- Email: [email protected]
Deacylation of benzylpenicillin by immobilized penicillin acylase
BIOCHIMIE, 1980, 62, 317-321.
Deacylation of benzylpenicillin by immobilized penicillin acylase. M. D. LI,LLY, S. W. CARLEYSMITH ( ' ) and P. DUNNILL.
Department of Chemical and Biochemical Engineering, University College London, Enghmd.
Rdsum~.
Summary.
Les pr6parations de p6nicilline acylase immobilis6es ont montr6 de meilleures activit6s (par unit6 de volume) que les pr6parations de cellules immobilis6es.
Immobilized penicillin acylase preparations h a v e much hiqher activities per unit volume than immobilized cell preparations. Many parameters of the deacylation reaction are dependent on pH and both reactant and one of the products, 6-aminopenicillanic acid, are acid and alkali labile. Acid is produced as result of the deacylation reaction and must be neutralised. The influence of these pH effects on the design of the catalyst and the reactor is discussed.
De nombreux param~tres de la r6action de d6acylation sont d6pendants du pH. L'influence de ces effets de pH sur la conception d u catalyseur et du r6acteur est discut6e.
Introduction. 6 - A m i n o p e n i c i l l a n i c acid has been p r o d r c e d c o m m e r c i a l l y for some years by the de a c y l a t i o n of b e n z y l p e n i c i l l i n u s i n g i m m o b i l i z e d p e n i c i l l i n acylase [11. In c o n t r a s t to most o t h e r co,mmercial p r o c e s s e s using i m m o b i l i z e d e n z y m e s or imm o b i l i z e d cells, the d e a c y l a t i o n of b e n z y l p e n i c i l lin results in the f o r m a t i o n of an acid, p h e n y l a c e tic acid, w h i c h a.t the n o r m a l o p e r a t i n g p H is dissociated. To m a i n t a i n tire reaction pH in the desired range it is t h e r e f o r e n e c e s s a r y to add alkali or use large a m o u n t s of a b u f f e r i n g salt. Thus the design and o p e r a t i o n of an i m m o b i l i z e d p e n i c i l l i n acylase r e a c t o r is greatly influenced I)y the effect of p H on the r e a c t i o n p a r a m e t e r s and the p r o p e r ties of the i m m o b i l i z e d enzyme, r e a c t a n t and l)roducts. Some of these effects are discussed in this paper.
Materials and Methods. Penicillin aeylase (partially-purified from Escherichia eoli), benzylpenieillin an.d 6-aminopenicillanic acid (6-APA) were supplied by Beecham Pharmaceuticals (U.K. Division). Immobilized penicillin acylase was prepared by covalent binding of the enzyme to
(') Present address : Beecham Pharmaceuticals (U.K. Division), Worthing, England.
Amberlite XAD-7 beads (420-850 p+m, Robin and Haas (UK) Ltd, Croydon, England) [2]. The enzyme forms a thin layer within the bead, the depth of which can be controlled by adjusting the reaction conditions [3]. Both soluble and immobilized penicillin acylase were assayed at 37°C in a pH-stat as described previously.
Results. Comparison of activities o[ immobili:ed penicillin acylase preparations. T h e l a c t a m rings of b e n z y l p e n i c i l l i n and 6-APA are both acid and alkali-labile. It is essential ther e f o r e that the acid p r o d u c e d d u r i n g the d e a c y l a tion r e a c t i o n is r a p i d l y n e u t r a l i s e d w i t h o u t causing regions of h i g h a l k a l i n i t y w i t h i n the reactor. E v e n u n d e r the most f a v o u r a b l e c o n d i t i o n s a finite rate of c h e m i c a l h y d r o l y s i s of the l a c t a m r i n g occurs and t h e r e is a c o n s i d e r a b l e i n c e n t i v e to re~ duce the r e s i d e n c e times of r e a c t a n t and p r o d u c t s in the reactor. This may be a c h i e v e d by u s i n g a h i g h c a t a l y t i c a c t i v i t y / u n i t v o l u m e of reactor. It is of interest t h e r e f o r e to c o m p a r e the a c t i v i t i e s of both i m m o b i l i z e d p e n i c i l l i n acylase and cells c o n t a i n i n g the e n z y m e (table I). As e x p e c t e d , t h e r e is a large d i f f e r e n c e b e t w e e n the a c t i v i t i e s of the i m m o b i l i z e d e n z y m e p r e p a r a t i o n s and the i m m o bilized cells. It is also w o r t h noting that the activity yield, tile m e a s u r e d a c t i v i t y of lhe p r e p a r a tion d i v i d e d by the amount of e n z y m e a c t i v i t y
M. D. Lilly and coll.
318
used in the i m m o b i l i z a t i o n p r o c e d u r e , is either s i m i l a r to or higher than for tim e n z y m e p r e p a r a tions, p a r t l y because of the larger particle sizes of the e n t r a p p e d cell p r e p a r a t i o n s .
Effect of pH on the reaction equilibrium. The deacylation of b e n z y l p e n i c i l l i n can be represented b y A~--P+Q+H
E[lect of pH on the activity and stability of penicillin acylase. The i n i t i a l activity of soluble p e n i c i l l i n acylase on 200 mM b e n z y l p e n i c i l l i n was greatest at pH 8 (figure 1). The a s y m m e t r i c pH-activity profile is s i m i l a r to those d e s c r i b e d by W a r b u r t o n [10] and Kutzbach a n d R a u e n b u s c h [11]. Berezin et al. [12] and S z e n t i r m a i [13] r e p o r t e d more s y m m e t r i c a l profiles. The m e a s u r e d pH activity profile of Am-
TABLE I.
Immobilization of penicillin acylase.
E. colt Enzyme
Immobilization
Support
Catalyst
method
Polyacrylamide Polyacrylamide E p o x y resin DEAE-cellulose S e p h a d e x G200 Cellulose t r i a c e t a t e GMA/EDMA copolymer CM-cellulose Amberlite XAD7
Entrapment Entrapment Entrapment Triazine CNBr Entrapment Glutaraldehyde Glutaraldehyde Glutaraldehyde
Aetivily yield
Retained acti-
t per cent)
vity (per cent}
Activity (')
Reference
-40 40 -47 35 39 82 77
---45 --42 82 89
0.33 0.88 4.8 35 225 146 (+) 70(+)(25oC) 272 70
4 5 5 6 7 8 9 2 2
(*) mol/min/ma or tonne wet weight. (+) mol/min/tonne dry weight. All enzyme assays were done at 37°C unless otherwise stated.
w h e r e A, P, Q and H are b e n z y l p e n i c i l l i n , 6-APA, p h e n y l a c e t i c acid and h y d r o g e n ion respectively. The e q u i l i b r i u m constant, K', can be w r i t t e n (ass u m i n g activity coefficients to be u n i t y ) as, - -
berlite XAD-7 p e n i c i l l i n acylase varies greatly dep e n d i n g on the c o n c e n t r a t i o n s of b e n z y l p e n i c i l l i n a n d buffer used.
[P] [Q] [H] K ~
or
c~
[A] [P] LQ]
log K' = log
[A]
1980,
62,
n ° 5-6.
1.0
oC¢
pH.
Solutions of 10' 1rim b e n z y l p e n i c i l l i n were i n c u b a ted at 37°C w i t h p e n i c i l l i n acylase at various fixed pH values in the pH-stat apparatus. A t t a i n m e n t of r e a c t i o n e q u i l i b r i u m was i n d i c a t e d by the cessation of alkali uptake. 'l-Tie degree of c o n v e r s i o n of b e n z y l p e n i c i l l i n to 6-APA was calculated from the m o l a r uptake of alkali after c o r r e c t i n g for the partial p r o l o n a t i o n of 6-APA. The e q u i l i b r i u m conversions for an i n i t i a l b e n z y l p e n i c i l l i n c o n c e n t r a tion of 10 mM are s h o w n in figure 1. Using the exp e r i m e n t a l l y derived value of K' (3 × 10~7 MU), values for the e q u i l i b r i u m c o n v e r s i o n s at various pH values were calculated for 200 mM b e n z y l p e n i c i l 1in a n d are also s h o w n in tigure 1, w h i c h shows that at this higher c o n c e n t r a t i o n high yields of 6-APA can only be o b t a i n e d at pH values above 7.5. BIOCHIMIE,
¢,
(L, . _ O
>,
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¢~0.5 Et::3
/
klJ 0::
4
t
I
I
I
I
I
5
6
7 pH
8
9
10
FIG. 1 . - The influence of pH on the equilibrium conversion of 10 mM (O) and 200 mM ( - - - - ) benzylpeni-
cillin to 6-aminopenieillanie acid at 37°C. The effect of pH on the rate of enzymic deacylation (expressed as a proportion of the maximal rate) is also shown (Q).
Tim influence of pH on the stability of Amberlite XAD-7 p e n i c i l l i n acylase at 37°C has been described elsewhere [14]. The n l a x i m u m half-life
I m m o b i l i z e d penicillin acylase : benzyIpenicillin (943 hours) w a s at pH 350 h o u r s at p H 8.
6.6 and
fell to about
Internal p H changes in Amberlite XAD-7 penicillin acylase. T h e .effects of p H d e s c r i b e d so far h a v e been c o n c e r n e d w i t h the bulk pH. W i t h the e n z y m e i m m o b i l i z e d in A m b e r l i t e XAD-7, w h i c h has a po-
1.0
/
z o
,/ /," \1
>0.5 z o
u
;,
/
/
I
1
319
W h e n the curves in figure 2 are s u p e r i m p o s e d by r e p l o t t i n g w i t h all the i n i t i a l rates set equal to tile initial rate of c u r v e 3, t h e i r shapes can be c o m p a r e d m o r e r e a d i l y (figure 3). T]~e d e g r e e of i n h i b i t i o n of the u n g r o u n d or u n b u f f e r e d i m m o b i l i z e d e n z y m e then a p p e a r e d to be less. T h e rate of r e a c t i o n f o r the g r o u n d m a t e r i a l w i t h buffer p r e s e n t w a s fast i n i t i a l l y b u t as p r o d u c t s q u i c k l y a c c u m u l a t e d , i n h i b i t i o n r a p i d l y d e c r e a s e d the r e a c t i o n rate. As p r o d u c t s w e r e f o r m e d w i t h the u n b u f f e r e d or u n g r o u n d e n z y m e p r e p a r a t i o n s , the r e a c t i o n rate fell r e l a t i v e l y m o r e s l o w l y f o r t w o reasons. First, as the rate of h y d r o g e n ion p r o d u c tion fell the p H rose i n s i d e the beads i n c r e a s i n g the a c t i v i t y of the i m m o b i l i z e d enzyme. S e c o n d l y , and m o r e i m p o r t a n t l y the p r o d u c t , 6-&PA, a c t e d as a buffer e n h a n c i n g the rate of h y d r o g e n ion t r a n s f e r to the s u r f a c e of the beads [15]. T h e net r e s u l t w a s that the r e a c t i o n profile was i n i t i a l l y s t r a i g h t e r (figure 3).
I
0
5 10 =15 TIME (T) Fro. 2. - - Progress curves for the conversion o[ 0.1 M benzylpenicillin to 6-APA, eatalysed by Amberlite XAD-7 penicillin acylase at 37°C and pH 8. The conditions for each curve were as follows : 1. unground immobilized enzyme with no added buffer ; 2. unground preparation with added phosphate buffer (0.1 M) ; 3. ground preparation with added phosphate buffer. The unit of time, x, is the time that would be required for eornplete eor~version of the benzylpenieillin if the rate of reaction remained eonstant at the initial rate of curve 3.
1.0
--
~
2
z
/
uJ 0.5 7 0 (..)
.
f
~
'
,
i
rous structure, w e h a v e s h o w n [2] that the p H inside the beads, w h e n b e i n g used in a reactor, fails to about p H 4.5, at w h i c h p H the rate of reaction in the beads is p r e d i c t e d to be about h a l f that o b s e r v e d at pH 8. Thus in the absence of buffer the r e a c t i o n at a buIk p H of 8 p r o c e e d s m o r e s l o w l y than e x p e c t e d (figure 2). A d d i t i o n of a buff e r i n g salt to the r e a c t o r c o n t a i n i n g i m m o b i l i z e d p e n i c i l l i n acylase caused a large i n c r e a s e in the rate of r e a c t i o n (figure 2). Observation of the imm o b i l i z e d e n z y m e beads c o n t a i n i n g a d s o r b e d pHi n d i c a t i n g dye in a r e a c t o r s h o w e d that the effect of a d d e d buffers w a s to raise the p H near the surface of the beads a b o v e 7, c o m p a r e d to 5 or less in the absence of buffer. H o w e v e r , even in the p r e s e n c e of buffer a p H g r a d i e n t still e x i s t e d in lhe beads w i t h the i n t e r i o r at about pH 5. Grind i n g of tile beads led to a f u r t h e r i n c r e a s e in the r e a c t i o n rate (iigure 2) and the r e s u l t i n g b a t c h r e a c t i o n profile w a s almost i d e n t i c a l in shape to that observed w i t h the soluble enzyme.
%
-
1~',"Y , ~ 3
0
0
BIOCHIMIE, 1980, 62, n ° 5-6.
deacylation.
I
1
t
i
2
i
I
i
3
TIME(T) FIe. 3. Progress curves for the conditions described in [igure 2. The curves have been superimposed by setting the initial slopes of all the curves to be equal to the initial slope of curve 3, -
-
Operational stability o[ the zyme.
immobilized
en-
W h e n the i m m o b i l i z e d e n z y m e w a s used in a four-stage c o n t i n u o u s s t i r r e d tank r e a c t o r w i t h o u t a d d e d buffer, the o p e r a t i o n a l stability w a s m u c h less than e x p e c t e d on the basis of the k n o w n storage s t a b i l i t y at 37°C [14]. In the p r e s e n c e of a large c o n c e n t r a t i o n (0.1 M) of p h o s p h a t e buffer the s t a b i l i t y w a s greater but still less t h a n expected. T h e m a i n reason for loss of a c t i v i t y w a s inactivation by alkali added to n l a i n t a i n a c o n s t a n t bulk pH. It seems likely that the e n z y m e close to or on the s u r f a c e of the beads w a s being e x p o s e d to l o c a l i z e d r e g i o n s of high pH. This loss can be
320
M. D. L i l l y a n d coll.
r e d u c e d b y i m p r o v i n g the m i x i n g in the r e a c t o r a n d / o r r e d u c i n g the c o n c e n t r a t i o n of the alkali feed. An alternative a p p r o a c h is to locate the enzyme l a y e r below the surface of the bead. W e have exam i n e d elsewhere [3] the rate of p e n e t r a t i o n of b o v i n e s e r u m albumen, w h i c h has a s i m i l a r molecular w e i g h t to l~enicillin acylase, into Amberlite X.~D-7 beads. Using this i n f o r m a t i o n we first i m m o b i l i z e d a t h i n l a y e r of BSA a d j a c e n t to the surface of beads a n d then i m m o b i l i z e d p e n i c i l l i n acylase as a layer b e n e a t h the BSA. T h i s modified i m m o b i l i z e d p e n i c i l l i n acylase a n d a p r e p a r a t i o n made in the n o r m a l w a y w e r e used i n three consecutive batch reactions a n d the activities remain i n g after each reaction w e r e m e a s u r e d (figure 4).
100 o
<
50
cO
E cE
1 2 3 Number of Batch Uses Fro. 4 . The proportion of the initial activity remaining after reuse in a batch reactor for immobilized penicillin acylase (Q) and immobilized penicillin acylase pretreated with bovine serum albumen (O). Batch reaction conditions, - - initial benzy]penicillin concentration, 0.2 M ; temperature, 37°C ; pH 8, maintained by addition of 5 N NaOH.
The r e a c t i o n c o n d i t i o n s were designed to cause r e a s o n a b l y fast loss of activity of the n o r m a l preparation. The location of the enzyme l a y e r below a BSA layer clearly reduces the rate of loss of activity.
Discussion. F r o m the results described in this p a p e r it is clear that pH has a m a r k e d effect on the perform a n c e and b e h a v i o u r of a reactor for tile deacylation of b e n z y l p e n i c i l l i n , ttigh c o n v e r s i o n s to BIOCHIMIE, 1980, 62, n ° 5-6.
6-APA can o n l y be achieved w h e n the bulk pH, at least at the e n d of the reaction, is close to p H 8, w h i c h is also the value for m a x i m u m e n z y m i c activity. However, this is n o t the o p t i m u m pH for enzyme stability a n d there is a positive advantage in a l l o w i n g the pH w i t h i n the beads to fall since the e n z y m e is then more stable. Even if the pH drops to 6 the e n z y m e w i l l be over twice as stable as at pH 8 a n d the activity w i l l only be 20 p e r cent l o w e r (figure 1). F o r the i m m o b i l i z a t i o n c o n d i t i o n s used, the l a y e r of b o u n d e n z y m e r e p r e s e n t s only a small p r o p o r t i o n of the total volume of the Amberlite XAD-7 beads. On a v o l u m e t r i c basis the activity is still m u c h h i g h e r t h a n those for i m m o b i l i z e d cells (table I) a n d the particles are a more m a n a geable size t h a n some of the other enzyme s u p p o r t materials listed i n table I. Although more enzyme c a n be i m m o b i l i z e d i n the i n t e r i o r of the Amberlite XAD~7 beads, it w o u l d not p a r t i c i p a t e effectively i n the d e a c y l a t i o n reaction. F u r t h e r m o r e , i n a large reactor, w h e r e good m i x i n g is more difficult, a h i g h e r catalytic activity per u n i t volume w o u l d i n c r e a s e the likelihood of d e n a t u r a t i o n of enzyme n e a r the surface due to localised high pH. As s h o w n i n figure 4 this loss can be greatly reduced b y p l a c i n g the enzyme layer b e n e a t h a surface l a y e r of i n e r t p r o t e i n . T h u s the designs of the catalyst and the reactor influence the pH profiles adjacent to a n d w i t h i n the catalyst particles. These profiles influence greatly the yield of 6-A~A a n d the o p e r a t i o n a l stability of the i m m o b i l i z e d enzyme, both of w h i c h are key factors i n terms of e c o n o m i c operation. REFERENCES. 1. Lilly, M. D. (1978) Pro¢. ]st European Congress of Bioteehnologll, Deehema Monographs, vo]. 82, 165. 2. Carleysmith, S. W., Dunnill, P. ~ Lilly, M. D. (1979) Bioteehnol. Bioenff., in press. 3. Carleysmith, S. W., Eames, M. B. L. ~ Lilly, M. D. (1979) Bioteehnol. Bioeng., in press. 4. Sato, T., Tosa, T. • Chibata, I. (1976) Eur. J. Appl. Microbiol., 2, 153. 5. Klein, J., Wagner, F., Washausen, P., Eng, H. Martin, C. K. A. (1978) Proc. 1st Eur. Congr. Biotechnol., part I, p. 190. 6. Warhurton, D., Balasingham, K., Dunnil], P. • Lilly, M. D. (1972) Biochim. Biophys. Acta, 284, 278. 7. Lagerl6f, E., Nathorst-Westfelt, L., EkstrSm, B. SjSberg, B., in <
I m m o b i l i z e d penicillin acylase : benzylpenicillin deacylation. 11. Kutzbach, C. • Rauenbuseh, E. (1974) Hoppe-Seyler's Z. Physiol. Chem., 354, 45. 12. Berezin, I. V., Klyesov, A. A., Margolin, A. L., Nys, P. S. ~ Savitskaya, E. M. (1976) Antibiotikii, 21, 519.
BIOCHIMIE, 1980, 6~, n ° 5-6.
321
13. Szentirmai, S. (1965) Aeta Microblologica Aeademiae Seientiarum Hungaricae, 12, 395. 14. Carleysmith, S. W. a Lilly, M. D. (1979) Biotechnol. Bioeng., 21, 1057. 15. Engasser, J. M. a Horvath, C. (1974) Biochim. Biophys. Acta, ~58, 178.
Deacylation of benzylpenicillin by immobilized penicillin acylase. M. D. LI,LLY, S. W. CARLEYSMITH ( ' ) and P. DUNNILL.
Department of Chemical and Biochemical Engineering, University College London, Enghmd.
Rdsum~.
Summary.
Les pr6parations de p6nicilline acylase immobilis6es ont montr6 de meilleures activit6s (par unit6 de volume) que les pr6parations de cellules immobilis6es.
Immobilized penicillin acylase preparations h a v e much hiqher activities per unit volume than immobilized cell preparations. Many parameters of the deacylation reaction are dependent on pH and both reactant and one of the products, 6-aminopenicillanic acid, are acid and alkali labile. Acid is produced as result of the deacylation reaction and must be neutralised. The influence of these pH effects on the design of the catalyst and the reactor is discussed.
De nombreux param~tres de la r6action de d6acylation sont d6pendants du pH. L'influence de ces effets de pH sur la conception d u catalyseur et du r6acteur est discut6e.
Introduction. 6 - A m i n o p e n i c i l l a n i c acid has been p r o d r c e d c o m m e r c i a l l y for some years by the de a c y l a t i o n of b e n z y l p e n i c i l l i n u s i n g i m m o b i l i z e d p e n i c i l l i n acylase [11. In c o n t r a s t to most o t h e r co,mmercial p r o c e s s e s using i m m o b i l i z e d e n z y m e s or imm o b i l i z e d cells, the d e a c y l a t i o n of b e n z y l p e n i c i l lin results in the f o r m a t i o n of an acid, p h e n y l a c e tic acid, w h i c h a.t the n o r m a l o p e r a t i n g p H is dissociated. To m a i n t a i n tire reaction pH in the desired range it is t h e r e f o r e n e c e s s a r y to add alkali or use large a m o u n t s of a b u f f e r i n g salt. Thus the design and o p e r a t i o n of an i m m o b i l i z e d p e n i c i l l i n acylase r e a c t o r is greatly influenced I)y the effect of p H on the r e a c t i o n p a r a m e t e r s and the p r o p e r ties of the i m m o b i l i z e d enzyme, r e a c t a n t and l)roducts. Some of these effects are discussed in this paper.
Materials and Methods. Penicillin aeylase (partially-purified from Escherichia eoli), benzylpenieillin an.d 6-aminopenicillanic acid (6-APA) were supplied by Beecham Pharmaceuticals (U.K. Division). Immobilized penicillin acylase was prepared by covalent binding of the enzyme to
(') Present address : Beecham Pharmaceuticals (U.K. Division), Worthing, England.
Amberlite XAD-7 beads (420-850 p+m, Robin and Haas (UK) Ltd, Croydon, England) [2]. The enzyme forms a thin layer within the bead, the depth of which can be controlled by adjusting the reaction conditions [3]. Both soluble and immobilized penicillin acylase were assayed at 37°C in a pH-stat as described previously.
Results. Comparison of activities o[ immobili:ed penicillin acylase preparations. T h e l a c t a m rings of b e n z y l p e n i c i l l i n and 6-APA are both acid and alkali-labile. It is essential ther e f o r e that the acid p r o d u c e d d u r i n g the d e a c y l a tion r e a c t i o n is r a p i d l y n e u t r a l i s e d w i t h o u t causing regions of h i g h a l k a l i n i t y w i t h i n the reactor. E v e n u n d e r the most f a v o u r a b l e c o n d i t i o n s a finite rate of c h e m i c a l h y d r o l y s i s of the l a c t a m r i n g occurs and t h e r e is a c o n s i d e r a b l e i n c e n t i v e to re~ duce the r e s i d e n c e times of r e a c t a n t and p r o d u c t s in the reactor. This may be a c h i e v e d by u s i n g a h i g h c a t a l y t i c a c t i v i t y / u n i t v o l u m e of reactor. It is of interest t h e r e f o r e to c o m p a r e the a c t i v i t i e s of both i m m o b i l i z e d p e n i c i l l i n acylase and cells c o n t a i n i n g the e n z y m e (table I). As e x p e c t e d , t h e r e is a large d i f f e r e n c e b e t w e e n the a c t i v i t i e s of the i m m o b i l i z e d e n z y m e p r e p a r a t i o n s and the i m m o bilized cells. It is also w o r t h noting that the activity yield, tile m e a s u r e d a c t i v i t y of lhe p r e p a r a tion d i v i d e d by the amount of e n z y m e a c t i v i t y
M. D. Lilly and coll.
318
used in the i m m o b i l i z a t i o n p r o c e d u r e , is either s i m i l a r to or higher than for tim e n z y m e p r e p a r a tions, p a r t l y because of the larger particle sizes of the e n t r a p p e d cell p r e p a r a t i o n s .
Effect of pH on the reaction equilibrium. The deacylation of b e n z y l p e n i c i l l i n can be represented b y A~--P+Q+H
E[lect of pH on the activity and stability of penicillin acylase. The i n i t i a l activity of soluble p e n i c i l l i n acylase on 200 mM b e n z y l p e n i c i l l i n was greatest at pH 8 (figure 1). The a s y m m e t r i c pH-activity profile is s i m i l a r to those d e s c r i b e d by W a r b u r t o n [10] and Kutzbach a n d R a u e n b u s c h [11]. Berezin et al. [12] and S z e n t i r m a i [13] r e p o r t e d more s y m m e t r i c a l profiles. The m e a s u r e d pH activity profile of Am-
TABLE I.
Immobilization of penicillin acylase.
E. colt Enzyme
Immobilization
Support
Catalyst
method
Polyacrylamide Polyacrylamide E p o x y resin DEAE-cellulose S e p h a d e x G200 Cellulose t r i a c e t a t e GMA/EDMA copolymer CM-cellulose Amberlite XAD7
Entrapment Entrapment Entrapment Triazine CNBr Entrapment Glutaraldehyde Glutaraldehyde Glutaraldehyde
Aetivily yield
Retained acti-
t per cent)
vity (per cent}
Activity (')
Reference
-40 40 -47 35 39 82 77
---45 --42 82 89
0.33 0.88 4.8 35 225 146 (+) 70(+)(25oC) 272 70
4 5 5 6 7 8 9 2 2
(*) mol/min/ma or tonne wet weight. (+) mol/min/tonne dry weight. All enzyme assays were done at 37°C unless otherwise stated.
w h e r e A, P, Q and H are b e n z y l p e n i c i l l i n , 6-APA, p h e n y l a c e t i c acid and h y d r o g e n ion respectively. The e q u i l i b r i u m constant, K', can be w r i t t e n (ass u m i n g activity coefficients to be u n i t y ) as, - -
berlite XAD-7 p e n i c i l l i n acylase varies greatly dep e n d i n g on the c o n c e n t r a t i o n s of b e n z y l p e n i c i l l i n a n d buffer used.
[P] [Q] [H] K ~
or
c~
[A] [P] LQ]
log K' = log
[A]
1980,
62,
n ° 5-6.
1.0
oC¢
pH.
Solutions of 10' 1rim b e n z y l p e n i c i l l i n were i n c u b a ted at 37°C w i t h p e n i c i l l i n acylase at various fixed pH values in the pH-stat apparatus. A t t a i n m e n t of r e a c t i o n e q u i l i b r i u m was i n d i c a t e d by the cessation of alkali uptake. 'l-Tie degree of c o n v e r s i o n of b e n z y l p e n i c i l l i n to 6-APA was calculated from the m o l a r uptake of alkali after c o r r e c t i n g for the partial p r o l o n a t i o n of 6-APA. The e q u i l i b r i u m conversions for an i n i t i a l b e n z y l p e n i c i l l i n c o n c e n t r a tion of 10 mM are s h o w n in figure 1. Using the exp e r i m e n t a l l y derived value of K' (3 × 10~7 MU), values for the e q u i l i b r i u m c o n v e r s i o n s at various pH values were calculated for 200 mM b e n z y l p e n i c i l 1in a n d are also s h o w n in tigure 1, w h i c h shows that at this higher c o n c e n t r a t i o n high yields of 6-APA can only be o b t a i n e d at pH values above 7.5. BIOCHIMIE,
¢,
(L, . _ O
>,
',...O
dd >
¢~0.5 Et::3
/
klJ 0::
4
t
I
I
I
I
I
5
6
7 pH
8
9
10
FIG. 1 . - The influence of pH on the equilibrium conversion of 10 mM (O) and 200 mM ( - - - - ) benzylpeni-
cillin to 6-aminopenieillanie acid at 37°C. The effect of pH on the rate of enzymic deacylation (expressed as a proportion of the maximal rate) is also shown (Q).
Tim influence of pH on the stability of Amberlite XAD-7 p e n i c i l l i n acylase at 37°C has been described elsewhere [14]. The n l a x i m u m half-life
I m m o b i l i z e d penicillin acylase : benzyIpenicillin (943 hours) w a s at pH 350 h o u r s at p H 8.
6.6 and
fell to about
Internal p H changes in Amberlite XAD-7 penicillin acylase. T h e .effects of p H d e s c r i b e d so far h a v e been c o n c e r n e d w i t h the bulk pH. W i t h the e n z y m e i m m o b i l i z e d in A m b e r l i t e XAD-7, w h i c h has a po-
1.0
/
z o
,/ /," \1
>0.5 z o
u
;,
/
/
I
1
319
W h e n the curves in figure 2 are s u p e r i m p o s e d by r e p l o t t i n g w i t h all the i n i t i a l rates set equal to tile initial rate of c u r v e 3, t h e i r shapes can be c o m p a r e d m o r e r e a d i l y (figure 3). T]~e d e g r e e of i n h i b i t i o n of the u n g r o u n d or u n b u f f e r e d i m m o b i l i z e d e n z y m e then a p p e a r e d to be less. T h e rate of r e a c t i o n f o r the g r o u n d m a t e r i a l w i t h buffer p r e s e n t w a s fast i n i t i a l l y b u t as p r o d u c t s q u i c k l y a c c u m u l a t e d , i n h i b i t i o n r a p i d l y d e c r e a s e d the r e a c t i o n rate. As p r o d u c t s w e r e f o r m e d w i t h the u n b u f f e r e d or u n g r o u n d e n z y m e p r e p a r a t i o n s , the r e a c t i o n rate fell r e l a t i v e l y m o r e s l o w l y f o r t w o reasons. First, as the rate of h y d r o g e n ion p r o d u c tion fell the p H rose i n s i d e the beads i n c r e a s i n g the a c t i v i t y of the i m m o b i l i z e d enzyme. S e c o n d l y , and m o r e i m p o r t a n t l y the p r o d u c t , 6-&PA, a c t e d as a buffer e n h a n c i n g the rate of h y d r o g e n ion t r a n s f e r to the s u r f a c e of the beads [15]. T h e net r e s u l t w a s that the r e a c t i o n profile was i n i t i a l l y s t r a i g h t e r (figure 3).
I
0
5 10 =15 TIME (T) Fro. 2. - - Progress curves for the conversion o[ 0.1 M benzylpenicillin to 6-APA, eatalysed by Amberlite XAD-7 penicillin acylase at 37°C and pH 8. The conditions for each curve were as follows : 1. unground immobilized enzyme with no added buffer ; 2. unground preparation with added phosphate buffer (0.1 M) ; 3. ground preparation with added phosphate buffer. The unit of time, x, is the time that would be required for eornplete eor~version of the benzylpenieillin if the rate of reaction remained eonstant at the initial rate of curve 3.
1.0
--
~
2
z
/
uJ 0.5 7 0 (..)
.
f
~
'
,
i
rous structure, w e h a v e s h o w n [2] that the p H inside the beads, w h e n b e i n g used in a reactor, fails to about p H 4.5, at w h i c h p H the rate of reaction in the beads is p r e d i c t e d to be about h a l f that o b s e r v e d at pH 8. Thus in the absence of buffer the r e a c t i o n at a buIk p H of 8 p r o c e e d s m o r e s l o w l y than e x p e c t e d (figure 2). A d d i t i o n of a buff e r i n g salt to the r e a c t o r c o n t a i n i n g i m m o b i l i z e d p e n i c i l l i n acylase caused a large i n c r e a s e in the rate of r e a c t i o n (figure 2). Observation of the imm o b i l i z e d e n z y m e beads c o n t a i n i n g a d s o r b e d pHi n d i c a t i n g dye in a r e a c t o r s h o w e d that the effect of a d d e d buffers w a s to raise the p H near the surface of the beads a b o v e 7, c o m p a r e d to 5 or less in the absence of buffer. H o w e v e r , even in the p r e s e n c e of buffer a p H g r a d i e n t still e x i s t e d in lhe beads w i t h the i n t e r i o r at about pH 5. Grind i n g of tile beads led to a f u r t h e r i n c r e a s e in the r e a c t i o n rate (iigure 2) and the r e s u l t i n g b a t c h r e a c t i o n profile w a s almost i d e n t i c a l in shape to that observed w i t h the soluble enzyme.
%
-
1~',"Y , ~ 3
0
0
BIOCHIMIE, 1980, 62, n ° 5-6.
deacylation.
I
1
t
i
2
i
I
i
3
TIME(T) FIe. 3. Progress curves for the conditions described in [igure 2. The curves have been superimposed by setting the initial slopes of all the curves to be equal to the initial slope of curve 3, -
-
Operational stability o[ the zyme.
immobilized
en-
W h e n the i m m o b i l i z e d e n z y m e w a s used in a four-stage c o n t i n u o u s s t i r r e d tank r e a c t o r w i t h o u t a d d e d buffer, the o p e r a t i o n a l stability w a s m u c h less than e x p e c t e d on the basis of the k n o w n storage s t a b i l i t y at 37°C [14]. In the p r e s e n c e of a large c o n c e n t r a t i o n (0.1 M) of p h o s p h a t e buffer the s t a b i l i t y w a s greater but still less t h a n expected. T h e m a i n reason for loss of a c t i v i t y w a s inactivation by alkali added to n l a i n t a i n a c o n s t a n t bulk pH. It seems likely that the e n z y m e close to or on the s u r f a c e of the beads w a s being e x p o s e d to l o c a l i z e d r e g i o n s of high pH. This loss can be
320
M. D. L i l l y a n d coll.
r e d u c e d b y i m p r o v i n g the m i x i n g in the r e a c t o r a n d / o r r e d u c i n g the c o n c e n t r a t i o n of the alkali feed. An alternative a p p r o a c h is to locate the enzyme l a y e r below the surface of the bead. W e have exam i n e d elsewhere [3] the rate of p e n e t r a t i o n of b o v i n e s e r u m albumen, w h i c h has a s i m i l a r molecular w e i g h t to l~enicillin acylase, into Amberlite X.~D-7 beads. Using this i n f o r m a t i o n we first i m m o b i l i z e d a t h i n l a y e r of BSA a d j a c e n t to the surface of beads a n d then i m m o b i l i z e d p e n i c i l l i n acylase as a layer b e n e a t h the BSA. T h i s modified i m m o b i l i z e d p e n i c i l l i n acylase a n d a p r e p a r a t i o n made in the n o r m a l w a y w e r e used i n three consecutive batch reactions a n d the activities remain i n g after each reaction w e r e m e a s u r e d (figure 4).
100 o
<
50
cO
E cE
1 2 3 Number of Batch Uses Fro. 4 . The proportion of the initial activity remaining after reuse in a batch reactor for immobilized penicillin acylase (Q) and immobilized penicillin acylase pretreated with bovine serum albumen (O). Batch reaction conditions, - - initial benzy]penicillin concentration, 0.2 M ; temperature, 37°C ; pH 8, maintained by addition of 5 N NaOH.
The r e a c t i o n c o n d i t i o n s were designed to cause r e a s o n a b l y fast loss of activity of the n o r m a l preparation. The location of the enzyme l a y e r below a BSA layer clearly reduces the rate of loss of activity.
Discussion. F r o m the results described in this p a p e r it is clear that pH has a m a r k e d effect on the perform a n c e and b e h a v i o u r of a reactor for tile deacylation of b e n z y l p e n i c i l l i n , ttigh c o n v e r s i o n s to BIOCHIMIE, 1980, 62, n ° 5-6.
6-APA can o n l y be achieved w h e n the bulk pH, at least at the e n d of the reaction, is close to p H 8, w h i c h is also the value for m a x i m u m e n z y m i c activity. However, this is n o t the o p t i m u m pH for enzyme stability a n d there is a positive advantage in a l l o w i n g the pH w i t h i n the beads to fall since the e n z y m e is then more stable. Even if the pH drops to 6 the e n z y m e w i l l be over twice as stable as at pH 8 a n d the activity w i l l only be 20 p e r cent l o w e r (figure 1). F o r the i m m o b i l i z a t i o n c o n d i t i o n s used, the l a y e r of b o u n d e n z y m e r e p r e s e n t s only a small p r o p o r t i o n of the total volume of the Amberlite XAD-7 beads. On a v o l u m e t r i c basis the activity is still m u c h h i g h e r t h a n those for i m m o b i l i z e d cells (table I) a n d the particles are a more m a n a geable size t h a n some of the other enzyme s u p p o r t materials listed i n table I. Although more enzyme c a n be i m m o b i l i z e d i n the i n t e r i o r of the Amberlite XAD~7 beads, it w o u l d not p a r t i c i p a t e effectively i n the d e a c y l a t i o n reaction. F u r t h e r m o r e , i n a large reactor, w h e r e good m i x i n g is more difficult, a h i g h e r catalytic activity per u n i t volume w o u l d i n c r e a s e the likelihood of d e n a t u r a t i o n of enzyme n e a r the surface due to localised high pH. As s h o w n i n figure 4 this loss can be greatly reduced b y p l a c i n g the enzyme layer b e n e a t h a surface l a y e r of i n e r t p r o t e i n . T h u s the designs of the catalyst and the reactor influence the pH profiles adjacent to a n d w i t h i n the catalyst particles. These profiles influence greatly the yield of 6-A~A a n d the o p e r a t i o n a l stability of the i m m o b i l i z e d enzyme, both of w h i c h are key factors i n terms of e c o n o m i c operation. REFERENCES. 1. Lilly, M. D. (1978) Pro¢. ]st European Congress of Bioteehnologll, Deehema Monographs, vo]. 82, 165. 2. Carleysmith, S. W., Dunnill, P. ~ Lilly, M. D. (1979) Bioteehnol. Bioenff., in press. 3. Carleysmith, S. W., Eames, M. B. L. ~ Lilly, M. D. (1979) Bioteehnol. Bioeng., in press. 4. Sato, T., Tosa, T. • Chibata, I. (1976) Eur. J. Appl. Microbiol., 2, 153. 5. Klein, J., Wagner, F., Washausen, P., Eng, H. Martin, C. K. A. (1978) Proc. 1st Eur. Congr. Biotechnol., part I, p. 190. 6. Warhurton, D., Balasingham, K., Dunnil], P. • Lilly, M. D. (1972) Biochim. Biophys. Acta, 284, 278. 7. Lagerl6f, E., Nathorst-Westfelt, L., EkstrSm, B. SjSberg, B., in <
I m m o b i l i z e d penicillin acylase : benzylpenicillin deacylation. 11. Kutzbach, C. • Rauenbuseh, E. (1974) Hoppe-Seyler's Z. Physiol. Chem., 354, 45. 12. Berezin, I. V., Klyesov, A. A., Margolin, A. L., Nys, P. S. ~ Savitskaya, E. M. (1976) Antibiotikii, 21, 519.
BIOCHIMIE, 1980, 6~, n ° 5-6.
321
13. Szentirmai, S. (1965) Aeta Microblologica Aeademiae Seientiarum Hungaricae, 12, 395. 14. Carleysmith, S. W. a Lilly, M. D. (1979) Biotechnol. Bioeng., 21, 1057. 15. Engasser, J. M. a Horvath, C. (1974) Biochim. Biophys. Acta, ~58, 178.