Polaina J., MacCabe A.P. (ed.). Industrial Enzymes. Structure, Function and Applications
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PENICILLIN ACYLASES 587
on the carbonyl carbon of the amide bond by the active site nucleophile, a covalent
acyl-enzyme complex is formed via a tetrahedral transition state in which the
negatively charged oxyanion is stabilised by H-bonds to oxyanion residues (N241
and A69 in the case of E. coli PAC) (Duggleby et al., 1995). After expulsion
of the leaving group from the active site, the acyl-enzyme is deacylated by water,
or another nucleophile, giving rise to the final transacylation product and the free
enzyme.
PAC can also be used in the catalytically reverse direction to condense an acyl
group with a -lactam nucleus. In this reaction an activated acyl donor, generally
an amide or methyl ester of a PAA derivative, acylates the enzyme at the active
site serine with the concomitant release of ammonia or methanol. The resulting
acyl-enzyme is then deacylated by a -lactam nucleophile such as 6-APA or
7-desacetoxycephalosporanic acid (7-ADCA). This yields a semisynthetic penicillin
or cephalosporin.
Mutation studies coupled with crystal structure data have demonstrated that a
number of amino acids within the protein have critical functions in either the
processing or catalytic reactions, or both. The K10 residue is involved in the
pH-dependent processing of the preproprotein (Lee et al., 2000). Residues involved
in catalysis by direct contact include R145, F146, S1, A127, N241 and
R263 in E. coli, along with the hydrophobic residues F146, F24 and F57
which are implicated in substrate specificity and acyl transfer (Alkema et al., 2002).
Sequence alignment studies of PAC proteins from sources other than E. coli have
been performed and in many cases homologous amino acids have been found giving
weight to the idea that the mode of action of PAC is the same across diverse
species.
The hydrolysis of Pen G to 6-APA proceeds at a slightly alkaline pH, whereas
the reverse reaction proceeds at acidic pH. It follows then that the overall reaction
is determined by pH and the relative concentrations of substrate and product.
Kheirolomoom et al. (2001) have demonstrated that PAC is inhibited by excess
substrate and the presence of both products, 6-APA and PAA. Non-competitive
inhibition by 6-APA and competitive inhibition by PAA were observed for
the ordered uni bi deacylation reaction in the forward direction. In the reverse
direction, Pen G acts as a mixed type inhibitor with respect to 6-APA at unsaturating
concentrations of PAA. This inhibition can be overcome with saturating amounts
of PAA. In addition, Pen G is a competitive inhibitor with respect to PAA at all
concentrations of 6-APA.
5. ENHANCING THE EXPRESSION OF PAC
Classical mutation and selection procedures have been applied to enhance the
production of PAC from many micro-organisms. Erarslan et al. (1991) performed
random mutagenesis induced by NTG or NMS followed by screening for overpro-
ducers using a Serratia marcescens overlay technique. A four-fold improved E. coli
ATTC 11105 mutant was obtained in this way. Another approach has been the
588 SPENCE AND RAMSDEN
isolation of mutants in the presence of amides as sole nitrogen sources. The rationale
for this is that PAC expression liberates nitrogen for growth. The addition of
glucose in the medium has resulted in the isolation of mutants which are resistant to
catabolite repression, leading to increased PAC activity (Shewale and Sivaraman,
1989). A succinate resistant mutant of P. rettgeri showing increased activity has
also been reported (Daumy et al., 1985).
Recombinant DNA technology has made it possible to clone and express the
pac gene from many sources. The gene has been cloned into multicopy vectors to
increase gene dosage. This has been achieved from species including E. coli (Oh
et al., 1987), Arthrobacter viscosus (Ohashi et al., 1989), P. rettgeri (Daumy et al.,
1986), Kluyvera citrophila (Barbero et al., 1986), Bacillus megaterium (Meevoo-
tisom and Saunders, 1987), A. faecalis (Verhaert et al., 1997), and Achromobacter
xylosoxidans (Cai et al., 2004). There are fewer examples of the cloning of the
pen V acylase gene since the pen G acylase gene remains the gene of choice for
exploitation. Segregational instability of recombinant plasmids in the absence of
selective pressure is widespread. To overcome this effect, the E. coli pac gene
has been cloned in pYA292, which is stable in strain 6212 in the absence of
challenge. The resultant clone produces higher PAC levels (Vohra et al., 2001).
The location of the mature PAC protein can determine the efficiency of production.
For example Ohashi et al. (1989) showed that expressing the A. viscosus pac
gene in E coli resulted in PAC production intracellularly, whilst expression of the
same gene in B subtilis resulted in extracellular production with higher levels of
activity. The penicillin acylases from gram negative bacteria accumulate within the
periplasmic space, whereas those from gram positive bacteria tend to be secreted.
Other organisms such as Bacillus sp have been shown to produce the enzyme
intracellularly (Rajendhran et al., 2002).
The transcription of the pac gene in E. coli is inefficient due to the non-
optimal disposition of bases present in the regulatory sequence. The translation of
the message is also relatively poor due to there only being four bases between
the ATG start codon and the ribosome binding site. PAC expression has been
enhanced by placing the pac gene under the control of a strong promoter and
optimising the spacing upstream of the translational start (Chou et al., 1999).
However, one of the drawbacks to this approach was the subsequent production
of inclusion bodies which reduced the level of PAC activity recovered. Since
active PAC is formed in the periplasmic space of E. coli, engineering of strains
to improve transport and folding would be expected to be beneficial to PAC
production. In this regard, Lin et al. (2001) have demonstrated that in order to
avoid solubility problems it is necessary to achieve balanced protein synthesis
flux throughout the gene expression steps (transcription, translation and post
translational modification) in addition to improvements in the efficiency of each
step. This has led to significant improvement in recombinant PAC production.
For the commercial manufacture of PAC, combinations of the aforementioned
approaches have been applied to production strains to improve the efficiency of the
process.
PENICILLIN ACYLASES 589
6. CONDITIONS FOR THE PRODUCTION, EXTRACTION
AND IMMOBILIZATION OF PAC
The most widely used organism is E. coli, containing either the endogenous gene
or a heterologous gene. Fermentations are performed in up to 250m
3
fed batch,
stirred fermenters. Seed cultures are grown at 37
C for a prescribed time, usually a
pH change indicates optimal time for transfer, then up to 10% seed can be used to
inoculate the production fermenter. After an initial growth period, the temperature
is reduced and carbon feeding is initiated. This is the trigger for PAC production.
The fermentation is continued until maximum activity is achieved and then the
fermentation is harvested.
The level of dissolved oxygen is required to be maintained at 15% or higher to
avoid repression of PAC. In addition, since glucose is a known repressor of PAC
activity when used as carbon source its rate of supply has to be limited. Alternative
carbon sources such as sucrose or glycerol are commonly used. The pH of the
culture needs to be maintained in the range 6.8 to 7.1 for optimal PAC production.
Likewise, the production temperature has to be maintained below 30
C. Since the
expression of the pac gene can be differentially modulated depending on the exact
nature of the plasmid constructed, diverse expression inducers can be used to switch
on the pac gene fused to a particular upstream sequence. Examples include galactose
(De Leon et al., 2003) and isopropyl-beta-D-thiogalactopyranoside (IPTG) (Wen
et al., 2005) to induce the lac promoter, and rhamnose (Deak et al., 2003) to switch
on the rhaBAD promoter.
Since the active PAC enzyme is localised in the periplasmic space in E. coli this
immediately affords a purification step as less than 5% of cellular proteins are found
in this compartment. The periplasmic fraction can be separated from the cytoplasmic
fraction using selective permeabilisation. Cell permeabilisation by osmotic shock in
combination with EDTA has been reported to yield 70% protein without affecting
cell viability (Neu and Heppel, 1965). Polar organic solvents and aqueous solutions
of ionic and non-ionic detergents have been used to permeabilise cells (Novella et al.,
1994). For example, on a laboratory scale, treatment with guanidine/EDTA has been
reported to give a 93% yield with a 25-fold purification (Novella et al., 1994. The use
of organic solvents as permeabilisation agents has also been demonstrated (De Leon
et al., 2003). The authors reported attempts with ten different solvents with varying
results. Solvents with dielectric constants below five and having high hydrophobicity
released the most active PAC protein. The production of reverse micelles using AOT
in water/hexane resulted in the selective release of the enzyme without cell breakage
(Bansal-Mutalik and Gaikar, 2003). More specific means of purification of PAC have
been demonstrated. Sanchez et al. (2001) achieved greater than 65–fold purification
by immobilized metal affinity chromatography (IMAC). The use of pseudo-affinity
chromatography to aid the purification of PAC has been expertly reviewed by Fitton
et al. (2001). The advantage of this technology is that it can be scaled up and the
matrices are suitable for sanitisation.
Whilst the permeabilisation methods outlined above can be applied to small scale
cultures, the release of PAC from industrial fermentations poses greater problems.
590 SPENCE AND RAMSDEN
To this end the large scale release of PAC usually involves physical disruption
followed by partial purification. After fermentation cells are harvested by centrifu-
gation or settled with flocculant. The concentrated cells are then homogenised and
cell debris removed. The extract is then purified by the method of choice. The most
general means of purification is chromatography and/or ammonium sulphate precip-
itation. The exact method depends on the given specific activity and the activity
required for industrial use as a biocatalyst. A typical method may involve a pH shift
and heating to selectively denature sensitive proteins leaving PAC active in the mix.
The use of two-phase affinity partitioning as a means of protein purification is
well documented. A polyethyleneglycol (PEG) derivative and salt system has been
shown to be effective in the purification of PAC (Gavasane and Gaikar, 2003).
The PEG derivative is selected on the basis of its expected interaction through
hydrophobic, electrostatic and biospecific effects. In most cases the ligand has a
structure analogous to the penicillin G substrate of the enzyme. Thus benzoate
and phenylacetamide derivatives are useful ligands. The salt phase usually contains
sodium sulphate or sodium citrate (Marcos et al., 1999).
Thermostability can be an important feature for enzymes used in industrial
processes as higher temperatures can be used to enhance reaction rates, shift thermo-
dynamic equilibria, increase reactant solubility and decrease the reaction viscosity
(Kazan and Erarslan, 1997). The catalytic performance of PAC increases at temper-
atures between 25
C and 50
C. However, the enzyme shows poor stability at
temperatures above 35
C and therefore an effective means of providing thermal
stability is highly desirable. Many methods of PAC thermostabilisation have been
studied. Thermal unfolding of penicillin acylases has been linked to their conforma-
tional mobility in water. This mobility can be reduced by diminishing the amount
of free water which can be achieved by the addition of stabilisers such as polyol
compounds (Erarslan, 1995), bisimidoesters (Erarslan and Ertan, 1995), neutral salts
or proteins. The stability of PAC was improved by up to 180% by the addition of
trehalose after thirty minutes incubation at 60
C (Azevedo et al., 1999).
Another approach to attaining thermostability is to isolate inherently thermostable
PAC proteins from natural sources. Examples of this include the cloning and
expression in E. coli of the pac gene from A. faecalis (Verhaert et al., 1997) and
Ac. xylosoxidans (Cai et al., 2004).
In order to be useful as a biocatalyst the PAC enzyme preparation has to be
active, robust and for economic reasons, re-usable. One of the most effective
ways to enhance stability for many enzymes is to immobilise the enzyme onto
a solid support. In addition, immobilisation may allow re-use of the catalyst and
thus increase cost effectiveness. A number of immobilisation methods have been
used in this context. Each method shows superiority to the more traditional use of
free cells, extracts or even immobilized whole cells. Immobilized enzyme prepa-
rations attain higher activity and specificity and show better control of contami-
nation. The methods used include adsorption, fibre entrapment, microencapsulation,
cross-linking, copolymerisation and covalent attachment (reviewed by Shewale and
Sivaraman, 1989).
PENICILLIN ACYLASES 591
The most common immobilisation methods include cross-linking and covalent
attachment. Glutaraldehyde is the usual cross-linking agent used. A 15-fold
improvement in thermostability was apparent after cross-linking with dimethy-
ladipimate (Erarslan and Ertan, 1995). The use of different physical forms of
chitosan (powder, particles or beads) to immobilise PAC either by adsorption
followed by reticulation with glutaraldehyde or by direct cross-linking to the
matrix pretreated with glutaraldehyde has been reported (Braun et al., 1989).
Singh et al. (1988) used double entrapment methodology for the immobilisation
of PAC on agar-polyacrylamide resins. A highest specific activity of 322U/g was
obtained by covalent binding of PAC onto vinyl copolymers (Dahl et al., 1985). At
present, commercial manufacturers such as Resindion supply epoxy group resins
such as Sepabeads
™
for use as immobilisation matrices for enzymes including
PAC. Sepabeads are porous spherical beads with outstanding mechanical stability
and extensive cross-linking. Multipoint covalent immobilisation of PAC from
K. citrophila stabilised the enzyme 10,000-fold compared to the soluble enzyme
from E. coli (Guisan et al., 1993).
A further means of PAC enzyme stabilisation is afforded by the production of
cross-linked enzyme crystals or CLECs (SynthaCLEC-PA, Altus Biologics). This
approach is unique in that it results in both stabilisation and immobilisation without
activity dilution. The protein matrix is both the catalyst and the support. The
crystals are produced by stepwise crystallisation of the purified enzyme followed
by molecular cross-linking to preserve the structure, resulting in a biocatalyst which
is extremely stable to both temperature and organic solvents. The stabilisation is a
consequence of both polar and hydrophobic interactions. The PAC CLEC has been
commercialised for both hydrolytic and synthetic activity use, and retains activity
over more than 1000 batches (Govardhan, 1999). Another novel preparation called
a cross-linked enzyme aggregate (CLEA) has also been reported (Cao et al., 2001).
CLEAs are prepared by slowly adding a precipitant such as ammonium sulphate
to the enzyme at low temperature. The aggregated enzyme is subsequently linked
with glutaraldehyde and is then available for use as a biocatalyst. The CLEA can
be used in aqueous media in both forward and reverse reactions. Unlike the CLEC
preparation, the CLEA enzyme need not be purified to near homogeneity.
7. INDUSTRIAL USES OF PAC
By far the most widespread use of PACs is in the production of 6-APA from both
Pen G and Pen V. Indeed, the annual worldwide production of 6-APA is greater
than 10,000 tonnes. Immobilized PAC enzymes mainly from E. coli, B. megaterium
and A. faecalis are available from a number of commercial suppliers. Reactions
are carried out at >5000L scale under controlled conditions, the pH being either
controlled at approximately 8.0, or slowly ramped from 7.0 to, depending upon the
catalyst, as high as 8.5. Exposure to high temperature >30
C and pH >80 is
minimised to reduce inactivation of the enzyme and retain high product yield of
the otherwise relatively unstable 6-APA.
592 SPENCE AND RAMSDEN
The use of PAC in large scale production of semisynthetic penicillins and
cephalosporins is also widespread. These processes are focused on the condensation
of an appropriate D-amino acid derivative with a -lactam nucleus in a PAC-
catalysed reaction (see Wegman et al., 2001). This involves the direct acylation of
nucleophiles such as 6-APA or 7-ADCA with free acids at low <70 pH.
Kinetically controlled synthesis involves an acyl group transfer reaction in which
activated acids, esters or amides are used as the acylating agents. The yield of
this type of reaction is dependant upon three different reactions carried out by
the enzyme: 1) the synthesis of the -lactam compound, 2) the hydrolysis of the
activated acyl donor, and 3) the hydrolysis of the product. There are many ways
to optimise such a reaction including optimising pH (Ospina et al., 1996), addition
of suitable solvents (Rosell et al., 1998) and the use of high concentrations of acyl
donor and nucleus (Kaasgard and Veitland, 1992). Using such controlled strategies
a number of different antibiotics are produced including amongst others the high
volume antibiotic products amoxicillin (Diender et al., 2000), ampicillin (Illanes
and Fajardo, 2001), cephalexin (Hernandez-Justiz et al., 1999) and cefazolin (Park
et al., 2000).
An alternative use of PAC is in peptide synthesis. The acylase can be used for
the protection and deprotection of amino groups of amino acids by direct enzymatic
synthesis and acyl group transfer reactions. For example PAC has been used as a
biocatalyst in the synthesis of the sweetener aspartame (Fuganti et al., 1986), and
further use has been in the preparation of D-phenyl dipeptides whose esters readily
undergo ring closure to the corresponding diketopiperazines. Such peptides are
used as food additives and as synthons for fungicidal, antiviral and anti-allergenic
compounds (Van Langen et al., 2000). In addition, PAC can hydrolyse phenylacetyl
derivatives of a number of peptides and resolve enantiomers of some organic
compounds (Shewale et al., 1990).
PACs have also been shown to resolve racemic mixtures of chiral compounds
including amino acids (Ng et al., 2001), -amino esters (Roche et al., 1999),
amines (Van Rantwijk et al., 2002) and secondary alcohols (Svedas et al., 1996) in
aqueous media, anhydrous organic media and in water-cosolvent mixtures. The pure
enantiomers which result are used as intermediates in the production of bio-active
compounds. Specific examples include the resolution of ethyl-3-amino-4-pentynoate
to generate the S isomer which is used in the synthesis of the anti-platelet agent
Xemilofiban (Topgi et al., 1999). Kinetic enantioselective acylation of the racemic
azetidine intermediate is catalysed by PAC during the synthesis of the cephalosporin
antibiotic Loracarbef, an analogue of Cefaclor (Cainelli et al., 1997). A further use
of immobilized PAC is in the hydrolysis of racemic iso-propylamide of mandelic
acid which is used in the preparation of functionalised cephalosporins such as
cefamandole and cefonicid (Rocchietti et al., 2002).
In the biocatalytic synthesis of ampicillin and cephalexin the acyl chain donor is
(R)-phenylglycine amide. The resolution of racemic phenylglycinonitrile is carried
out in aqueous media by PAC to give the desired enantiomer (Chilov et al.,
2003). PAC can also be used in organic media, thus increasing substrate solubility.
PENICILLIN ACYLASES 593
Indeed, the production of cephalexin in ethylene glycol can be performed with
high substrate concentrations and small amounts of enzyme (Illanes et al., 2005).
Another example is that of the enantioselective acylation of the L-enantiomers of
the methyl esters of phenylglycine and 4-hydroxyphenylglycine (Basso et al., 2000).
This process leads to the isolation of the pure D-enantiomer which is used in the
preparation of -lactam antibiotics.
8. CONCLUDING REMARKS
Penicillin acylase has been studied and exploited for more than fifty years. The gene
encoding it has been cloned and sequenced from a number of species and used to
further the understanding not only of prokaryotic expression control but also protein
maturation and folding. The crystal structure of the enzyme has been solved and the
chemistry of cleavage of its substrate is well understood. Bulk production and use
of PAC is an important contributor to the wide availability and low price of many
penicillin and cephalosporin antibiotics. Exploitation of this industrial enzyme is
continuing in a number of applications, with additional substrates and products, and
genetic modifications to enhance both the enzyme’s activity and its properties. It
is clear that this family of enzymes will continue to play an important role in the
pharmaceutical and bioprocess world for some considerable time.
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