Influence of polyhydric compounds on the pH stability of penicillin G acylase obtained from a mutant of Escherichia coli ATCC 11105

Process Biochemistry, Vol. 31, No. 7, pp. 691-697, 1996

Copyright © 1996Elsevier Science Ltd Printed in Great Britain. All rights reserved 0032-9592/96 $15.00+ 0.00

FLSEVIER

PI i:S0032-9592

(96)00014-3

Influence of Polyhydric Compounds on the pH Stability of Penicillin G Acylase Obtained from a Mutant of Escherichia coli ATCC 11105 Dilek K a z a n a & A l t a n E r a r s l a n a'b * ' Laboratory of Enzyme and Fermentation Technology, Research Institute for Genetic Engineering and Biotechnology, The Scientific and Technical Research Council of Turkey, Marmara Research Centre, PO Box 21, 41470 Gebze-Kocaeli, Furkey ~'Section of Biochemistry, Department of Chemistry, Faculty of Arts and Sciences, Kocaeli University, 41300 IzmitKocaeli, Turkey ~Received 7 December 1995; accepted 10 February 1996)

Fhe effects of polyhydric compounds (PHC) on the stabilization of penicillin G acylase (PGA) obtained from a mutant of Escherichia coli ATCC 11105 against p H were studied. Glucose and sucrose in 1 M concentration and polyethylene glycols (PEG) at a molecular weight of 400, 4000 and 15 000 in 250, 150 and 50 mM concentrations, respectively, were used as PHC. The reactivation mechanisms of native and PHC containing PGA are considered to obey first order inactivation kinetics during prolonged incubation time interval ,~f enzyme solutions at different p H values in 40°C. PHC containing PGA preparations showed lower inactivation rate constants than native PGA preparations at the p H values studied. Lowest inactivation rates were obtained at p H 7.0. Inactivation rates were higher when the enzyme solutions were incubated at p H values above 7"0. Among the PHC compounds used, the sucrose, PEG 400 and PEG 4000 showed the highest enhancement to the p H ~tability of PGA. The addition of PHC into PGA solution did not cause any change in the p H optimum of enzyme in penicillin G (Pen G) hydrolysis reaction. Copyright © 1996 Elsevier Science Lid

pH values (4.0-7.0) the enzyme also catalyses the N-acylation of 6-APA with the analogues of phenyl acetic acid (PAA) to produce the corresponding semi-synthetic penicillins such as ampicillin, amoxycillin and oxacillin. 1 Other useful properties of PGA include the hydrolysis of phenyl acetyl derivatives of a number of peptide molecules and the resolution of phenyl acetyl derivatives of certain organic compounds. 1 The long term stability of the enzyme is the major concern in its industrial applications. The enzyme is never used under conditions of maximum reaction rate in industrial processes due to

INTRODUCTION

Penicillin acylase (PGA) (EC 3.5.1.11) is an enzyme currently used on an industrial scale for the production of 6-aminopenicillanic acid (6-APA) and 7-amino-3-deacetoxy cephalosporanic acid (7-ADCA) from penicillin G (Pen G) and cephalosporin G (Cep G), respectively, by hydrolytic cleavage of the acyl side chain at slightly alkaline pH (7.5-8.5). The specificity and catalytic properties of the enzyme are interesting and potentially important in other applications of biocatalysis. At lower to neutral * To whom correspondence should be addressed. 691

692

D. Kazan, A. Erarslan

inhibitions caused by hydrogen ions, products and substrates. Among these factors, pH is particularly important, because the poor stability of the enzyme towards pH changes is an important problem for its application in the synthesis of semi-synthetic penicillins. Many enzymes rapidly and irreversibly loose their catalytic activity at pH values far from the optimal pH range of enzymatic reaction. The stabilization of enzymes against pH inactivation depends on many factors such as temperature, ionic strength and the chemical nature of the buffer, concentration of various preservatives, chemical modifications on protein molecules in order to increase its rigidity, etc. In our previous work, we observed four-fold stabilization of PGA against pH inactivation by chemical cross-linking of enzyme molecule with dimethyladipimidate (DMA). 2 On the other hand, the use of various additives that have the ability to enhance the stability of protein molecules has been extensively studied) Enzyme stability is greatly influenced by the presence of water molecules in its micro environment. Additives that strengthen the hydrophobic interactions in side protein molecules by reducing the amount of free water are described as stabilizing agents. 4 Since the unfolding of enzymes is linked to their conformational mobility in water, it may be hindered by reducing the water activity of enzyme solutions by the addition of water binding compounds such as sugars or PEG. 5 Most of the work appearing in the literature related to the stabilization of PGA is focused on the thermostabilization of the enzyme. 6-1° Little or no work related to the pH stabilization of PGA has appeared in the literature. 2"11 The objective of this research was to investigate the inactivation kinetics and stabilization of PGA at pH values far from its optimal pH in the presence of PHC. MATERIALS AND METHODS Chemicals Pen G and 6-APA were kindly provided by Unifar Chemical Ltd (Istanbul, Turkey). DEAE-cellulose used for enzyme purification and PEG compounds at various molecular weights were obtained from Sigma (U.S.A.). All other chemicals were analytical grade and

supplied either by Merck AG (Germany) or Sigma. Microorganism A mutant of Escherichia coli ATCC 11105 was obtained by chemical mutagenesis of parent cells treated with N-methyl-N'-nitro-N-nitrosoguanidine (NTG), as described elsewhere, 12 and used throughout this work. The mutant strain is four-fold more productive in PGA production than the parent strain and is deposited in the culture collection of the Research Institute for Genetic Engineering and Biotechnology, Marmara Research Centre. Media and culture collections The production of PGA by cultivation of E. coil cells in a jar fermenter (Biostat E, Braun Melsungen GmbH, Germany) was carried out under the same medium and culture conditions described previously.13 The fermentation temperature, pH and dissolved oxygen concentration were 28°C, 7-0 and 10% of saturation, respectively. PGA synthesis was induced after 6 h of incubation by continuous feeding of phenyl acetic acid (PAA) into the fermenter to a final concentration of 0.3% (w/v). Enzyme purification Intracellular PGA was extracted from the mutant strain after cell disruption and purified by DEAE-cellulose ion exchange chromatography followed by preliminary precipitation steps as described previously. 12 Determination of enzyme activity The hydroxyl amine method of Batchelor et a l ) 4 was used to determine enzyme activity during purification. Due to the relatively poor sensitivity of this method, however, the p-dimethylaminobenzaldehyde (PDAB) method 15 was used instead of the hydroxyl amine method for activity measurements during kinetic investigations. One unit of enzyme activity is defined as the amount of enzyme required to produce 1 pmol of 6-APA per min at 40°C and pH 8.0 from 15 mM Pen G in 50 mM phosphate buffer. Protein measurement Protein contents of enzyme solutions were measured by the Coomassie Blue binding method

Polyhydriccompoundsandpenicillin G acylase using bovine serum albumin (BSA) as the standard.16' 17 Estimation of inactivation rate constants at different pH values Inactivation rate constants (ki) of PGA at different pH values, in media containing sucrose, glucose and various PEG compounds of dif!'erent molecular weights were estimated according to first order inactivation kinetics described previously. ~° In order to prepare the mzyme solutions at different pH values 0.05 ml ~nzyme solution in 10 mM phosphate buffer at ~H 7.0 (specific activity: 28.28 U mg -1, protein ,.'oncentration: 0-273mg ml -~) was added to ).45 ml and 50 mM buffer solutions containing glucose, sucrose and PEG compounds at different pH values between 4 and 9. At pH values 4.0 and 5.0 acetate buffer, at pH values 6.0, 7.0, ~-0, 9.0 phosphate buffer, at pH values 9.4 ~odium carbonate-bicarbonate buffer solutions were used. These 0.5 ml enzyme solutions at different pH values were incubated for different time intervals at 40°C. Thereafter 0.5 ml 30 mM Pen G solution in 50 mM phosphate buffer pH 8.0 was added and the mixture incubated for 3 min at 40°C. The activities of enzyme solutions were measured by the PDAB method and tci values were calculated from the slope of the linear equation of first order inactivation kinetics Ln[Ei/Eo]=-kit [Ei: initial activity of inactivated form of enzyme (the residual activity of enzyme after pH treatment), Eo: initial activity of enzyme before pH treatment, t: time (min)]. The expression of the level of pH stabilization The level of pH stabilization of the PGA preparations having different PHC was expressed as a stabilization factor (SF) and calculated with the formula indicated below: SF= [half-life

time]PHC_PGAmixture/

[half-life time]native PGA. The SF value of native PGA is always found to be 1.0 according to this calculation. Half-life time is the time required for 50% inactivation of enzyme activity and calculated from the Ln[Ei/Eo]=-kit linear equation by placing Eo=2Ei.

693

RESULTS AND DISCUSSION pH inactivation kinetics of PGA in the presence of polyhydric compounds In our previous work, we reported nearly twoto four-fold thermostabilization of PGA by glucose and sucrose in 1 M concentration. 1° In our recently completed work, we observed seven- to 20-fold stabilization of PGA against increasing tempratures by PEGs having 400, 4000, 6000, 10000 and 15 000 molecular weight when they are used in 250, 150, 150, 100 and 50mM concentrations, respectively. We assumed that polyol compounds and PEGs may also increase the stabilization of PGA against extreme pH values. On the other hand, no loss of the initial activity of PGA was observed at pH interval between 4-0 and 9.0 in our recently published research. 2 However, at pH values below 4.0 and above 9.0 sharp decreases were observed. 2 For this reason, the effect of polyhydric compounds (PHC) compounds on the pH stability of PGA was studied in the pH interval between 4.0 and 9.0. The irreversible inactivation mechanism of both native and additive containing PGA at pH extremes, is considered to be a two-step process. The first step results in the formation of a transient state of the enzyme, then in the second step, the transient form is inactivated by first order kinetics upon prolonged exposures at different pH values. Thus, the inactivation rate constants (kj values) of the enzyme at different pH values are obtained from the slope of linearly regressed Ln[Ei/Eo]=-kit line when Ln[Ei/Eo] is plotted vs t (E~ and Eo are the activity of the inactivated and native forms of enzyme, respectively). Inactivation plots of both native and PHCcontaining PGA preparations at pH values 4.0, 7.0 and 9.0 are shown in Fig. 1A-C, respectively. Inactivation rate constants (ki) were estimated from the slopes of these linearly regressed lines and given in Table 1. ki values of sucrose-, glucose- and PEG-containing PGA preparations were always lower than that of the native PGA at all pH values studied. This indicates the stabilizing effect of polyhydric compounds on the PGA molecule against pH. Sucrose and glucose showed the higher half-life times than PEG compounds (Table 1). Highest SF values were obtained with sucrose, PEG400 and PEG4000 at pH 8.0 as given in Table 2.

D. Kazan, A. Erarslan

694

These findings indicate that sucrose, PEG400 and PEG4000 are better stabilizers of PGA against pH than any of the other PHC used.

Free energies (AGO for the inactivation of PGA in the presence of PHC We have calculated the free energies for the inactivation of the native and PHC-containing PGA preparations by using the equation

AGi = - R T Ln(kih/kBT) evaluated at different pH values (ki: inactivation rate constant, kB: Boltzmann constant, h: Planck's constant and T: absolute temperature). Results are given in Table 2. A G i values of PHC-containing compounds were higher than that of native PGA at all pH values studied. The highest A G i values were obtained in sucrose-, PEG400-, and PEG4000-containing PGA preparations. These

0 O

Glucose, r = -0.9897

-0.1

a

-0.2

A

PEG 400, r = -0.9963

-0.3

v

PEG 4000, r = -0.9894

o

PEG 15000, r -- -0.9990

-0.4

Sucrose, r -- -0.9935

Native, r -- -0.9999 -0.5 0

30

60

90

120

150

180 210

240

Time, (rain)

-0,02 -0,04

,

o

Glucose, r = -0.9937

o

Sucrose, r = -0.9939 PEG 400, r = 0.9899

-0,08

v

-0,1

*

o

-0,12

-0,14

, I ,,

0

30

I,,,

60

PEG 15000, r = -0.9820 Native, r = -0.9940

,

90

PEG4000, r = -0.9910

120 150 180 210 240 270 300

Time, (rain) 0 o

Glucose,

r = -0.9909

D

S~rose,

r =

-0,1 -0.9804

-0,2

A

-0,3

v

PEG 4000, r = -0.9904

-0,4

o

PEG 15000, r = -0.9923

-0,5

*

Native, r = -0.9933

0

20

40

60

80

PEG 400, r -- -0.9996

100 120 140 160 180

Time, (rain) Fig. 1. Inactivation of native and PHC-containing P G A preparations at pH values 4-0 (A), 7.0 (B) and 9.0 (C). The apparent inactivation rate constant for each pH is calculated from the slope of these lines.

Polyhydric compounds and penicillin G acylase

695

Table 1. Inactivation rate constants (ki) and half-life times (HL) of native and polyhydric compounds containing penicillin

G acylase (PGA) preparations at different pH values

pH 4"0

pH 5.0

pH 6"0

pH 7.0

pH 8.0

pH 9"0

ki × 10 2 HL ki x 10 2 HL ki × 10 -e HL ki × 10 2 HL k i x 10 2 HL ki × 10 -2 HL (rain 1) (rain) (min 1) (min) (min 1) (min) (min i) (min) (rain 1) (rain) (min -1) (min) ~atwe,(A) k+glucose k+sucrose k+PEG400 k+PEG4000 k+PEGI5000

0.193 0.157 0.098 0.116 0-140 0.145

359 441 71)7 597 495 478

0.121 0.045 0-030 0.041 0-047 0.060

573 1540 2310 1690 1475 1155

0.092 0.029 0.019 0.030 0-036 0.056

753 2389 3647 2310 1925 1238

0-040 0.009 0.008 0.011 0.010 0.020

1733 7700 8663 6300 6299 3465

0.189 0.040 0.025 0.024 0.025 0.090

367 1733 2772 2887 2772 770

0.860 0-452 0.182 0.353 0.240 0.284

81 153 380 196 289 244

Fable

2. Stabilization factor (SF) and activation free energies of inactivation (AGi) values of native and polyhydric :ompounds containing penicillin G acylase (PGA) preparations at different pH values

pH 4"0

Native, (A) ~, + glucose a.+sucrose .~ + PEG400 a, + PEG4000 +PEG15000

pH 6"0

pH 7"0

pH 9"0

AGi (kc al/mol)

SF

AGi (kcal/mol)

SF

AGi (kcal/mol)

SF

AGi (kcal/mol)

SF

AGi (kc al/mol)

SF

AGi (kcal/mol)

1.00 1.23 1.97 1.66 1.38 1.33

24.79 24.93 25.22 25.11 24.99 24.97

1.00 2.69 4.03 2.95 2.57 2-02

25.08 25.70 25.96 25.76 25.67 25.53

1.00 3.17 4.79 3.07 2.56 1.64

25.26 25.97 26.24 25.96 25.84 25.57

1.00 4.45 5.00 3.63 3.63 2.00

25.78 26.70 26.78 26.58 26.64 26.21

1.00 4.72 7.55 7-86 7-55 2.10

24.81 25.76 26-07 26.09 26.07 25.27

1.00 1.89 4-70 2.42 3.57 3-01

23.87 24.26 24.83 24.42 24.66 24.56

[]

PF_,G4000

[] Saero~

n Glucose]

o

-4

-4

-5

-5

-6

-6

.... i

-7

....

5

-8

-9

-9

-10

-10 0.1

0.15

0.2

0.25

0.3

0.05

1/pH

PF_,O400

' ' '~'

' ' ....

, , , ~ , ,

.~,,,,

A PEGI5000

, ....

' ' ' 'fi,

-7

-8

0.05

pH 8"0

SF

I o Native

.5

pH 5"0

0.1

0.15

....

0.2

, ....

0.25

0.3

1/pH

Fig. 2. Graphical representation of an Arrhenius-type model applied to describe the effect of pH on the first order inactivation rate constants of native and PHC-containing PGA at 40°C.

findings also indicate that sucrose, PEG400 and PEG4000 are better stabilizers of PGA against pH than any of the other PHC used. Effect of pH on the inactivation rate constants of PHC-containing PGA preparations

The effect of pH on the first order inactivation rate constants (k~) of native and PHC-contain-

ing PGA preparations was studied by fitting the values to an Arrhenius-type model described by Ospina et al. 18 In this model, k~ values change with pH according to the k~=A exp[B/ pHI equation. The constants of this equation can be calculated from the slope and intercept values of its linear form obtained as: Ln ki-Ln A - ( B / p H ) when Ln k~ is plotted against ki

696

D. Kazan, A. Erarslan

Table 3. The A and B constants for each polyhydric compounds and native PGA obtained from Fig. 2 p H < 7"0 Enzyme and additives

Native PGA, (A) A + sucrose A+glucose A + PEG400 A + PEG4000 A + PEG15000

'

p H > 7"0

A

B

A

B

7.53 × 10-5 3"73 × 10-6 3"21 × 1 0 - 6 7"36 × 1 0 - 6 5-56 × 10 6 2"44 x 10-5

13.41 22.31 24.95 20"42 22"42 16.49

324 56.3 223 273 83.1 28"5

-95.46 - 95-37 - 120"53 - 105.37 - 97"05 - 82"94

I

'

I

'

I

~

I

'

!

'

I

0

,

I

i

I

'

I

i

--0- Ghra)se

90

--ffl-- Sucrose

80

- d ~ PEG 400 ---O- PEG 4000

70

--O- PEG 15000 60

Native 50

I

3

I

4

t

I

5

i

I

,

6

I

7

,

I

8

i

I

9

10

i

I

11

,

I

12

t

13

pH Fig. 3. pH-activity profile of native and PHC-containing PGA preparations. Enzyme preparations were mixed with

15 mM Pen G solution prepared in 50 mM phosphate buffer in various pH values and incubated at 40°C for 3 min. Activities were measured by the PDAB method.

1/pH [k~: inactivation rate constant (min-1), A and B: constants of Arrhenius-type model]. Lowest ki values for native and PHC-containing P G A preparations were obtained at p H 7.0. Ln k~ values obtained below and above p H 7.0 were fitted by linear regression separately as shown in Fig. 2. Two different values for each of the constants A and B were calculated from the slopes of these fitted lines and results are given in Table 3. We could find only the report of Ospina et al. m in the P G A literature describing the effect of p H on the inactivation rate constant of PGA. They have investigated the storage stability of immobilized E. coli P G A at 37°C in different p H values and observed the lowest ki value at p H 7.5. Consequently, they have calculated the A and B constants for immobilized P G A by

fitting the k~ values obtained below and above p H 7.5. We found higher A and lower B values than those of Ospina et al. 18 by fitting the ki values of soluble P G A obtained at p H values below 7.0. A and B constants found in our work by fitting the ki values obtained at p H values above 7.0 were lower and higher, respectively, than the results of Ospina et al. T h e p r o p o s e d equations may be used to predict the behaviour of the enzyme u n d e r different reaction conditions.

Effect of P H C on the optimal pH of PGA

The addition of P H C did not cause any change on the optimal p H of PGA. The o p t i m u m p H for Pen G hydrolysis by P G A in all cases was 8.0 (Fig. 3).

Polyhydric compounds and penicillin G acylase

REFERENCES 1. Shewale, J. G., Despande, B. S., Sudhakaran, V. K. & Amberkar, S. S., Penicillin acylases: applications and potentials. Process Biochem., 25 (1990) 97-103. 2. Kazan, D., Ertan, H. & Erarslan, A., Stabilization of penicillin G acylase against pH by chemical crosslinking. Process Biochem., 31 (1996) 135-40. 3. Schmid, R. D., Stabilized soluble enzymes. In Advances in Biochemical Engineering, Vol. 12, ed. T. K. Ghose, A. Fiechter & N. Blakebrough. Springer, Berlin, 1979, pp. 41-127. 4. Klibanov, A. M., Stabilization of enzymes against thermal inactivation. In Advances in Applied Microbiology, Vol. 29, ed. A. I. Laskin. Academic Press, New York, 1983, pp. 1-28. 5. Larreta-Garde, V., Xu, Z. F. & Thomas, D., Behaviour of enzymes in the presence of additives. In Enzyme Engineering, Vol. 9, ed. H. W. Blanch, A. M. Klibanov. Academic Press, New York, 1988, pp. 294-8. 6. Alvaro, G., Lafuente, R. F., Blanco, R. M. & Guis~n, J. M., Immobilization-stabilization of penicillin acylase from Escherichia coli. Appl. Biochem. Biotechnol., 26 (1990) 181-95. 7. Lafuente, R. F., Rosell, C. M., Alvaro, G. & Guis~in, J. M., Additional stabilization of penicillin G acylaseagarose derivatives by controlled chemical modification with formaldehyde. Enzyme Microb. Technol., 14 (1992) 489-95. 8. Erarslan, A. & Koger, H., Thermal inactivation kinetics of penicillin G acylase obtained from a mutant derivative of Escherichia coli ATCC 11105. J. Chem. Technol. Biotechnol., 55 (1992) 79-84. 9. Erarslan, A., The effect of polyol compounds on the thermostability of penicillin G acylase obtained from

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697

a mutant of Escherichia coli ATCC 11105. Process Biochem., 30 (1994) 133-9. Erarslan, A. & Ertan, H., Thermostabilization of penicillin G acylase obtained from a mutant of Escherichia coli ATCC 11105 by bisimidoesters as homobifunctional cross-linking agents. Enzyme Microb. Technol., 17 (1995) 629-35. Rio, G. D., Rodriguez, M. E., Munguia, M. E., Munguia, A. L. & Sober6n, X., Mutant Escherichia coli penicillin acylase with enhanced stability at alkaline pH. Biotechnol. Bioeng., 48 (1995) 141-8. Erarslan, A., Terzi I., Giiray, A. & Bermek, E., Purification and kinetics of penicillin G acylase from a mutant strain of Escherichia coli ATCC 11105. J. Chem. Technol. Biotechnol., 51 (1991) 27-40. Erarslan, A. & GiJray, A., Fermentation of penicillin G acylase by a mutant strain of Escherichia coli ATCC 11105. Do~a-Tr. J. Biol., 15 (1991) 167-74. Batchelor, F. R., Chain, E. B., Hardy, T. L., Mansford, K. R. L. & Rolinson, G. N., 6-Aminopenicillanic acid. III. Isolation and purification. Proc. Roy. Soc., B, 154 (1961) 498-508. Shewale, G. J., Kumar, K. K. & Ambekar, G. R., Evaluation and determination of 6-aminopenicillanic acid by p-dimethylaminobenzaldehyde. Biotechnol. Techniques, 1 (1987) 69-72. Spector, T., Refinement of the coomassie blue method of protein quantitation. Anal Biochem., 86 (1978) 142-6. Sedmak, J. J. & Grossberg, S. E., A rapid, sensitive and versatile assay for protein using Coomassie Brilliant Blue G-250. Anal, Biochem., 74 (1977) 544-52. Ospina, S. S., Lopez-Munguia, A., Gonzalez, R. L. & Quintero, R., Characterization and use of a penicillin acylase as biocatalyst. J. Chem. Tech. Biotech., 53 (1992) 205-14.