaying function and a periodic sine function;
the integration is performed over each half-period of the
sine wave that was developed by Rasmuson (1985). The
curves calculated by the numerical method with Danckwerts
boundary conditions are in agreement with those of Eqs.
(21)–(29) for a first-order reaction; thus, the accuracy of the
numerical method is tested by the analytical solution.
As evident from Figure 1, the whole time courses of the
exit concentrations can be approximately divided into two
parts: (i) the dynamic zone and (ii) the (pseudo-)steady-state
zone. At the dynamic zone, the exit concentrations increase
sharply with time, then the (pseudo-)steady state is reached
where the time courses of the exit concentrations do not
change (␶1 ⳱ ⬁) or change slowly (␶1 ⳱ 10; the enzyme
deactivation is a rather slow process when compared to the
mean residence time of substrates in the reactor). Usually, a
continuous resolution process would be carried out at the
(pseudo-)steady-state zone.
As shown in Figure 1, the exit concentrations of the R-
and S-enantiomer increase with time at pseudo-steady state
Figure 1. Typical time courses of exit concentration y by orthogonal
collocation method at E ⳱ 20, Bi ⳱ 10, Pe ⳱ 50, Dr ⳱ 1.4, and [␾S(0)]2
⳱ 10. Open circles: analytical solution of Eqs. (21)–(29); dashed lines:
first-order reaction; solid lines: Michaelis–Menten kinetics.
XIU, JIANG, AND LI: MASS TRANSFER LIMITATIONS IN A FIXED-BED REACTOR
for ␶1 ⳱ 10. The inhibition of substrates decreases the
reaction rate for Michaelis–Menten kinetics compared with
that of the first-order reaction, leading to lower conversion
of the substrates than those of the first-order reaction.
Generally, optical purity (expressed as the enantiomeric
excess of the remaining substrate, eeS, or the product excess,
eeP) is recognized as the target parameter in the resolution
process. The enantiomeric excess of the remaining substrate
(eeS), and the product excess (eeP) is calculated by:
XS − XR
eeS = (33)
2 − XS − XR
XS − XR
eeP = (34)
XS + XR
in which X j represents the conversion of R- and S-
enantiomers at the exit of the fixed-bed reactor. At the
(pseudo-)steady state, Xj is defined as:
Xj = 1 − 共yj⬁ 兲␨=1 (35)
The conversion of racemate is:
XS + XR
␩= (36)
2
Figure 2 demonstrates the relationship between Dr, ␩,
and eeS (or eeP) for shape factor p ⳱ 2, 1, and 0, and ␨ ⳱
1, ␧b ⳱ 0.5, and ␧p ⳱ 0.5 at the steady state for the first-
order reaction. The racemate conversion, ␩, and the enan-
tiomeric excess of the remaining substrate, eeS, increase
with increasing Dr, whereas the product excess, eeP, de-
creases with increasing Dr. The bed-length parameter, Dr, is
the ratio of the residence time of the substrates in the reactor
to the intraparticle diffusion time of the substrates in the
porous support. It is a major adjustable parameter to decide
the racemate conversion ␩ whereas the other operating con-
ditions should be specified. For the specified levels of op-
tical purity of the desired product, there always exists an
optimal value of Dr for which the largest yield of racemate
Figure 2. Effects of Dr and shape factor, p, on eeP, eeS, and ␩ at E ⳱ 10,
Bi ⳱ 10, Pe ⳱ 100, and [␾S(0)]2 ⳱ 1 for a first-order reaction at the
steady state. Solid lines: p ⳱ 2; dashed lines: p ⳱ 1; dotted lines: p ⳱ 0.
33
can be obtained. If the E value is given, the optimal oper-
ating resolution process indicates that the more reactive S-
enantiomer is almost completely converted, whereas most
of the less reactive R-enantiomer remains at the exit of the
fixed-bed reactor. This process can be initiated by adjusting
the bed-length parameter, Dr.
The shape factor, p, has a significant influence on ␩, eeS,
and eeP, as shown in Figure 2. In the following subsections
we focus on the case of p ⳱ 2; other conditions are speci-
fied at ␨ ⳱ 1, ␧b ⳱ 0.5, and ␧p ⳱ 0.5.
New Solutions to Predict Enzyme Effective
Enantioselectivity, Eeff, Optical Purity, and Yield
of Desired Product for First-Order Reaction
To predict the optical purity and yield of the desired product
in a racemate kinetic resolution process, Chen et al. (1982)
first derived the following equation that quantitatively de-
scribes the relationship among E, ␩, and eeS (or eeP) for the
homogeneous kinetic resolution process with irreversible
uni–uni competitive Michaelis–Menten kinetics in a batch
reactor:
ln关共1 − ␩兲共1 − eeS兲兴
=E 共for intrinsic reaction兲
ln关共1 − ␩兲共1 + eeS兲兴
(37a)
and:
ln关1 − ␩共1 + eeP兲兴
=E 共for intrinsic reaction兲
ln关1 − ␩共1 − eeP兲兴
The equation just expressed has the same form as Eq.
(37); therefore, Eq. (37) is also suitable for describing the
intrinsic enzyme-catalyzed kinetic resolution process in a
fixed-bed reactor.
The enantiomeric ratio, E, is generally accepted as a pa-
rameter for quantification of the enantioselectivity of the
enzyme. For an immobilized enzyme-catalyzed kinetic
resolution process in a fixed-bed reactor, the mass-transfer
resistances limit the reaction rate more extensively for the
more reactive S-enantiomer than for the less reactive R-
enantiomer, resulting in an overall decrease in effective ste-
reoselectivity of the enzyme. Extensive analogous expres-
sions can be developed for an immobilized enzyme-
catalyzed kinetic resolution process. Similar to Eq. (40), we
have:
ln yS ln关共1 − ␩兲共1 − eeS兲兴
= = Eeff (41a)
ln yR ln关共1 − ␩兲共1 + eeS兲兴
ln yS ln关1 − ␩共1 + eep兲兴
= = Eeff (41b)
ln yR ln关1 − ␩共1 − eep兲兴
where Eeff is the effective enantiomeric ratio (or the enzyme
effective enantioselectivity), as proposed by Matson and
Lopez (1990).
Matson and Lopez (1990) extended the work of Chen et
al. (1982) to a membrane bioreactor for a first-order reac-
tion, and found the quantitative formula for Eeff in which
␾j(0) is involved:

When the mass-transfer limitations in the kinetic resolu-

(37b)

公
冋 1
−
1
tanh␾S共0兲 ␾S共0兲 册
冋 册
Eeff = E 共for p = 2兲
tion process can be neglected, based on Eqs. (1)–(8), at 1 1
−
(pseudo-)steady state the mass balance equation for a fixed- tanh␾R共0兲 ␾R共0兲
bed reactor with ideal plug flow can be written as: (42)

−
dyj
d␨
= 共1 − ␧b兲
Vmj␳pXEL
Kmju0 冉 冊
exp −
t
t1 j
R
Evidently, from Eq. (42), if ␾j(0) → 0, then Eeff will de-
generate into E.
共for intrinsic reaction in a fixed-bed reactor) For immobilized enzyme-catalyzed kinetic resolution of
(38) racemate in a batch reactor, Xiu et al. (2000) derived the
following expression for Eeff:
Therefore, we have:
dyS RS yS ␩GS
=E =E (39) Eeff = E (43)
dyR RR yR ␩GR

Considering yS0 ⳱ yR0 ⳱ 1 for a racemic mixture, integra- in which the global effectiveness factor ␩Gj is (Goto et al.,
tion of Eq. (39) yields: 1990):

冋 册冋 册
ln yS ln关共1 − ␩兲共1 − eeS兲兴
= 3 1 1
ln yR ln关共1 − ␩兲共1 + eeS兲兴 −
␾j共0兲 tanh␾j共0兲 ␾j共0兲
=E 共for intrinsic reaction in a fixed-bed reactor)
冋 册冋 册
␩ Gj = 共for p = 2兲
(40a) ␾j共0兲 1 1
− +1
Bi tanh␾j共0兲 ␾j共0兲
ln yS ln关1 − ␩共1 + eep兲兴 (44)
=
ln yR ln关1 − ␩共1 − eep兲兴
=E 共for intrinsic reaction in a fixed-bed reactor) For an immobilized enzyme-catalyzed kinetic resolution
(40b) process in a fixed-bed reactor, based on Eq. (32), we have:

34 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 74, NO. 1, JULY 5, 2001

 ln共yj⬁兲␨=1 =
Pe
2
− ln 冋冉 Pe ␦j
+
4␦j Pe 冊
sinh ␦j + cosh ␦j 册 Mass-Transfer Limitations on Enzyme Effective
Enantioselectivity and Optical Purity and Yield of
Desired Product
(45)
The results calculated by Eq. (37) are shown in Figure 3 (for
Thus, it is convenient to obtain the expression of Eeff from intrinsic reaction, solid lines). When enzyme enantioselec-
Eq. (45): tivity is favored (e.g., E ⳱ 100), almost 50% of the enan-
Pe
2
− ln 冋冉 Pe ␦S
+
4␦S Pe 冊
sinh ␦S + cosh ␦S 册 tiopure product will be obtained, whereas almost 50% of the
enantiopure substrate will remain at the end of the resolu-
tion process. For lower enantioselectivity (e.g., E ⳱ 5), the
冋冉 冊 册
Eeff = (46)
Pe Pe ␦R required enantiomeric purity of the remaining substrate may
− ln + sinh ␦R + cosh ␦R
2 4␦R Pe be reached at a higher conversion of racemate, but it is not
possible to obtain an enantiopure product.
Eq. (41), with Eq. (46), quantitatively describes the rela-
For an immobilized enzyme-catalyzed kinetic resolution
tionship between the racemate conversion (␩), the optical
process in a fixed-bed reactor, the optical purity and yield of
purity and yield of the desired product (eeS and eeP), and the
the desired product will decrease due to mass-transfer limi-
model parameters (E, ␾j, Bi, Pe, and Dr) at steady state
tations, as shown in Figure 3. The dashed lines represent the
without enzyme deactivation in the exit of the fixed-bed
results calculated by Eqs. (10)–(17) with the orthogonal
reactor for a first-order reaction.
If Pe → ⬁ (where the effect of axial dispersion can be
neglected), the exit concentration at steady state in the
fixed-bed reactor could be simplified to:

冋
共yj⬁兲␨=1 = exp − 共1 + p兲St 1 − 冉 Bi
Bi + ␭j 冊册 共Pe → ⬁兲
(47)
Then Eq. (46) could be degenerated into the following con-
cise mathematical expression:
Bi
1−
Bi + ␭S ␭S共Bi + ␭R兲
Eeff = = 共Pe → ⬁兲 (48)
Bi ␭R共Bi + ␭S兲
1−
Bi + ␭R
If p ⳱ 2, Eq. (48) is the formula proposed by Xiu et al.
(2000) [i.e., Eq. (43)].
Furthermore, under the assumption of Bi → ⬁ (absence of
the external mass-transfer resistance), the expression of
(yj⬁)␨⳱1 and Eeff can be simplified to:

冋
共yj⬁兲␨=1 = exp − 共1 + p兲 冉 冊
1 − ␧B
␧B
␧pDr␭j 册
共Bi → ⬁ and Pe → ⬁兲 (49)
and:
␭S
Eeff = 共Bi → ⬁ and Pe → ⬁兲 (50)
␭R
If p ⳱ 2, Eq. (50) is the formula proposed by Matson and
Lopez (1990) [i.e., Eq. (42)].
The difference between Eq. (46) and its derived equations Figure 3. (a) Mass-transfer limitations on eeP and eeS as a function of the
[Eq. (48) and (50)] should be noted. Eeff is dependent on the racemate conversion, ␩, at steady state for Bi ⳱ 100, Pe ⳱ 100, and
bed-length parameter, Dr, if Eq. (46) is adopted; Dr is a [␾S(0)]2 ⳱ 1. Solid lines: Eq. (37) (Chen et al., 1982); dashed lines:
major adjustable parameter for deciding the racemate con- Michaelis–Menten kinetics with ␤j ⳱ 1; dotted lines: first-order reaction
version, ␩, as shown in Figure 2. Based on Eq. (48) and Eq. [Eq. (41) with Eq. (46)]. (b) Mass-transfer limitations on eeP and eeS as a
function of the racemate conversion, ␩, at steady state for Bi ⳱ 10, Pe ⳱
(50), Eeff is independent of the bed-length parameter, Dr; in 20, and [␾S(0)]2 ⳱ 10. Solid lines: Eq. (37) (Chen et al., 1982); dashed
this case, Eeff has the same form for different types of re- lines: Michaelis–Menten kinetics with ␤j ⳱ 1; dotted lines: first-order
actors. reaction [Eq. (41) with Eq. (46)].

XIU, JIANG, AND LI: MASS TRANSFER LIMITATIONS IN A FIXED-BED REACTOR 35

collocation method for Michaelis–Menten kinetics, whereas
the dotted lines represent the results of Eqs. (41) and (46)
for a first-order reaction. Mass-transfer limitations should
be negligible for an immobilized enzyme-catalyzed kinetic
resolution process in a fixed-bed reactor if [␾S(0)]2 ⱕ 1, Bi
ⱖ 100, and Pe ⱖ 100, as shown in Figure 3a; in this case,
the dashed and dotted lines are close to the solid lines for E
⳱ 100 and E ⳱ 5.
Otherwise, if mass-transfer limitations exist (e.g., Bi ⳱
10, Pe ⳱ 20, and [␾S(0)]2 ⳱ 10, as shown in Fig. 3b), for
lower values of enantiomeric ratio (E ⳱ 5), the optical
purity of the desired product will decrease significantly due
to the mass-transfer limitations; in this case, the dashed and
the dotted lines are both well apart from the solid lines.
Figure 4 shows the effect of Thiele modulus, ␾S(0), on Figure 5. Effect of Bi on enzyme effective enantioselectivity, Eeff , at Pe
the enzyme effective enantioselectivity, Eeff, with E ⳱ 100 ⳱ 100, Dr ⳱ 1, and [␾S(0)]2 ⳱ 10 for steady state with E ⳱ 5 and E ⳱
and E ⳱ 5 at Bi ⳱ 100, Pe ⳱ 100, and Dr ⳱ 0.6 for the 100. Solid lines: Michaelis–Menten kinetics with ␤j ⳱ 1; dashed lines:
first-order reaction [Eq. (46)].
immobilized enzyme-catalyzed kinetic resolution process in
a fixed-bed reactor at steady state. Two cases are involved
in Figure 4: one for first-order reaction [Eq. (46); dashed
mass-transfer and axial dispersion limitations also give rise
lines], and the other for the competitive Michaelis–Menten
to a decrease in the enzyme effective enantioselectivity, Eeff
kinetics (␤j ⳱ 1.0; solid lines). The square of the Thiele
(especially at a larger value of [␾S(0)]2) for the first-order
modulus, ␾S2, has the physical interpretation of a first-order
reaction [Eq. (46); dashed lines] and for the competitive
reaction rate to the intraparticle diffusion rate (Bailey and
Michaelis–Menten kinetics (solid lines), as shown in Fig-
Ollis, 1986); it is a measure of whether the process is reac-
ures 5 and 6. Biot number, Bi, is the ratio of intraparticle
tion rate controlled (low ␾S) or diffusion rate controlled
diffusion resistance to external mass-transfer resistance, and
(high ␾S). It is evident from Figure 4 that only when
Peclet number, Pe, is the ratio of backmixing time to resi-
[␾S(0)]2 ⱕ 1, does the Eeff value approach the intrinsic one,
dence time. The Eeff value decreases sharply with decreas-
E, and Eeff decreases rapidly upon increasing the value,
ing Bi and Pe if both are <50. Again, we find that the effects
[␾S(0)]2, if [␾S(0)]2 > 1. The larger the E value, the more
of axial dispersion and external mass-transfer resistance on
serious the intraparticle diffusion limitation. The intrapar-
Eeff are more significant for a first-order reaction than for
ticle diffusion limitation on Eeff is more significant for a
Michaelis–Menten kinetics.
first-order reaction than that for Michaelis–Menten kinetics,
Figure 7 presents a typical example for an immobilized
because inhibition of substrates for Michaelis–Menten ki-
enzyme-catalyzed kinetic resolution process in a fixed-bed
netics results in a decrease in the special reaction rate of
reactor with E ⳱ 10, Bi ⳱ 5, Pe ⳱ 10, and [␾S(0)]2 ⳱ 5
enantiomers.
(solid line 4), along with results from the intrinsic reaction
Similar to intraparticle diffusion limitation, external
(solid line 1). If racemate conversion, ␩, is controlled at

Figure 4. Effect of [␾S(0)]2 on enzyme effective enantioselectivity, Eeff , Figure 6. Effect of Pe on enzyme effective enantioselectivity, Eeff , at Bi
at Bi ⳱ 100, Pe ⳱ 100, and Dr ⳱ 0.6 for E ⳱ 5 and E ⳱ 100. Solid lines: ⳱ 100, Dr ⳱ 1, and [␾S(0)]2 ⳱ 10 for steady state with E ⳱ 5 and E ⳱
Michaelis–Menten kinetics with ␤j ⳱ 1; dashed lines: first-order reaction 100. Solid lines: Michaelis–Menten kinetics with ␤j ⳱ 1; dashed lines:
[Eq. (46)]. first-order reaction [Eq. (46)].

36 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 74, NO. 1, JULY 5, 2001


Figure 7. Disassembly of mass-transfer limitations on enzyme effective
enantioselectivity, Eeff , for a particular resolution process at E ⳱ 10, ␤j ⳱
⬁ (first-order reaction; dashed lines), and ␤j ⳱ 1.0 (Michaelis–Menten
kinetics; solid lines) at steady state. (1) Intrinsic reaction; (2) Bi ⳱ 100, Pe
⳱ 100, and [␾S(0)]2 ⳱ 5; (3) Bi ⳱ 5, Pe ⳱ 100, and [␾S(0)]2 ⳱ 5; (4)
Bi ⳱ 5, Pe ⳱ 10, and [␾S(0)]2 ⳱ 5.
about 0.8 in order to approach 96% enantiomeric excess of
the remaining substrate, the ratio of Eeff/E will decrease
from 1 (solid line 1) to about 0.56 (solid line 4), in which an
approximately 30% reduction in Eeff could be found due to
intraparticle diffusion limitation (solid line 2), another 30%
due to the external mass-transfer limitation (solid line 3),
and the remainder due to axial dispersion (solid line 4).
Figure 7 also presents a comparison between the first-
order reaction (dashed lines) and the competitive Michae-
lis–Menten kinetics (␤j ⳱ 1.0; solid lines) for mass-transfer
limitations on enzyme effective enantioselectivity in a
fixed-bed reactor. Although the Eeff value decreases due to
mass-transfer limitations, the inhibition of the substrates is
more favorable to it at the same operating conditions, as
shown by comparison of the dashed lines with the corre-
sponding solid lines. In this case, however, the longer bed
length is needed for Michaelis–Menten kinetics to reach the
same desired optical purity as for the first-order reaction.
In conclusion, for more reactive enantiomers and moder-
ate enzyme enantioselectivity, it is possible that racemic
substrates would barely be separated in a fixed-bed reactor
due to the mass-transfer limitations in the resolution pro-
cess. With the parameters chosen in the aforementioned
examples, the parameters Bi and Pe should be controlled at
>50 to minimize the external mass-transfer and axial dis-
persion limitations on the enzyme effective enantioselectiv-
ity and the optical purity and yield of the desired product for
the continuous steady-state resolution process.
Furthermore, because the first-order reaction rate is the
most rapid for Michaelis–Menten kinetics, the mass-transfer
limitations on the enzyme effective enantioselectivity, Eeff,
are severe. Combining the results of Figures 3 to 7, we find
that:
Efirst-order
eff ⱕ EM−M
eff ⱕE (51)
where Efirst-order
eff is calculated by Eq. (46) or its derived Eq.
XIU, JIANG, AND LI: MASS TRANSFER LIMITATIONS IN A FIXED-BED REACTOR
(48) and (50), and EM−M
eff is the enzyme effective enantio-
selectivity for more complicated reaction kinetics (e.g.,
Michaelis–Menten kinetics). Thus, by the concise formula
derived from the first-order reaction we can predict the
mass-transfer limitations on the enzyme effective enantio-
selectivity for more complicated reaction kinetics in an im-
mobilized enzyme-catalyzed reactor.
Effects of Enzyme Deactivation on Enzyme
Effective Enantioselectivity and Optical Purity
and Yield of Desired Product
Due to longer time needed for a continuous resolution pro-
cess in a fixed-bed reactor, enzyme deactivation should be
considered. Apart from the inevitable mass-transfer limita-
tions, enzyme stability is one of the principle factors affect-
ing reactor productivity using immobilized enzymes. En-
zymes suffer irreversible deactivation from a variety of
causes, such as pH, temperature, ionic strength, and the
presence of toxic impurities. There have been many studies
on enzyme deactivation (Chen and Wu, 1987; Erarslan and
Kocer, 1992; Palazzi and Converti, 1999; Simon et al.,
1986; Toscano et al., 1994). In this work, enzyme activity
was assumed to be an exponentially decaying function, as
shown in Eq. (1).
A comparison of the time course between ␶1 → ⬁ and
␶1 ⳱ 10 was also presented in Figure 1. If enzyme activity
remains constant during the whole operation period, a con-
tinuous steady-state kinetic resolution process can be
reached, and the exit concentrations of S- and R-enantiomer
will not change. Otherwise, the exit concentrations will in-
crease as a result of enzyme deactivation as time proceeds,
and the optical purity will change continuously for a given
value of Dr, as shown in Figure 8. In contrast to the effect
of enzyme deactivation on ␩ and eeS, enzyme deactivation
has no negative effect on the enzyme effective enantiose-
lectivity (Eeff) and product excess (eep); that is, it favors
them due to the continuous decreases in the special reaction
rates of enantiomers.
Figure 8. Effect of enzyme deactivation (␶1 ⳱ 30, dashed lines; ␶1 ⳱ ⬁,
solid lines) on the optical purity of the desired product (eeP, eeS), racemate
conversion, ␩, and enzyme effective enantioselectivity, Eeff, for Michaelis–
Menten kinetics (␤j ⳱ 1.0) at E ⳱ 10, Bi ⳱ 10, Pe ⳱ 20, Dr ⳱ 2.5, and
[␾S(0)]2 ⳱ 10.
37
 It should be noted that, if the enzyme deactivation con-
stant increases, the mode of operation necessitates the av-
eraging of conversions over a certain period of operation,
beyond which the falling of conversions are no longer ac-
ceptable.
CONCLUSIONS
The immobilized enzyme-catalyzed kinetic resolution of
racemate in a fixed-bed reactor has been studied theoreti-
cally using an analytical solution for a first-order reaction
and the orthogonal collocation method for Michaelis–
Menten kinetics in which reaction kinetics could be coupled
with intraparticle diffusion, external mass transfer, and axial
dispersion.
The time course of the exit concentrations can be pre-
dicted by the analytical solution for a first-order kinetic
resolution process and by the orthogonal collocation method
for the complex kinetic resolution process. At a specified
level of optical purity, the analytical solution and the or-
thogonal collocation method can be used to predict the
value of the optimal bed length.
Intraparticle diffusion resistance, external mass-transfer
resistance, and axial dispersion were shown to reduce en-
zyme effective enantioselectivity, leading to an overall de-
crease in optical purity for a continuous steady-state reso-
lution process. A new foundation has been provided to
evaluate the mass-transfer limitations of enzyme effective
enantioselectivity.
The inhibition of substrates to the enzyme-catalyzed re-
action will decrease racemate conversion and optical purity,
but will favor the enzyme effective enantioselectivity com-
pared with the situation for a first-order reaction in the same
operating conditions. Enzyme deactivation will also reduce
the racemate conversion and optical purity of the remaining
substrate, but does not have a negative effect on enzyme
effective enantioselectivity or product excess, but rather it
favors them due to the continuous decrease in the special
reaction rates of enantiomers.
The authors thank two anonymous reviewers for helpful com-
ments.
NOMENCLATURE
Bi Biot number, defined as kf R/Deff
C substrate concentration in bulk phase (kg/m3)
c substrate concentration in porous support (kg/m3)
Dax longitudinal dispersion coefficient (m2/s)
Deff effective diffusivity (m2/s)
Dr bed-length parameter, defined as ␧bDeff L/␧PR2u0
eeP product excess, defined as (XS − XR)/(XS + XR)
eeS enantiomeric excess of the remaining substrate, defined as (XS −
XR)/(2 − XS − XR)
E enantioselectivity of the enzyme, defined as VmSKmR/VmRKmS
Eeff enzyme effective enantioselectivity, defined by Eq. (41)
kf external liquid–solid mass-transfer coefficient (m/s)
Km Michaelis–Menten constant (kg/m3)
L length of column (m)
38 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 74, NO. 1, JULY 5, 2001
p shape factor, p ⳱ 2 for sphere, p ⳱ 1 for cylinder, and p ⳱ 0
for slab
Pe Peclet number based on reactor length, defined as u0L/Dax␧b
r radial coordinate inside support matrix (m)
R particle radius for p ⳱ 2 and 1, and half thickness for p ⳱ 0 (m)
Rj dimensionless reaction rate of substrate
St Stanton number, defined as BiDr␧P(1 − ␧b)/␧b
t time (s)
t1 time constant of enzyme deactivation (s)
u0 the interstitial fluid velocity in the column, (m/s)
v reaction rate of substrate [kg/(kg of enzyme) (s)]
Vm maximum specific reaction rate per unit enzyme [kg/(kg of en-
zyme) (s)]
x dimensionless concentration, defined as c/C0
X individual conversion, defined by Eq. (35)
XE enzyme loading per unit support [kg/(kg of support)]
y dimensionless concentration, defined as C/C0
Z axial distance from column entrance (m)
Greek letters
␤ dimensionless Michaelis–Menten constant, defined as Km/C0
␧b bed porosity (m3/m3)
␧p support porosity
␩ racemate conversion, defined by Eq. (36)
␩G global effectiveness factor
␨ dimensionless axial distance, defined as Z/L
␰ dimensionless radial distance, defined as r/R
␳p support density (kg/m3)
␶ dimensionless contact time, defined as u0t/L␧b
␶1 dimensionless time constant of enzyme deactivation
␾R Thiele modulus for R-enantiomer, defined as ␾S/√E
␾R(0) ␾R in the case of ␶1 → ⬁, defined as ␾S(0)/√E
␾S Thiele modulus for S-enantiomer, defined as ␾S(0)exp(−␶/2␶1)
␾S(0) ␾S in the case of ␶1 → ⬁, defined as R√VmS␳pXE/KmSDeff
Subscripts
j R-, S-
R R-enantiomer
S S-enantiomer
0 initial
References
Bailey JE, Ollis DE. 1986. Biochemical engineering fundamentals, 2nd ed.
New York: McGraw-Hill.
Chen CS, Fujimoto Y, Girdaukas G, Sih CJ. 1982. Quantitative analyses of
biochemical kinetic resolutions of enantiomers. J Am Chem Soc
104:7294–7299.
Chen KC, Wu JK. 1987. Substrate protection of immobilized glucose
isomerase. Biotechnol Bioeng 30:817–824.
Erarslan A, Kocer H. 1992. Thermal inactivation kinetics of penicillin G
acylase obtained from a mutant derivative of Escherichia coli ATCC
11105. J Chem Technol Biotechnol 55:79–84.
Froment GF, Bischoff KB. 1990. Chemical reactor analysis and design,
2nd ed. New York: John Wiley & Sons.
Garcia T, Coteron A, Martinez M, Aracil J. 2000. Kinetic model for the
esterification of oleic acid and cetyl alcohol using an immobilized
lipase as catalyst. Chem Eng Sci 55:1411–1423.
Goto M, Smith JM, McCoy BJ. 1990. Parabolic profile approximation
(linear driving-force model) for chemical reactions. Chem Eng Sci
45:443–448.
Indlekofer M, Brotz F, Bauer A, Reuss M. 1996. Stereoselective biocon-
versions in continuous operated fixed bed reactors: Modeling and pro-
cess optimization. Biotechnol Bioeng 52:459–471.
Indlekofer M, Reuss M, Barth S, Effenberger F. 1993. Kinetic analysis and
 simulation studies for lipase-catalysed resolution of racemic 2-methyl-
1-pentanol. Biocatalysis 7:249–266.
Li P, Xiu GH, Jiang L. 2000. Adsorption and desorption of phenol on
activated carbon fibers in a fixed bed. Sep Sci Technol.
Marrazzo WN, Merson RL, McCoy BJ. 1975. Enzyme immobilized in a
packed-bed reactor: Kinetic parameters and mass transfer effects. Bio-
technol Bioeng 17:1515–1528.
Matson SL, Lopez JL. 1990. Multiphase membrane reactors for enzymatic
resolution: Diffusional effects on stereoselectivity. In: Sikdar SK, Bier
M, Todd P, editors. Frontiers in bioprocessing. Boca Raton, FL: CRC
Press. p 391.
Palazzi E, Converti A. 1999. Generalized linearization of kinetics of glu-
cose isomerization to fructose by immobilized glucose isomerase. Bio-
technol Bioeng 63:273–283.
Raghavan NS, Ruthven DM. 1983. Numerical simulation of a fixed-bed
adsorption column by the method of orthogonal collocation. AIChE J
29:922–925.
Rakels JLL, Romein B, Straathof AJJ, Heijnen JJ. 1994. Kinetic analysis
of enzymatic chiral resolution by progress curve evaluation. Biotech-
nol Bioeng 43:411–422.
Rasmuson A. 1985. Exact solution of a model for diffusion in particles and
longitudinal dispersion in packed beds: Numerical evaluation. AIChE
J 31:518–519.
Rasmuson A, Neretnieks I. 1980. Exact solution of a model for diffusion
in particles and longitudinal dispersion in packed beds. AIChE J
26:686–690.
Rosen JB. 1952. Kinetics of a fixed bed system for solid diffusion into
spherical particles. J Chem Phys 20:387–394.
Simon LM, Kotorman M, Szajani B, Boross L. 1986. Preparation and
XIU, JIANG, AND LI: MASS TRANSFER LIMITATIONS IN A FIXED-BED REACTOR
characterization of immobilized glucose-phosphate isomerase. En-
zyme Microb Technol 8:222–226.
Smith JM. 1981. Chemical engineering kinetics, 3rd edition. New York:
McGraw-Hill.
Straathof AJJ, Jongejan JA. 1997. The enantiomeric ratio: Origin, deter-
mination and prediction. Enzyme Microb Technol 21:559–571.
Straathof AJJ, Rakels JLL, Heijnen JJ. 1992. Kinetics of the enzymatic
resolution of racemic compounds in bi–bi reactions. Biocatalysis
7:13–27.
Suzuki M, Smith JM. 1971. Kinetic studies by chromatography. Chem Eng
Sci 26:221–235.
Toscano G, Pirozzi D, Maremonti M, Greco G. 1994. Solid-state enzyme
deactivation in air and in organic solvents. Biotechnol Bioeng
44:682–689.
Tsai SW, Lin JJ, Chang CS, Chen JP. 1997. Enzymatic synthesis of (S)-
ibuprofen ester prodrug from racemic ibuprofen by lipase in organic
solvents. Biotechnol Prog 13:82–88.
van Tol JBA, Jongejan JA, Duine JA. 1995. Description of hydrolase-
enantioselectivity must be based on the actual kinetic mechanism:
Analysis of the kinetic resolution of glycidyl (2.3-epoxy-1-propyl)
butyrate by pig pancreas lipase. Biocatal Biotrans 12:99–117.
Xiu GH, Jiang L, Li P. 2000. Mass-transfer limitations for immobilized
enzyme-catalyzed kinetic resolution of racemate in a batch reactor. Ind
Eng Chem Res 39:4054–4062.
Xiu GH, Li P. 1999. Linear chromatographic elution and breakthrough
curves: Quasi-lognormal distribution function and its derived equa-
tions. Chem Eng Sci 54:377–387.
Zheng YZ, Gu TY. 1996. Analytical solution to a model for the startup
period of fixed-bed reactors. Chem Eng Sci 51:3773–3779.
39