Enzyme and Microbial Technology 25 (1999) 378 –383

Interaction of penicillin V acylase with organic solvents:

catalytic activity modulation on the hydrolysis of penicillin V
Miguel Arroyo*, Raquel Torres, Isabel de la Mata, M. Pilar Castillón, Carmen Acebal
Departamento de Bioquı́mica y Biologı́a Molecular I, Facultad de Ciencias Biológicas, Universidad Complutense de Madrid,
28040 Madrid, Spain

Received 15 October 1998; received in revised form 24 March 1999; accepted 15 April 1999

Abstract

The modulation of hydrolytic activity of penicillin V acylase (EC 3.5.1.11) from Streptomyces lavendulae by organic solvents is reported.
On one hand, the addition of water-soluble cosolvents increases the catalytic activity up to a critical concentration of the non-aqueous
solvents, yet further increase of the latter leads to protein denaturation. For alcohols and aprotic polar solvents, there are linear correlations
between the critical concentration of water miscible cosolvent (at which enzyme deactivation does not begin to take place) and the dielectric
constant of the cosolvents added. On the other hand, water-immiscible solvents can show activating or inhibitory effects that may be related
to interactions between the structure of the solvent and the enzyme. © 1999 Elsevier Science Inc. All rights reserved.

Keywords: Penicillin V acylase; Hydrolysis; Organic solvents; Dielectric constant; Enzyme denaturation

1. Introduction which cosolvent to choose to avoid significant loss of ac-

tivity, or in other cases, what is the highest concentration of
Penicillin V acylase (PVA) catalyzes the hydrolysis of a given organic solvent that the enzyme can endure [5]. In
penicillin V to yield 6-amino penicillanic acid (6-APA) and the hydrolysis of penicillin V, we propose some rules for the
phenoxyacetic acid. PVA is important to the pharmaceutical selection of water-miscible solvents to enhance PVA activ-
industry due to its potential in the production of 6-APA, ity, and a quantitative criterion for the determination of the
which is the key intermediate of semisynthetic penicillins highest cosolvent concentration in the medium to mantain
[1]. In fact, PVA is involved in the 12.5% of the total the increased activity without significant deterioration of the
6-APA produced enzymatically worldwide [2]. Screening enzymes performance.
of cultures producing penicillin V acylases have shown that
these enzymes are produced by bacteria, actinomycetes,
yeasts, and fungi [3]. Streptomyces lavendulae is one of 2. Materials and methods
those bacteria known to produce PVA [4]. In the present 2.1. Enzyme production and partial purification
article, we report that certain solvents can increase or di-
minish the hydrolytic activity of PVA from S. lavendulae. Penicillin V acylase (PVA) was produced from S. lav-
We have also studied the effects of different water-miscible endulae (ATCC 13664), which was grown fermentatively
and water-immiscible organic solvents on catalytic activity under the conditions described by Batchelor et al. [4]. The
and enzyme stability. The use of water/organic cosolvent harvest broth was clarified by centrifugation at 4°C. Then
binary mixtures containing dissolved enzymes has been a the enzyme was precipitated by using 2 vol of acetone, and
restricted approach in biocatalysis, when one has to decide redissolved in 10 mM potassium phosphate buffer, pH 8.0,
containing 1 M NaCl.

2.2. Enzyme activity

* Corresponding author. Tel.: ⫹34-91-394-4150; fax: ⫹34-91-394-
4672.
For enzyme assays, we used the potassium salt of phe-
E-mail address: [email protected] (M. Arroyo) noxy methyl penicillin (PVK) from Sigma (St. Louis, MO,
USA). Fifty ␮l of the semipurified PVA solution was mixed

0141-0229/99/$ – see front matter © 1999 Elsevier Science Inc. All rights reserved.
PII: S 0 1 4 1 - 0 2 2 9 ( 9 9 ) 0 0 0 5 7 - 5
 M. Arroyo et al. / Enzyme and Microbial Technology 25 (1999) 378 –383 379

with 85 ␮l of water, 15 ␮l of 1 M potassium phosphate

buffer (pH ⫽ 8.0), and 150 ␮l of a PVK solution (45 mg/ml)
in water. The reaction mixture was incubated for 30 min at
40°C under gentle shaking. At the end of incubation, the
reaction was stopped by addition of 0.9 ml of a 20% (v/v)
acetic acid solution. The reaction mixture was centrifuged
and an aliquot of 0.9 ml was processed for estimation of
6-APA by addition of 300 ␮l of a 5% (p/v) solution of
p-dimethyl-amino benzaldehyde (Sigma) in methanol [6].
One activity unit is defined as the amount of enzyme pro-
ducing 1 ␮mol/min of 6-APA under the conditions defined.
We used an enzyme solution that had an activity of 0.3 U/ml
in all the experiments.

2.3. Effect of organic solvents

Fig. 1. Effect of alcohol concentration on the hydrolysis of penicillin V by
We examined the effect of organic solvents on penicillin PVA from Streptomyces lavendulae.
V acylase activity at different solvent concentrations. The
solvents were incorporated into 50 ␮l of enzyme solution
before the addition of penicillin V. The volume of water was (v/v) solvent concentration, but it was reduced from 180%
corrected for the added solvent so that the volume remained to 106% at the same concentration of glycerol. The other
150 ␮l. The enzyme activity, in contact with the solvent, alcohols enhanced PVA activity among these values.This
was measured after the addition of 150 ␮l of PVK solution, behavior may be attributed to the nucleophilic strength of
so that the solvent concentration (% v/v) was referred to a the alcohols, which depend on their chemical structures.
final reaction volume of 300 ␮l. All the experiments were Isopropanol is the most nucleophilic alcohol, whereas glyc-
carried out in triplicate. The measure of solvent hydro- erol is the lowest. Then there may be higher or lower PVA
phobicity was the log P value, where P is the partition activation, depending on the nucleophilicity of the alcohol
coefficient for the solvent between octanol and water. (isopropanol ⬎ methanol ⬎ ethanol ⬵ propanol ⬵ 1,3
The log P values provided were taken from, or calculated propanediol ⬵ 1,4 butanediol ⬎ glycerol). Enzyme activa-
on the basis of Rekker [7]. Dielectric constants (⑀) at tion with alcohols and polyols also depends on the concen-
20°C, water immicible at 20°C and surface tension values tration of alcohol in the buffer, indicating that the nature (its
were taken from the monographs by Reichardt [8] and physicochemical properties) of the cosolvent may be in-
Riddick et al. [9]. volved as well. Some authors have also reported a similar
activation of penicillin V acylases from other sources at
moderate concentrations of alcohols, such as methanol, pro-
3. Results and discussion panol, isopropanol, and glycols [12,13].
2) The addition of aprotic polar solvents increased PVA
3.1. Effects of water-miscible solvents on PVA activity activity to a maximal rate that was different depending on

The presence of an enzyme in a water-organic cosolvent

mixture may affect its catalytic properties and stability [10].
In our case, PVA activity was modulated in the presence of
different concentrations of alcohols (Fig. 1), polyols (Fig.
2), and aprotic polar solvents (Fig. 3). Taking into account
the nature of the solvent, the alteration in PVA activity can
be discussed separately.
(1) The alcohols and polyols may interfere in the enzy-
matic reaction, increasing the rate of hydrolysis. In penicil-
lin G acylase from Escherichia coli, the deacylation of the
intermediate acyl-enzyme is affected by the action of water
[11]. If we suppose a similar mechanism for PVA from S.
lavendulae, the acyl-enzyme could be attacked by other
competing nucleophiles, such as alcohols, to release the
product in a faster manner, thus enhancing the catalytic
activity. As shown in Table 1, the highest relative hydroly- Fig. 2. Effect of diols and glycerol concentration on the hydrolysis of
sis rate was achieved in the presence of isopropanol at 5% penicillin V by PVA from Streptomyces lavendulae.
380 M. Arroyo et al. / Enzyme and Microbial Technology 25 (1999) 378 –383

Table 2
Effect of aprotic polar solvents in the hydrolysis of penicillin V by PVA
from Streptomyces lavendulae

Solvent (5%) ␧ Log P Relative

hydrolysis (%)

Nil 78.54 — 100

Dimethylsulfoxide 46.5 ⫺1.35 170
1,4 Dioxane 2.2 ⫺1.10 167
Dimethylformamide 37.0 ⫺1.00 161
Acetonitrile 35.9 ⫺0.34 149
Acetone 20.7 ⫺0.24 156
Tetrahydrofurane 7.6 0.49 132
Pyridine 12.9 0.71 75

larity and hydrophobicity, may be changed. In this situation,

Fig. 3. Effect of aprotic polar solvent concentration on the hydrolysis of electrostatic, hydrophobic and other interactions of PVA
penicillin V by PVA from Streptomyces lavendulae.
with the substrate are intensified, thus influencing the effec-
tiveness of catalysis. The critical point arrives when one has
the nature of the solvent and its concentration in the medium to determine the optimal concentration of solvent in the
(Fig. 3). At the same solvent concentration (Table 2), we binary mixture with the enzyme displaying maximal activity
could find a correlation between the log P value of the and being stable. There are several examples in the literature
aprotic solvent and the activity enhancement (Fig. 4). The wherein solvent physicochemical properties such as dielec-
more polar solvents (with lower log P value) favored the tric constant, dipole moment and hydrophobicity are related
hydrolysis of penicillin V. So, the striking stimulatory effect to various effects on enzyme activity and stability [14,15]. It
of some solvents such as dimethyl sulfoxide, dioxane and has been suggested that electrostatic forces are one of the
dimethylformamide (DMF) could be attributed to an in- factors correlating protein structure and function [16]. En-
creased solubility of 6-APA and phenoxyacetic acid and zymes are more flexible in solvents with high ⑀ value than
thereby decreased diffusional limitations. This fact was in those with low ⑀ [17–19]. A higher flexibility can facil-
confirmed when we observed that the stimulatory effect itate enzyme-substrate interactions, thus increasing the cat-
of these solvents gradually diminished when the substrate alytic activity [20]. In water-solvent mixtures, all these
concentration was reduced two and four times. It should interactions will also depend on the solvent concentration
be noted that pyridine at 3% concentration increased the before reaching its denaturing effect. In view of this, we
rate of hydrolysis by 22%, and at 5% concentration and have found a linear correlation between the dielectric con-
above a deleterious effect was observed, thus it was not stant and the maximal concentration of the solvent that PVA
included in the curve fitting. can accept without any loss of enzymatic activity. The
solvents were divided in two groups: alcohols and aprotic
polar solvents (Fig. 5). On one hand, those solvents with
3.2. Optimization of the biocatalytic process in water-
organic solvent systems

At low cosolvent concentration (⬍20%), the general

properties of the medium, such as dielectric constant, po-

Table 1
Effect of alcohols in the hydrolysis of penicillin V by PVA from
Streptomyces lavendulae

Solvent (5%) ␧ Log P Relative

hydrolysis (%)

Nil 78.54 — 100

Methanol 32.7 ⫺0.76 160
Ethanol 24.3 ⫺0.25 148
Propanol 20.3 0.28 155
Isopropanol 18.3 0.05 181
1,3 Propanediol 35.0 ⫺1.66 152
1,4 Butanediol 30.2 ⫺0.80 155
Glycerol 42.5 ⫺3.04 106 Fig. 4. Effect of log P of aprotic polar solvents on the hydrolysis of
penicillin V by PVA from Streptomyces lavendulae.
 M. Arroyo et al. / Enzyme and Microbial Technology 25 (1999) 378 –383 381

caused by the action of solvents, introducing a new param-

eter called denaturation capacity (DC). This parameter in-
dicates the denaturing strength of an organic solvent in a
scale from 0 to 100. In our case, the more denaturing
solvents for PVA were pyridine and tetrahydrofurane (Fig.
3), which reduced drastically its activity at 25% (v/v) sol-
vent concentration. In the DC scale of Khmelnitsky, tetra-
hydrofurane has the highest DC value (DC ⫽ 100), meaning
that this solvent exerts the strongest denaturing effect on
proteins. The DC scale does not include pyridine, which is
even more deleterious for PVA than tetrahydrofurane in
water/organic cosolvent mixtures. It should be addressed
that glycerol and diols are among the solvents possessing
the lowest values of DC (DC ⬍ 50), and did not decrease
PVA activity at high solvent concentration (Fig. 2). Similar
Fig. 5. Optimization of the solvent concentration for penicillin V hydro- results have been reported by Kim and Lee [23], who
lysis in monophasic systems.
studied the effect of various organic solvents on penicillin
acylase-catalyzed synthesis of pivampicillin and ampicillin
in water-solvent mixtures.
low ⑀ values (pyridine, tetrahydrofurane) should be added to
the medium at low concentration (less than 5%) if we 3.3. Effects of water-immiscible solvents on PVA activity
wished PVA to display enhanced activity. On the other
hand, solvents with high ⑀ values (dimethyl sulfoxide, glyc- Aqueous-organic solvent two phase systems can offer
erol) can be used at higher concentrations without any different advantages in contrast to water-miscible cosol-
deleterious effect. In addition to not changing drastically the vents, such as higher retention of enzyme activity, easier
⑀ of the medium, diols and glycerol are mild denaturants, product recovery, and decreased substrate and/or product
and that explains why PVA is highly active and stable at inhibition. The effects of several water-immiscible organic
25% solvent concentration, whereas it is inactive at the solvents at 5% (v/v) concentration on PVA activity are
same concentration of tetrahydrofurane or pyridine. summarized in Table 3. We stated that some solvents di-
In monophasic water-organic solvent systems, enzymatic minished PVA activity, whereas others enhanced or main-
activity is normally lost at high solvent concentration tained it. Trying to explain this behavior, we could not find
(⬎20%) by replacement of water in the protein’s hydration correlation between any physicochemical parameter of the
surface layer by the organic solvent [21]. Displacements of solvents tested and the rate of hydrolysis. The loss of en-
bound water molecules by organic cosolvents results in a zyme activity in two-liquid phase systems may take place at
dramatic change of protein structure, leading to denatur- the interface between the two phases or by a purely dis-
ation and thus to inactivation. Khmelnitsky et al. [22] have solved solvent effect. Several studies to date have shown
cast some light on the regularities of protein denaturation both interfacial and dissolved solvent inactivation of differ-

Table 3
Effect of water-immiscible solvents in the hydrolysis of penicillin V by PVA from Streptomyces lavendulae

Solvent (5%) Solubility in water (%) Surface tension ␧ Log P Relative hydrolysis
at 20°C (dynes/cm) (%)

Nil — — 78.54 — 100

L-Butanol 7.45 24.6 17.1 0.80 107
L-Hexanol 0.706 24.5 12.1 1.80 91
L-Heptanol — — — 2.30 53
L-Octanol 0.0538 26.06 10.3 2.90 22
Heptane — 20.35 1.92 4.40 127
Octane 0.0142 21.77 1.95 4.90 51
Isooctane 0.0055 18.85 1.95 4.50 122
Decane — 23.92 1.99 5.60 43
2-Octanone 0.11 14.1 11.2 2.40 22
Isopropylether 0.87 17.34 3.88 1.70 125
Dichloromethane 1.32 28.12 9.08 1.72 94
Chloroform 0.822 27.3 4.806 2.01 103
1,2 Dichloroethane 0.81 32.23 10.6 1.63 87
1,1,1 Trichloroethane — 25.67 3.4 2.50 153
382 M. Arroyo et al. / Enzyme and Microbial Technology 25 (1999) 378 –383

Fig. 6. Effect of water-immiscible solvent concentration on the hydrolysis Fig. 7. Effect of chlorinated solvent concentration on the hydrolysis of
of penicillin V by PVA from Streptomyces lavendulae. penicillin V by PVA from Streptomyces lavendulae.

ent enzymes, but the results do not correlate with any Once again, small differences in the structure of the solvent
solvent physicochemical parameter [24,25]. The possible produced different actions on enzymatic activity. Whereas
role of surface tension effects on two-liquid phase systems chloroform and trichloroethane (three atoms of Cl) en-
has been suggested [26,27] to explain the different denatur- hanced PVA activity, dichloromethane, and 1,2 dichloro-
ant capacities of some solvents of identical hydrophobicity. ethane had an inhibitory effect at higher concentration.
We could check that PVA deactivation or activation were In view of this, the direct binding of the solvent on
not related to logP, the dielectric constant or the interfacial specific binding sites of the enzyme [29] should be consid-
tension of the solvent (Table 3). ered as a contributing factor that determines PVA activity.
It is obvious that no single solvent parameter can predict On this basis, some possible molecular interactions between
the behavior of PVA in these systems. However, there is penicillin G acylase from E. coli and organic solvents have
clear evidence of trends that are specific for penicillin V been suggested recently [23].
acylase from S. lavendule:
(1) It is known that the larger the size of the enzyme
molecule, the greater the contacting area with the interface, Acknowledgments
favouring its deactivation [24]. PVA must be a small en-
zyme that can only be deactivated by 5 solvents out of 14 Financial support from the Comision Interministerial de
(Table 3). Ciencia y Tecnologia (QUI97-00490) is gratefully acknowl-
(2) PVA activity was not altered by the concentration of edged.
water-immiscible solvents but it was even increased with
higher concentration of some solvents such as heptane and
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