J. Chem. Tech. Biotechnol. (1992), 53, 205-214

Characterization and Use of a Penicillin Acylase Biocatalyst S. Sonia Ospina Departamento de Farmacia, Instituto de Biotecnologia, Universidad Nacional de Colombia, Apartado Aereo 14990, Bogota, Colombia

Agustin Lopez-Munguia,* Rosa Luz Gonzalez & Rodolfo Quintero Instituto de Biotecnologia, Universidad Nacional Autonoma de Mexico, Apartado Postal 510-3, Cuernavaca, Mor., 62271, Mexico (Received 28 March 1991 ; revised version received 1 July 1991 ; accepted 13 September 1991)

Abstract : A complete characterization of a penicillin acylase biocatalyst is presented, including the determination of physicochemical and kinetic parameters. Stability studies are detailed in terms of both storage temperature and pH as well as operational stability after 150 batch reactions of two hours duration each. An Arrhenius-type model was used to simulate the effect of pH on biocatalyst stability. A kinetic model is proposed to describe batch and continuous stirred tank reactors and to predict the long-term behavior of the process.

Key words : penicillin acylase, characterization, stability, reactors.

se,

NOTATION Constant in eqn ( 2 ) (s-l) 6-aminopenicillanic acid concentration (mmol dm-3) Accumulated productivity (g 6-APA (g cat)-' h-') Constant in eqn (2) (dimensionless) Batch productivity (g 6-APA (g cat)-' h-l) Continuous stirred tank reactor First-order enzyme deactivation constant (s-l) 6-APA non-competitive inhibition constant (mmol dm-3) Michaelis-Menten constant (mmol dm-7 PAA competitive inhibition constant (mmol dm-3) Substrate inhibition constant (mmol dm-3) Phenylacetic acid concentration (mmol dm-s) Potassium salt of penicillin G concentration (mmol dm-7 PGK concentration (mmol dm-7 Corrected PGK concentration (mmol dm-3)

* To whom correspondence

should be addressed.

J . Chem. Tech. Biotechnol. 0268-2575/92/$05.00

so t ui

Vm,,

X

x Y

r, 8

PGK concentration at equilibrium (mmol dm-3) Initial PGK concentration (mmol dm-3) Time (min) Initial reaction rate (mmol dm-3 min-l) Maximum initial rate in Michaelis-Menten equation (mmol dm-3) min-') PGK conversion (dimensionless) Corrected PGK conversion (dimensionless) Accumulated yield (g of 6-APA (g biocata1yst)-1) Batch yield (g of 6-APA (g biocata1yst)-' per batch) Residence time (min)

1 INTRODUCTION

Penicillin amidase (PA) E.C. 3.5.1.1 1 hydrolyses penicillin G (PG) or penicillin V (PV) to yield 6-aminopenicillanic acid (6-APA) and phenylacetic acid (PAA). Penicillin acylase is one of the few immobilized enzymes in use at the industrial scale, with annual sales of $6 million,' below those of glucose isomerase with $20

205 0 1992 SCI. Printed in Great Britain

206

S. S. Ospina, A . Lopez-Munguia, R. L. Gonzalez, R. Quintero

million dollars. The annual production of 5 tons of various penicillin acylase biocatalysts supports the world market of approximately 4500 tons of 6-APA which is the raw material for semisynthetic penicillins.' The enzyme is produced by a wide variety of microorganisms even after eliminating those which also exhibit b-lactamase activity. The microorganisms most frequently reported in the literature for PG-acylase are strains of Escherichia ~ o l i , Proteus ~ . ~ r u t g ~ r i ,Bacillus ~ meguterium6 and, for PV-acylase, Fusarium oxisporium' and Kluyveru citrophila.' The penicillin acylase from E. coli has been extensively studied and most industrial biocatalysts use this enzyme, The enzyme is a periplasmic protein produced by growth of the organism at pH 7 and 2430°C in a medium which includes PAA as an inducer. Two important conditions for the process are the requirement for low oxygen transfer and the absence of fermentable sugars.' After biomass production, penicillin acylase biocatalysts may take various forms either as free or immobilized microorganisms'"~l 1 or as immobilized enzyme, after partial purificati~n.'~-'~ In Table 1 the main industrial

penicillin acylase biocatalysts are presented, together with certain of their properties (reaction pH and temperature, type of industrial reactor employed, halflife, productivity and initial activity of biocatalyst). Kinetic data from laboratory reports have been recently presented in a review by Shewale and Sivaraman." However, kinetic information concerning industrial biocatalysts is rare. A general agreement exists that the enzyme is inhibited by excess substrate in addition to the two products. Kinetic information concerning PA biocatalysts is reported in Table 2, which includes the biocatalyst ' Semacylase' of Novo Industries. The ease of pH control has placed the batch reactor as the best operational alternative for PG hydrolysis."j However, deficiencies in the design of the pH control system and in the mixing conditions often cause a shortening of the life of biocatalysts. Productivity and yield are the fundamental criteria to determine the industrial feasibility of a PA biocatalyst. However, in spite of the importance of the product, there are few reports in the literature concerning the effect of temperature and pH on PA biocatalysts.

TABLE 1

Properties of Industrial Penicillin Acylase Biocatalysts Biocatalyst

Company

Reactor

pH

PC

Activity ( U g-'1

(h)

1000

't

Productivity (kg 6-APA kg eat-')

Reference

25&300

28

757

15

29

-

PGacylase E. coli (Eupergit PCA)

Rohm Pharma

STRa

8.0

31

100-150

PGacylase E. coli (Sephadex (3200)

Astra Alab AB

RPR*

7.8

35

20&250

PGacylase recombinant E . coli (Polyacrylamide)

Boehringer Mannheim

STR

8.9

28

310-360

100&1500

1000

PGacylase E. coli PGacylase B. megarerium (polyacrylonitrile fibers)

Beecham Toyo Jozo

RPR MCPR'

7-8 8.4

-

37

200

200w000 1200

100&2000 500-700

PGacylase E. coli (cellulose) PGacylase E. coli (amherlite)

Hindustan Antibiotics LTDA Orsabe Mexico

STR

7.8-8

37

600

STR

8.0

37

180

PGacylase E. cnli

Cipan Portugal

RPR

7.9

PGacylase E. coli cephalosporin C

Cipan Portugal

PGacylase PGacylase

600 30 cycles (4 months)

I00

15

-

2248 1090

2 2

-

30 31

RPR

8.0

Sclavo Italy

-

7.8

28

I50

500 cycles (4 months)

400

32

Sclavo Italy

-

7.8

28

150

355

32

RPR STR

7.0 7.5

35

60

300 cycles (4 months) 2000 1000-2000

219 (24

25 21

PVacylase (Novozym) Novo Novo PVacylase (Semacylase)

"

-

-

STR: Stirred tank reactor. RPR: Recirculated packed reactor. Multicolumn packed reactor.

-

-

Characterization and use of a penicillin acylase biocatalyst

207

TABLE 2 Kinetic Properties of Penicillin Acylase Biocatalysts Biocatalyst

Trc)

Pff

Knl

I(,,, (nmol dm-7

KP,,

lu,

Reference

fmmof dm-3) (mmol dm-3)

~

Free PA PA in Chitosan PA in Amberlite XAD-7 PA in DEAE-cellulose Semacylase (Novo) PA in activated silica gel

37 37 37 37 37 -

7.8 8.0 7.8 8.0 7.0 7.8

002-1.1 2.22 5-7 0.63 10.0 0.39

In the present article, we describe several characteristics of a PA biocatalyst produced at pilot-plant scale. The biocatalyst stability is studied in terms of pH, temperature and enzyme re-use. A kinetic model is proposed to describe data from batch and continuous reactors and to stimulate the expected performance of the process.

2 MATERIALS AND METHODS

0.24-1 5

0.249

13G270

-

-

-

-

9.0 200.0 -

-

4.6 50 (FA) -

-

250 -

4,12, 15,24 12 3 26 27 14

Chemical Co., USA) were used as standards and for inhibition studies. 2.3 Storage stability The biocatalyst (0.5 g) was stored in 50cm3 of 0.1 rnol dm-3 phosphate buffer without glycerol at 37, 45 and 49°C and pH values in the range 6.0-8.0. Samples of biocatalyst were withdrawn and assayed for PA activity as defined in Section 2.2 and returned to the storage conditions.

2.1 Biocatalyst preparation The biocatalyst was provided by Genin (Mexico D.F.) through a United Nations project'? and has an activity of 160-1 80 U g-l catalyst (wet weight), particle size of 100-200 pm; density of 1.02 g cm-3 and a porosity of 0.5. This biocatalyst is produced with PA from E. coli after extraction and purification by ion exchange and immobilization by covalent linkage with an epoxyacrylic resin. The biocatalyst is stored refrigerated with 30% (w/v) glycerol. The wet biocatalyst is prepared after vacuum filtration and washing with 0.03 rnol dm-3 phosphate buffer pH 7.8 and has a water content of about 75 % (w/w). For enzymatic reactions, the potassium salt of penicillin G, PGK (Hoechst, Germany) was typically used at 10 YO(w/v) concentration with the amount of biocatalyst defined as either 'enzyme load' (units of penicillin acylase activity per gram of penicillin) or as 'enzyme concentration ' (units of penicillin acylase activity per cm3 of reaction volume).

2.2 Enzyme activity

Enzyme activity was determined by incubating 10 mg of biocatalyst for 5 min in 3 cm3 of 2 YO(w/v) PGK solution in 0.1 rnol dm-3 phosphate buffer pH 7.8 at 37°C and following 6-APA formation with paradimethyl aminobenzaldehyde.18 One activity unit is defined as the amount of enzyme producing 1 pmol min-l of 6-APA under the conditions defined. PAA and 6-APA (Sigma

2.4 Operational stability

Two-hour reactions at 37°C with pH controlled between 7.45 and 7.55 (using 2 mol dm-3 NH,OH) were carried out sequentially (5 per day during 30 days). The final conversion and the amount of added base were recorded. The reactor volume was 50 cm3 with 10 % (w/v) PGK in 0.03 mol dm-3 phosphate buffer pH 7.5 and an enzyme load of 120 U g-' of PGK. The reactor was agitated with a low shear glass impeller of in-house design. After every 25 batches, the activity of the biocatalyst was determined.

2.5 Kinetic properties Initial rate measurements were carried out in a recirculated packed-bed differential reactor containing 100 mg of biocatalyst. The reaction volume was 30 cm3 of 10% (w/v) PGK solution in 0.1 mol dm-3 phosphate buffer pH 7.8 and the recirculation flow rate was 70 cm3 min-l. External diffusional limitations were determined to be negligible at this flow rate. The equilibrium constant was measured from the reverse reaction under the same reaction conditions found at the end of hydrolysis from 10% (w/v) PGK (0.268 mol of 6-APA and PAA, 120 U g-' PGK) by following the amount of PGK using an HPLC system (Spectra Physics Modular 8700B) equipped with a 220 nm UV detector and a p-Bondapack C,, column. The elution system consisted of a linear gradient from 90% (v/v) NaH,PO, (0.05 rnol ~ m - ~ and ) 10% (v/v)

S . S. Ospina, A . Lopez-Munguia, R. L. Gonzalez, R.Quintero

208 methanol to 50 YO(v/v) NaH,PO, and 50 % (v/v) methanol. 2.6

Continuous reaction

A continuous stirred tank reactor (CSTR) was employed for continuous hydrolysis of PGK. The substrate solution was sequentially pumped from a storage solution at 4°C to a tank at 37°C and to a 50 cm3 CSTR. The pH was controlled in the reactor by addition of 2 mol cm-R NH,OH and the volume was maintained constant by use of a second pump.

0 60

3 RESULTS AND DISCUSSION

I

I

I

I

65

7.0

7.5

80

85

PH

3.1 Effects of temperature and pH on biocatalyst activity The activity profile as a function of reaction temperature was examined first. The optimum reaction temperature occurred at 49"C, which is in agreement with various reports for free and immobilized penicillin a c y l a ~ e s . ' ~ , ' ~ From these results and in accordance with the Arrhenius model, an activation energy of 4-59 x lo4 J mo1-l was determined. This value is comparable to other activation values, which include data for the free enzyme.,, 1 7 . 1 9 In the PA reaction, pH is a critical parameter having a direct effect on the stability of the biocatalyst, PGK and 6-APA.2".21 Maximum activity was found in the pH range 7.8-8.0 at 49°C. However, when temperature was decreased to 37"C, the pH range for activity increased to 7.0-8.0 even though the activity was only 60% of the maximum (data not presented). For this reason, most PA biocatalysts are used at temperatures lower than 40°C as shown in Table I . To further characterize the effect of temperature, three experiments to measure storage stability were carried out at 37, 45 and 49°C and at pH values of 7.5 and 7.8, respectively. At 49°C (half-life of 28 h at pH 7.5) the activity of the biocatalyst was observed to be lost rapidly when compared to the stability at 37°C (half-life of 2880 h at pH 7.5). It was found that, even at 37"C, the half-life decreases to 880 h when the pH is increased to 7.8. In consideration of these preliminary results and with the requirement for pH regulation during the reaction, the effect of pH on biocatalyst stability was studied in further detail.

Fig. 1. Effect of storage pH on the half-life of the PA biocatalyst at 37°C in 0.1 mol dm-3 phosphate buffer. Average of three measurements.

where E is the enzyme activity and k is the first-order deactivation constant, in this case a function of the storage pH. With regard to pH, the stability profile is sharper than the activity profile both having an optimum value at pH 7.5. Under optimal operating conditions (37°C and strict pH control at 7.5), the extrapolated halflife of the biocatalyst is 2880 h, within the range of those reported in Table 1. The effect of pH on the first-order deactivation constant can be fitted to an Arrhenius-type model as follows: k = A exp [B/pH] where and B = 37.1 for pH < 7.5

A = 1.64 x and A

=

3.26 x 10' and B = - 137.98 for pH > 7.5

5

3.2 Effect of pH on biocatalyst stability Storage stability experiments were performed at 37°C and in a pH range of 6.0 to 8.5. The results shown in Fig. 1 are presented in terms of half-life, considering that first-order deactivation kinetics was observed in all cases, i.e. dE -- = k(pH) E dt

-9 011

I 012

I 013

I

I

015

014

I

016

I

1 -

PH

Fig. 2. Graphical representation of an Arrhenius-type model applied to describe the effect of pH on the first-order deactivation constants of the PA biocatalyst stored at 37°C.

Characterization and use of a penicillin acylase hiocatalyst

209

100

90

(%) 80

70

I

I

I

I

I

I

I

I

I

15

30

45

60

75

90

105

120

135

150

Batch number

Fig. 3. Operation stability experiments with the PA biocatalyst; 150 batch reactions (each of 2 h duration) were performed at 10% (w/v) PGK 0.03 rnol drnP phosphate buffer pH 7.5, at 37°C with pH control at 7.5 using 2 mol dm-3 NH,OH. Initial biocatalyst activity was 170 U g-' with 3.4 g of biocatalyst in 50 cm3 of reaction volume. (-0- activity; -7- conversion.)

which are the constants fitted to the data shown in Fig. 2. No reports exist in the literature of the effect of pH on the stability of PA industrial biocatalysts. Therefore, the proposed equations may be used to predict the behavior of the biocatalyst under different reaction conditions, including the frequent problems of imperfect mixing found in industrial reactors.

3.3 Operational stability From the results shown above, storage stability data for PA do not necessarily provide information on the real operational stability of the biocatalyst. We therefore carried out 150 batch reactions of 2 h each at 37°C and with pH regulation at pH 7.5. These results are shown in Fig. 3. In batch number 76, a sudden drop in biocatalyst activity was caused by an undefined contaminant in the water. For this reason, a half-life of 11 55 h was calculated by extrapolation of the data from the first 75 reactions, less than half the time predicted from the storage stability experiments where the pH was maintained constant at pH 7.5. During the batch reactions, the pH oscillated between approximately pH 7.45 and pH 7.55. This observation demonstrates the need for a rigorous pH control system, including good mixing, fast response time of electrodes and good distribution of diluted alkali in order to protect PA biocatalysts from pH deactivation. Nevertheless, the half-life obtained is within an order of magnitude of those shown in Table 1. Many enzymes have a higher operational stability than expected due to the stabilizing effect of the substrate. However, in this case such behavior was not observed due to the denaturing effect caused by the repeated changes in pH. 3.4 Kinetic behavior

In order to develop a rational design of the 6-APA production process, initial rate experiments were performed to determine the classical enzyme kinetic par17

ameters. PA is generally recognized to be inhibited by substrate" described by the following model : u. =

'

Vmax S Km+S+S2/Ks

(3)

Taking the inverse of eqn (3):

where Km and Vmax are the Michaelis-Menten parameters and K, is the substrate inhibition constant. From data at low substrate concentrations presented in Fig. 4(a), the values for Km and VmaXof the biocatalyst were found to be 4.17 mmol dm-3 and 170 U g-' biocatalyst, respectively. In the same figure, the substrate inhibition is evident. Therefore, in Fig. 4(b) a modified plot is used to determine the inhibition constant ( K J , neglecting the first term of the equation at high substrate concentrations. K, was found to be 413 mmol dm-3, a high value when compared to those reported in Table 2. In Fig. 5 a plot of initial rates in the presence of PAA at low substrate concentrations, shows competitive inhibition behavior by the product, with an inhibition constant (Kpaa)of 68.6 mmol dm3. In order to determine the non-competitive inhibition constant (Kapa)of the other product (dAPA), the method of measuring activity had to be modified since this is based upon the appearance of 6-APA. The low concentration method reported by Balasingham er aL4 and S a n d ~ v a l , yielded '~ no inhibition in the range of 6-APA concentrations studied. We therefore measured the inhibition constant by the following procedure. The reaction was monitored by following the amount of NH,OH (2 mol dm-3) needed to maintain the pH at 7.5 during the initial rate conditions. In the presence of 6-APA the initial rate is given by:

ui =

Vmax S

( )]:"[

(5)

( K m + s ) I+-

C T B 53

S . S . Ospina, A . Lopez-Munguia, R. L. Gonzalez, R. Quintero

210

I

200

100

300

m

I

400

1/S (mol-’dm3 ) I I00

I 200

I 300

400

1/S (mol-’dm3)

Fig. 5. Lineweaver-Burk representation of the experiments performed for the determination of the competitive inhibition constant of PAA in the PA reaction (37°C in 0.1 mol d m - $ phosphate buffer p H 7.5). Initial PAA concentration (% (w/v)): -B-, 1.5; -V-, 0.75; -n-, 0.

1.5

1

10 0 10

0 20

0 30

0 40

s( mol dm-3) Fig. 4. Michaelis-Menten kinetics for the PA biocatalyst at 37°C in 0.1 mol dm-3 phosphate buffer pH 7.5 for initial rate experiments. (a) Lineweaver-Burk representation ; (b) modification of the Lineweaver-Burk representation to determine the substrate inhibition constant.

0

6

3

where [6-APA] is the added initial concentration of the product. Considering the concentration of 6-APA to be in excess, this may be considered constant during the initial rate experiment. Then, eqn ( 5 ) for a batch reactor, of substrate conversion becomes: expressed in

(a,

12

9

15

Time ( m i n )

Fig. 6 . Graphical determination of the non-competitive inhibition constant for 6-APA, based on a plot of the integrated model: data for batch reactions with 10% (w/v) PGK in 50 cmYof 0.03 mol dm-3 phosphate buffer pH 7.5, pH regulation at p H 7.5 using 2 mol d m 3 NH,OH, 37°C and with enzyme load of 60 U g-’ PGK. Initial 6-APA concentration (YO(w/v)): -0-, 3.6; -B-, 2.4; -A- 1.8. 1.2; 0.6. -0-,0. ,

I

-up,

-a-

7

,

21 1

Characterization and use of u penicillin acyluse biocatalyst

If the substrate concentration during the experiment is sufficiently high to ensure saturating zero-order kinetics, but still far from excess substrate inhibition, then S % K,,, and eqn (6) may be simplified to:

( +-)]:a“‘

Vma,t = sox 1

or rearranging :

According to eqn (8), a plot of X versus t for the initial rate conditions should yield a straight line from which the inhibition constant (Kapa)may be determined. These results are shown in Fig. 6 for different 6-APA concentrations. From the gradient of the straight lines within Fig. 6, a value for Kapaof 100.7 mmol dm-3 was determined. After determination of the inhibition constants, different kinetic models as proposed in the literature were used to describe the experimental data. The best correlation was obtained from the mechanism : E-PAA

+ bAPA

E-PGK-PGK

I?

I?

PAA

PG K

+

JT E-PAA - 6-APA

E

+

+

P G K Z

E-PGK

+

1 T PAA

6-APA

+

+ E+6-APA+PAA

+ bAPA

?L

J? E-6-APA

E-PGK-&APA

3.5 Penicillin acylase reactors

Batch reactions in agitated tanks were carried out with a constant enzyme concentration (4.8 U ~ m - and ~ ) a PGK concentration range of 2-10% (w/v). In Fig. 7 the evolution of these reactions and the prediction of eqn (9) is shown. A good correlation is obtained at low substrate concentrations. Deviations occur at substrate concentrations higher than 8 % w/v, especially at the end of the reactions where the model predicts higher conversions than those observed. In these experiments, the ‘load’ of enzyme (U g-’ penicillin) decreases with increasing substrate. Furthermore, when loads lower than 80 U g-’ penicillin were used, high final conversions were never attained, even at long incubation times. In experiments carried out at constant enzyme load (thus varying the enzyme concentration) conversions higher than 93% were obtained at 12% (w/v) penicillin. Nevertheless at 15 YO(w/v) penicillin the final conversion decreased to 88 %. The recommended initial substrate concentration is thus between 10 and 12% (w/v). Fed-batch reactions were carried out in order to avoid excess substrate inhibition. However, no significant advantages were found in this reaction system : batch reactions with 15 % (w/v) penicillin, after numerical solution of eqn (9) with a V, of 12 mmol dm-3 min-I, predicts a reaction time of 130 min for 95 % conversion. If the inhibition effect of substrate is eliminated, the reaction time decreases to 1 15 min, while, if the product inhibition is removed, the reaction time decreases to 51 min. It is evident that the effect of product inhibition is stronger than substrate inhibition. Fed-batch mode therefore offers only a marginal advantage.

which is described by the following equation :22 ,,,a*

L

[PAA] [6-APA] [PAA] [6-APA]

+- Kapa vi={Km(l+r +

Kpaa Kapa

(9) The equation does not consider the reversibility of the reaction. The amount of penicillin formed at pH 7.5, 37°C and initial products concentration of 0.268 mol dm-3 was determined by the HPLC method described previously. An equilibrium constant of 6295 mmol dm-3 was found, which corresponds to a 97 YO conversion. Equation (9) was corrected considering that :

s = s- s,,

(10)

--

0

50

100

150

zoo

Reaction time ( m i n )

Fig. 7. PGK hydrolysis in batch reactors with the PA biocatalyst at different substrate concentrations as described by the corrected triple inhibition model. Reaction conditions : 4.8 U ~ r n - ~37°C; ; pH regulation at 7.5 using 2 mol dm-3 NH,OH. PGK concentration (% (w/v)): -0-.2 ; -0-, 4;

-A-, 6 ; -0-,8; -m-,

10. 17-2

S . S . Ospina, A . Lopez-Munguia, R. L. Gonzalez, R. Quintero

212

'--I

0

0

50

100

150

I

I

I

ZOO

400

600

-.-,

Fig. 8. PGK hydrolysis in a continuous stirred tank reactor at different substrate concentrations. Reaction conditions : enzyme load 120 U g-' PGK; 37°C; pH regulation at 7.5 using 2 mol dm-3 NH,OH. PGK concentration (% (w/v)): -Up, 5 ; ---O--, 8; 10.

A similar situation is found for the continuous stirred tank reactor (CSTR) which is described by:

where vi is obtained from eqn (9) and 9 is the residence time. Experiments carried out for three PGK concentrations and a constant enzyme load of 120 U g-' penicillin are shown in Fig. 8. A conversion higher than 93% was obtained (200 min residence time) only at the lowest substrate concentration (5 % w/v)). Equation (12) is suitable for predicting the reactor behavior only at PGK concentrations lower than 8 % (w/v). Increasing the enzyme load did not significantly increase the final conversion for experiments carried out at 10 % (w/v) penicillin. In the CSTR, the enzyme is constantly exposed to a high concentration of both products, thus being strongly inhibited. This eliminates the CSTR alternative when the productivity is compared with the results obtained in batch reactors. On the other hand, the pH stability results presented earlier render the use of the plug flow reactor inadequate for this reaction.

I

II

800

1000

1.

Batch number

200

Residence time (min)

I

Fig. 9. Computer simulation of the effect of the number of successive batch reactions on the yield, productivity and stability of PA biocatalyst, together with the batch reaction time needed for 95 YO conversion. Reaction conditions : 10 YO(w/v) PGK; enzyme load 120 U g-' PGK; 37°C. The pH was considered to be constant at pH 7.5.

batch yield of the biocatalyst, equal to 0.787 g of 6-APA (g biocatalyst)-l (from 10 YO (w/v) penicillin, 95 YO conversion and an enzyme load of 120 U g-' penicillin), after each batch simulation the following parameters were also derived:

Y (accumulated yield) = Y,, x number of batches BP (batch productivity)

=

ytl batch reaction time

AP (accumulated productivity)

=

Y accumulated time

These results are shown in Fig. 9. When the biocatalyst was used during one half-life, a yield of 510 kg 6-APA (kg biocdtalyst)-' was obtained. This is in the range of yields reported for the biocatalysts presently available, with the additional advantage of decreased reaction times (in the order of 2.5 h). For an extended biocatalyst use of two half-lives, the yield increases to 780 kg 6-APA (kg biocatalyst)-'. However, the required reaction time increases to 5 h. This is not recommended when considering the stability of substrate and products.

3.6 Process productivity 4

In order to evaluate biocatalyst productivity, a simulation program was carried out which considered the use of batch reactors with the results already shown. For each batch, carried out at 37°C with constant pH of 7.5, the time required to reach 95 % conversion was obtained from the solution of eqn (9) with 10% (w/v) penicillin for each batch. For each differential time increase during the integration process, the decrease in biocatalyst activity was determined according to a first-order deactivation model with a half-life of 1155 h (from experimental sequential batches). Considering Y,, the

CONCLUSIONS

In terms of productivity, stability and yield, the biocatalyst presented here is adequate for the 6-APA production process. The kinetic behavior of the biocatalyst is well described by the proposed model which allows for an improved design and the possibility to predict performance under different temperature-pH conditions. The common procedures to conduct enzymatic reactions may also be described. Clear evidence of the importance of pH on the process has been presented. Such results demonstrate that adequate control

Characterization and use of a penicillin ucylase hiocatalyst

of industrial reactors is essential for an extended use of penicillin acylase biocatalysts.

ACKNOWLEDGEMENTS This project was partially financed by the United Nations Program for Industrial Development, under contract DP/RLA/83/003. T h e authors express their gratitude t o Genin staff: Lorena Pedraza, Maria Elena Rodriguez, Fernando Gonzalez a n d t o Alfred0 Martinez and Mario Car0 for pilot plant production of the biocatalyst.

REFERENCES 1. Burk, K., Mauz, O., Noetzel, S. & Sauber, K., New synthetic carriers for enzyme coupling. Die Angewandte Makrom~)lekul~~re Chemie, 157 (1988) 105-21.

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