Bioprocess Engineering 9 (1993) 3742. Bioprocess Engineering. 9 Springer-Verlag ... by specific diffusional barriers at polymer-solution inter- face [9, 10], and/or ...
Bioprocess Engineering 9 (1993) 3742
BioprocessEngineering 9 Springer-Verlag 1993
Kinetic behavior of penicillin acylase immobilized on acrylic carrier J. Bryjak and A. Noworyta, Wroclaw, Poland
Abstract. The usefulness of Lilly's kinetic equation to describe penicillin G hydrolysis performed by immobilized penicillin acylase onto the acrylic carrier has been shown. Based on the experimental results characteristic kinetic constants have been estimated. The effect of noncompetitive inhibition of 6-amino penicillanic acid has not been found. Five components of reaction resistance have been defined. These components were also estimated for the reaction of the native enzyme as well as the Boehringer preparation. List of symbols CE g/m 3 Cp, CQ mol/m3 Cs mol/m 3 Cso mol/m3 KA mol/m 3
Kis
mol/m3
Kip
mol/m 3
KiQ k3
mol/m 3 mol/g/min
R(1) R(2) R(3) R(4) R(5) r =
--
dCs/dt mol/m 3min t ~(i)
enzyme concentration product concentrations substrate concentration initial substrate concentration constant which defines the affinity of a substrate to the enzyme substrate inhibitory constant PhAA inhibitory constant 6-APA inhibitory constant constant rate of dissociation of the active complex concentrational component of reaction resistance resistance component derived from substrate affinity resistance component due to the inhibition of the enzyme by substrate resistance component due to the inhibition of the enzyme by PhAA resistance component due to inhibition of the enzyme by 6-APA
butyl acrylate and glycol dimethacrylate of 40% crosslinking appeared to be the most suitable choice for that purpose since it bound a high amount of proteins with high activity. We have also determined the dependence of the protein binding yield on the immobilization conditions as well as initiated experimental studies on the hydrolysis of benzylpenicillin (PG) in a batch reactor. In this paper we report experimental results on the kinetics of the latter process. The interactions between enzyme and substrate are commonly defined by the Michaelis-Menten's equation. The proper determination of kinetic parameters is especially important in the case when the products are simultaneously enzyme inhibitors. It is noteworthy that the use of immobilized enzyme also might lead to substantial changes in the reaction kinetics. These changes are caused by specific diffusional barriers at polymer-solution interface [9, 10], and/or the various affinities of reactants to the polymeric carrier. In addition, the different local reactant concentrations might influence this process. Determination of kinetic parameters requires the use of more complex experimental procedures and the use of sophisticated mathematical kinetic models. Thus, the determined kinetic constants are often called "effective" or "apparent".
min
rate of reaction reaction time relative resistance of reaction
1 Introduction
The industrial application of immobilized penicillin acylase (AP) [1-4] promotes research to find a new, stable form of the enzyme. We have recently reported the experimental results on immobilization of penicillin acylase on insoluble polyacrylate supports [5-8]. The copolymer of
2 Materials and methods
The kinetic studies were carried out using the experimental set-up shown in Fig. 1. Thermostated reactor (1) of 1000cm 3 volume was equipped with stirrer (2) of 100-200 rpm. This speed ensured appropriate stirring without a significant effect on carrier grains. PH-electrode (3) was connected with a microcomputer which controlled p H level. For this purpose signal was transmitted to the volumetric p u m p (4). The p u m p supplied the reactor with ammonia water from the tank (5). Temperature was measured by means of a thermistor (6). The changes of p H and temperature during the process were continuously monitored.
38
Bioprocess Engineering 9 (1993) As seen from this scheme the exact determination of kinetic parameters requires the experimental determination of 17 reaction rate constants. Since this is virtually impossible the experiments are usually limited to the determination of several constants, which are in fact the resultant values from several reaction rate constants. Lilly et al. [11] used the standard Cleland equation for the description of penicillin G hydrolysis:
#
l
I (
5
31 I
I pdnter I
/l
t'-~-
C2
Fig. 1. Laboratory setup for determination of reaction kinetics
The experiments were carried out by varying the amount of enzymatic preparation and substrate concentration up to the practical depletion of substrate. The reaction progress was monitored by the determination of the 6-APA concentration. The samples of the reaction mixture were collected at 5 or 10 rain intervals and 6-APA concentration was determined by the technique described in [5-7]. In comparison, identical kinetic studies have been performed using native enzyme and gel immobilized penicillin acylase of Boehringer supplied by Tarchomin Pharmaceutical Plant from Warsaw.
3
Kinetic
equation
Enzymatic hydrolysis of penicillin G (PG) constitutes a concurrent-successive system in which the main reaction of hydrolysis resulting in formation of 6-APA and phenyl acetic acid (PhAA) may be commonly accompanied by: -
noncompetitive enzyme inhibition by substrate (S); noncompetitive enzyme inhibition by 6-APA (Q); competitive enzyme inhibition by PhAA (P).
The reaction scheme is given below.
KAc
k3 CE Cs KA
(ES)
S
P 5 --
k7 kl
S
E
E S). k2
k12
(EO)
S
k13
(ESO,)
C s Cp
+ Kip
In spite of introduced simplifications, this form of the equation is the most commonly used for the description of the reaction kinetics in the case of both native or immobilized enzymes [13-17]. The use of this equation for hetcrogenic systems is limited only to those cases in which the diffusional mass transfer resistance is negligible. Our previous studies have indicated that the low value of a specific surface area of acrylic carrier and the additional glutaraldehyde crosslinking of surface amino groups cause the enzyme to be located mainly out of the grain at the carrier's surface [7]. Thus, one can make the assumption that the studied reaction proceeds under kinetically controlled conditions. In order to verify this assumption the influence of temperature on the reaction rate was determined. The experimental results summarized in Fig. 2 clearly demonstrate that the temperature effect on the reaction rate is identical when both native and immobilized enzymes were used. Thus, it can be concluded that the reaction proceeds in the kinetically controlled regime. Based on this conclusion Eq. (1) can be applied to describe the rate of the reaction catalyzed by the enzyme immoblized on the acrylic carrier. Consequently, the estimation of five constants (k3, KA,Kip, KiQ, K~s) in this equation is required.
k6
k17
CQ
(1)
EP
k16
K A Cp
Cs+KA+~s+~Pipp+~CQ4 Kip Kiq
k3
P+Q+E
J. Bryjak and A. Nowryta: Kinetic behavior of immobilized penicillin acylase
\
,%
allow to estimate the values of inhibitory constants for products, Kip and Kio, based on the values of k3, Ir A and Kis previously found.
m
5 Discussion of the model equation
o =
g g ~--2 0
3.2
.... s;-
20
3.s 1/T.IO3 3.6
3;
2;ocl;
Temperature
Fig. 2. Influence of temperature on the reaction rate O native enzyme 9 acrylic preparation At the initial stage of the reaction, where the concentration of the products is negligible, the reaction rate equation can be simplified to: r =
39
k3 CE Cs Cs2 . C s + K A -b - Kis
(2)
This equation is commonly used for the determination of k3, KA and Kis constants.
4 Determination of reaction constants In order to determine k3, KA, and Kis values a subset of data which applies to the initial stage of the reaction, was analyzed. Using nonlinear regression the values of the above constants were determined from integrated form of Eq. (2):
1 E
f -- k3 CE Cso -- C s + K A In
+ ~Kis(C~o - C2)
In order to compare results, similar studies, as those described above, have been carried out using native enzyme and gel-immobilized enzyme produced by Boehringer. The reaction constants estimated for all the studied systems are given in Table 1. Since these values were obtained by means of multivariables regression function, the data requires some careful interpretation. The values of the reaction rate constants (k3) obtained for both immobilized enzymes (Boehringer and our preparation) are very similar. This is quite surprising since each preparation represents a completely different way of enzymes immobilization. It is also difficult to explain the higher value of k3 found for immobilized enzymes in comparison to the native one. Affinity of the substrate to the immobilized enzyme is lower (higher KA value) than those found for the native one. It may result from the lower accessibility of the substrate to the binding site(s) of the enzyme. Substrate inhibition, represented by Kis, is absent in the case of the native enzyme. This is in agreement with the results obtained by Park [14, 15]. It was also not observed in the case of Boehringer preparation. In the case of our preparation a slight inhibition of enzyme activity by the substrate has been observed. It may result from the sorption of substrate by acrylic carrier [8]. In all the cases PhAA appeared to act as competitive inhibitor and its affinity (Kip) to the enzyme was very close to each other. The highest affinity of this product has been the highest in the case of enzyme immobilized on the acrylic carrier whereas the lowest for Boehringer prepara-
,
(3)
Table 1. Values of constants of kinetic Eq. (l)
then fitting of all experimental data into the integrated form of the Eq. (1):
Constant
Cso KA KiQ + Ki p
]r 1 0 3 - -
1
I(
t= ~
1
KA Kip
I x (Cso
x
- Cs) +
C~o - c~ 2
Cso xKAln Cs ]
+
/(is
1
2KA Cso
KipKio )
KA
KA )
Ki p + KipK i
Cso
Cso
Kis
Cs2o ) Kit,
+ -igi + K ; k i o ; (4)
Kio
tool g min mol m3 mol mS mol
m3
tool
m3
Native enzyme
Acrylic preparation
Boehringer preparation
0.781
1.26
1.25
1.34
4.29
6.79
> 10ts
652
> 1015
26.8
20.9
41.5
32.7
>1015
>1015
40
Bioprocess Engineering 9 (1993)
tion. On the contrary the inhibition by the second product -6-APA-was observed only in the case of the native enzyme. The favorable characteristics of our enzyme preparation include: Prolonged activity, high thermostability, appropriate activity in a wide range of pH-values, as well as satisfactory mechanical properties [7, 8]. The results presented in this paper indicate that kinetic characteristics of this preparation is also better than the native enzyme. In Fig. 3 the representative example of the dependence of reaction time (t) on the degree of conversion (~) is shown. The overall time of reaction is significantly shorter if the acrylate immobilized-enzyme is used rather than the native one. If compared with the Boehringer preparation, this time is strongly dependent on the initial concentration of the substrate. Thus, for low substrate concentrations (Cso < 30 kg/m 3) our preparation appeared advantageous over the Boehringer one, while the use of higher concentrations of the substrate resulted in a faster reaction catalysed by the Boehringer preparation. It is due to the existence of inhibition by the substrate in the case of acrylate immobilized penicillin acylase. In Fig. 4 representative values of initial reaction rates r(0) versus initial substrate concentration are presented. As seen from this figure the significant inhibition of acrylate-bound enzyme was observed for higher substrate concentrations. In order to perform a quantitative analysis of the influence of certain reaction components on its kinetics the Eq. (1) was rewritten into the following form:
1.0 tool m 3 m[n
CE:1000g/m 3
B
0.9
o !
~0.8
--
0.7 N
c 0.6
I 20 z,0 60 mo[/m 3 Reactant concentration Cso
100
Fig. 4. Influence of reactant concentration Cso on initial reaction rate r(0); A acrylic carrier, B-Boehringer prapration, N-native enzyme
The denominator in this equation describes the resistance R of the reaction: r=
Ymax
(6)
R
where
rmax = k 3 C E
Thus, five characteristic components R(1) to R(5) of this resistance R were isolated, and they are as follows: R(1) = 1 ; concentrational component; KA
R(2) = ~
; component derived from substrate affinity;
Cs R(3) = ~ ; component due to the inhibition of the enF= k3 CE KA
1+
Cs
+
K A Cp
K A CQ
+ Ki--FG + KZ
K A Cp CQ
Cp
s + KipK,oCs + Kip
(5) 20O
r
_
_
160
R = ~ R (i)
2
E 120 Cso =50mo[/m 3 CE=1000g/m 3
ao 4,0
0.2 0.4. 0.6 0,8 Degree of conversion c~
C~o - c~" I 2KiQ ] ; comof the enzyme by . , component due
to inhibition of the enzme by 6-APA. The total resistance R is given by:
min
o
zyme by substrate; C~o - c~ I
