Assay of penicillin acylase in organic media

Summary The synthetic substrate 6-nitro-3-(phenylacetamido) benzoic acid (NIPAB) is an appropriate substrate for assaying penicillin acylase activity in reversed micellar systems of Aerosol - OT in isooctane. Accumulation of 6-nitro-3-aminobenzoic acid (NABA) produced by the enzymatic hydrolysis of NIPAB, followed by the increase in absorbance at 405 nm, was linear at 4 to 20 mM for up to 30 minutes and 15 °C to 40 °C.Abbreviations PA penicillin acylase (penicillin amidohydrolase EC 3.5.1.11) - AOT Aerosol OT (sodium bis- (2-ethylhexyl) sulfosuccinate) - NIPAB 6-nitro-3-(phenylacetamido)-benzoic acid - NABA 6-nitro-3-aminobenzoic acid - BSA bovine serum albumin     

Chromogenic analogues of penicillin dihydroF and penicillin K for the continuous spectrophotometric determination of aliphatic penicillin acylase activity

The synthesis of 2-nitro-5-[(hexanoyl)-amino]-benzoic acid and 2-nitro-5-[(octanoyl)-amino]-benzoic acid as chromogenic substrates for the determination of aliphatic penicillin acylase activity is described. During enzymatic hydrolysis, the released chromophore, 2-nitro-5-amino-benzoic acid, was detected at 405 nm. Penicillin acylase from Streptomyces lavendulae had an appreciable activity towards these substrates, which can then be used to detect penicillin acylases able to cleave hexanoyl and octanoyl residues off synthetic amides as well as penicillin dihydroF and penicillin K, their natural analogues.     

Substrate specificity of penicillin acylase from Streptomyces lavendulae

The kinetic parameters of several substrates of penicillin acylase from Streptomyces lavendulae have been determined. The enzyme hydrolyses phenoxymethyl penicillin (penicillin V) and other penicillins with aliphatic acyl-chains such as penicillin F, dihydroF, and K. The best substrate was penicillin K (octanoyl penicillin) with a k(cat)/K(m) of 165.3 mM(-1) s(-1). The enzyme hydrolyses also chromogenic substrates as NIPOAB (2-nitro-5-phenoxyacetamido benzoic acid), NIHAB (2-nitro-5-hexanoylamido benzoic acid) or NIOAB (2-nitro-5-octanoylamido benzoic acid), however failed to hydrolyse phenylacetil penicillin (penicillin G) or NIPAB (2-nitro-5-phenylacetamido benzoic acid) and penicillins with polar substituents in the acyl moiety. These results suggest that the structure of the acyl moiety of the substrate is more determinant than the amino moiety for enzyme specificity. The enzyme was inhibited by several organic acids and the extent of inhibition changed with the hydrophobicity of the acid. The best inhibitor was octanoic acid with a K(i) of 0.8 mM. All the results, taking together, point to an active site highly hydrophobic for this penicillin acylase from Streptomyces lavendulae.     

Penicillin Acylase of Pseudomonas melanogenum KY 3987

Partially purified penicillin acylases (EC 3.5.1.11) were prepared from Pseudomonas melanogenum KY 3987 and Kluyvera citrophila KY 3641 capable of synthesizing d(–)-α-amino-benzylpenicillin (APc) from 6-aminopenicillanic acid (6-APA) and phenylglycine methyl ester. As the cell-free extract of P. melanogenum contained high levels of penicillinase (EC 3.5.2.6), the acylase was separated completely from the penicillinase by use of Sephadex column chromatography or electrofocusing. The most salient property of the P. melanogenum penicillin acylase was its substrate specificity to penicillin substrates: it could form 6-APA only from APc but not from penicillin G, penicillin V and p-aminobenzylpenicillin, whereas the K. citrophila acylase acted on all of these penicillins. The P. melanogenum enzyme is hence considered a novel type of penicillin acylase.     

A method for screening penicillin G acylase-producing bacteria by means of 2-nitro-5-phenylacetaminobenzoic acid test paper

A simple, rapid assay for screening penicillin G acylase-producing bacteria is presented. The method is based on the formation of yellow 2-nitro-5-aminobenzoic acid by penicillin G acylase acting on 2-nitro-5-phenylacetaminobenzoic acid (NIPAB). NIPAB test paper is briefly applied to bacterial colonies on the agar surface, which are subsequently scored individually on the paper by color; bright yellow indicates the presence of penicillin G acylase, natural color its absence. The present method is suitable not only for screening penicillin G acylase-production by a variety of bacteria but also for detection from a large number of transformant colonies of clones containing a gene encoding for the enzyme.     

Chainia Penicillin V Acylase: Strain Characteristics, Enzyme Immobilization, and Kinetic Studies

Aerobic cultures of an actinomycete were found to produce penicillin V acylase (PVA) (PA, EC-3.5.1.11) extracellularly. The presence of L-2-3 diamino-propionic acid in cell wall and formation of sclerotia on culture media led to its identification as Chainia, a sclerotial Streptomyces. Partially purified acylase was adsorbed on kieselguhr and entrapped in polyacrylamide gel. The immobilized preparation proved effective with respect to retention of enzyme and enzyme activity even after 15 successful cycles. The pH optimum for crude enzyme was in the range of pH 7.5–8.0, and for the (NH4)₂ SO₄ fraction it was pH 8.5. The immobilized enzyme showed maximal activity at pH 9.5. The optimum temperature for acylase activity was at 55°C. The crude enzyme, ammonium sulfate fraction, and immobilized enzyme showed K _m value for penicillin V of 6.13 mM, 14.3 mM, and 17.1 mM, respectively. Received: 11 December 1997 / Accepted: 9 April 1998     

Thermal inactivation of immobilized penicillin acylase in the presence of substrate and products

Inactivation of immobilized penicillin acylase has been studied in the presence of substrate (penicillin G) and products (phenylacetic acid and 6-aminopenicillanic acid), under the hypothesis that substances which interact with the enzyme molecule during catalysis will have an effect on enzyme stability. The kinetics of immobilized penicillin acylase inactivation was a multistage process, decay constants being evaluated for the free-enzyme and enzyme complexes, from whose values modulation factors were determined for the effectors in each enzyme complex at each stage. 6-Aminopenicillanic acid and penicillin G stabilized the enzyme in the first stage of decay. Modulation factors in that stage were 0.96 for penicillin G and 0.98 for 6-aminopenicillanic acid. Phenylacetic acid increased the rate of inactivation in both stages, modulating factors being -2.31 and -2.23, respectively. Modulation factors influence enzyme performance in a reactor and are useful parameters for a proper evaluation. (c) 1996 John Wiley & Sons, Inc.     

Enzymatic splitting of penicillin V for the production of 6-APA using immobilized penicillin V acylase

Penicillin V acylase from Fusarium sp. SKF 235 was immobilized on several cation-exchange resins, of which Amberlite CG-50 was preferred. Maximum activity of the immobilized penicillin V acylase was 250 to 280 IU/g dry beads. The pH and temperature optima of the enzyme shifted from 6.5 to 6.8 and 55°C to 60°C, respectively, as a result of immobilization. However, the K _m for penicillin V remained at 10mm. Parameters for producing 6-aminopenicillanic acid were investigated and the immobilized penicillin V acylase was used for 68 cycles in a stirred tank reactor.     

Immobilization of permeabilized whole cell penicillin G acylase from Alcaligenes faecalis using pore matrix crosslinked with glutaraldehyde

The activity of penicillin G acylase from Alcaligenes faecalis increased 7.5-fold when cells were permeabilized with 0.3% (w/v) CTAB. The treated cells were entrapped by polyvinyl alcohol crosslinked with boric acid, and crosslinked with 2% (v/v) glutaraldehyde to increase the stability. The conversion yield of penicillin G to 6-aminopenicillanic acid was 75% by immobilized system in batch reaction. No activity was lost after 15 cycles and about 65% enzyme activity was retained at the end of the 31th cycle.     

Conjugation of penicillin acylase with the reactive copolymer of N-isopropylacrylamide: a step toward a thermosensitive industrial biocatalyst

Conjugation of penicillin acylase (PA) to poly-N-isopropylacrylamide (polyNIPAM) was studied as a way to prepare a thermosensitive biocatalyst for industrial applications to antibiotic synthesis. Condensation of PA with the copolymer of NIPAM containing active ester groups resulted in higher coupling yields of the enzyme (37%) compared to its chemical modification and copolymerization with the monomer (9% coupling yield) at the same NIPAM:enzyme weight ratio of ca. 35. A 10-fold increase of the enzyme loading on the copolymer resulted in 24% coupling yield and increased by 4-fold the specific PA activity of the conjugate. Two molecular forms of the conjugate were found by gel filtration on Sepharose CL 4B: the lower molecular weight fraction of ca. 10(6) and, presumably, cross-linked protein-polymer aggregates of MW > 10(7). Michaelis constant for 5-nitro-3-phenylacetamidobenzoic acid hydrolysis by the PA conjugate (20 microM) was found to be slightly higher than that of the free enzyme (12 microM), and evaluation of V(max) testifies to the high catalytic efficiency of the conjugated enzyme. PolyNIPAM-cross-linked PA retained its capacity to synthesize cephalexin from d-phenylglycin amide and 7-aminodeacetoxycephalosporanic acid. The synthesis-hydrolysis ratios of free and polyNIPAM-cross-linked enzyme in cephalexin synthesis were 7.46 and 7.49, respectively. Thus, diffusional limitation, which is a problem in the industrial production of beta-lactam antibiotics, can be successfully eliminated by cross-linking penicillin acylase to a smart polymer (i.e., polyNIPAM).     

Immobilization of penicillin acylase on acrylic carriers

Penicillin acylase obtained from E. Coli (E. C. 3.5.1.11) was covalently bound via glutaric aldehyde to acrylic carriers crosslinked with divinylbenzene or ethylene glycol dimethacrylate. The best enzymatic preparation was obtained by using ethyl acrylate/ ethylene glycol dimethacrylate copolymer. 1 cm³ of the carrier bound 6.4 mg of protein, having 72% activity in relation to the native enzyme. The preparation lost only 10% of its initial activity after 100 d of storage at 4°C. A negligible effect of immobilization on the enzyme activity at different temperatures or pH as well as significant increase of the stability of the immobilized enzyme at elevated temperatures were observed.Abbreviations BA butyl acrylate - AE ethyl acrylate - PA penicillin acylase - 6-APA 6-aminopenicillanic acid - EGDMA ethylene glycol dimethacrylate - DVB divinylbenzene     

Specificity of Penicillin Acylase of Fusarium and of Penicillium chrysogenum

Extracts containing penicillin acylase were obtained by shaking the mycelium of Fusarium avenaceum and of Penicillium chrysogenum in 0.2 M sodium acetate or sodium chloride solution. The optimum pH for conversion of penicillin V into 6-aminopenicillanic acid (6-APA) by the enzyme of Fusarium was about 7.5, and the reaction velocity was increased by a rise in temperature from 27 to 37 C. Penicillin G and penicillins with an aliphatic side chain were cleaved much less readily than was penicillin V. With the enzyme preparation obtained from a nonpenicillin-producing strain of P. chrysogenum, the reaction rate was higher at pH 8.5 than at pH 7.5 and pH 6.5. The acylase of P. chrysogenum hydrolyzes penicillin V more readily than penicillin G. In a series of aliphatic penicillins, the amount of 6-APA formed through the action of this enzyme increased with the number of carbon atoms of the side chain. Penicillins with a glutaryl or an adipyl group as side chain were unaffected by the enzyme of Fusarium and of Penicillium. No reaction was observed upon incubation of penicillin N (with a D-aminoadipyl side chain) or isopenicillin N (with an L-aminoadipyl side chain) with Fusarium and Penicillium extract. When the carboxy group of the side chain of these penicillins was esterified, formation of 6-APA was observed upon incubation with Penicillium extract, whereas no 6-APA or only very small amounts were obtained by acylase of Fusarium.     

Isolation and characterization of a new strain of Achromobacter sp. with beta-lactam antibiotic acylase activity

A bacterial strain producing a -lactam antibiotic acylase, able to hydrolyze ampicillin to 6-aminopenicillanic acid more efficiently than penicillin G, was isolated from soil and characterized. The isolate was identified as Achromobacter sp. using the phenotypic characteristics, composition of cellular fatty acids and 16S rRNA gene sequence. The enzyme synthesis was fully induced by phenylacetic acid (PAA) at a concentration of 2 g l^–1. PAA at concentrations up to 12 g l^–1 had no negative effect on the specific activity of acylase and biomass production, but slowed down the specific growth rate. Benzoic or 4-hydroxyphenylacetic acids can also induce synthesis of the enzyme. The inducers were metabolized in all cases. Acylase activity in cell-free extracts was determined with various substrates; ampicillin, cephalexin and amoxicillin were hydrolyzed 1.5- and 2-times faster than penicillin G. A high stability of acylase activity was observed over a wide range of pH (5.0–8.5) and at temperatures above 55°C.     

Cephalosporin C acylase and penicillin V acylase formation by Aeromonas sp. ACY 95

Aeromonas sp. ACY 95 produces constitutively and intracellularly a penicillin V acylase at an early stage of fermentation (12 h) and a cephalosporin C acylase at a later stage (36 h). Some penicillins, cephalosporin C and their side chain moieties/analogues, phenoxyacetic acid, penicillin V and penicillin G, enhanced penicillin V acylase production while none of the test compounds affected cephalosporin C acylase production. Supplementation of the medium with some sugars and sugar derivatives repressed enzyme production to varying degrees. The studies on enzyme formation, induction and repression, and substrate profile suggest that the cephalosporin C acylase and penicillin V acylase are two distinct enzymes. Substrate specificity studies indicate that the Aeromonas sp. ACY 95 produces a true cephalosporin C acylase which unlike the enzymes reported hitherto hydrolyses cephalosporin C specifically.The authors are with Research and Development, Hindustan Antibiotics Limited, Pimpri. Pune 411 018, India     

Cloning and characterization of penicillin V acylase from Streptomyces mobaraensis

We report on the molecular cloning and characterization of penicillin V acylase (PVA) from an actinomycete, Streptomyces mobaraensis (Sm-PVA), which was originally isolated as an acylase that efficiently hydrolyzes the amide bond of various N-fatty-acyl-l-amino acids and N-fatty-acyl-peptides as well as capsaicin (8-methyl-N-vanillyl-6-nonenamide). In addition, the purified Sm-PVA hydrolyzed penicillin V with the highest activity (k(cat)) among the PVAs so far reported, penicillin G, and 2-nitro-5-phenoxyacetamide benzoic acid. The BLAST search revealed that the Sm-PVA precursor is composed of a polypeptide that is characteristic of enzymes belonging to the beta-lactam acylase family with four distinct segments; a signal sequence (43 amino acids), an alpha subunit (173 amino acids), a linker peptide (28 amino acids), and a beta subunit (570 amino acids). The mature, active Sm-PVA is a heterodimeric protein with alpha and beta subunits, in contrast to PVAs isolated from Bacillus sphaericus and B. subtilis, which have a homotetrameric structure. The amino acid sequence of Sm-PVA showed identities to PVA from S. lavendulae, N-acylhomoserine lactone-degrading acylase from Streptomyces sp., cyclic lipopeptide acylase from Streptomyces sp., and aculeacin A acylase from Actinoplanes utahensis with 68, 67, 67, and 41% identities, respectively.     

pH stability of penicillin acylase from <Emphasis Type="Italic">Escherichia coli</Emphasis>

The inactivation kinetics of penicillin acylase from Escherichia coli have been investigated over a wide pH range at 25 and 50 degrees C. The enzyme was very stable in neutral solutions and quickly lost its catalytic activity in acidic and alkaline solutions. In all cases, the inactivation proceeded according to first order reaction kinetics. Analysis of the pH dependence of enzyme stability provides evidence that stable penicillin acylase conformation is maintained by salt bridges. Destruction of the salt bridges due to protonation/deprotonation of the amino acid residues forming these ion pairs causes inactivation by formation of the unstable "acidic" EH(4)(3+), EH(3)(2+), EH(2)(+) and "alkaline" E(-) enzyme forms. At temperatures above 35 degrees C penicillin acylase apparently undergoes a conformational change that is accompanied by destruction of one of these salt bridges and change in the catalytic properties.     

A postfermentative stage improves penicillin acylase production by a recombinant E. coli

Active penicillin acylase is formed only after an in vivo post-translational processing of the polypeptide precursor. Such a maturation process is rare in procaryotes. In this work, incubation under aerated conditions, of whole recombinant E. coli cells after glucose depletion and growth cessation, i.e., during the postfermentative stage, consistently resulted in 2- to 4-fold increases in penicillin acylase activity. Such results suggest that penicillin acylase maturation occurs to a high extent even during the postfermentative stage. Accordingly, the effect of different incubation conditions, during the postfermentative stage, on penicillin acylase was determined. Incubation under anaerobic conditions resulted only in a 1.27-fold increase of enzyme activity, with respect to the end of the batch culture, whereas a 3- and 4- fold increase occurred during incubation under dissolved oxygen concentrations of 100 and 43% (with respect to air sat.), respectively. Only a small negative effect, on the maturation process, was observed during incubation with acetate concentrations above 0.6 g/L. No effect of pH, in the range of 6.0 to 8.0, was observed.     

Enhanced production of penicillin V acylase from Streptomyces lavendulae

At 28 °C, Streptomyces lavendulae produced high levels of penicillin V acylase (178 IU/l of culture) when grown on skim milk as the sole nutrient source for 275 h. The enzyme showed catabolite repression by glucose and was produced in the stationary phase of growth. Penicillin V was a good inducer of penicillin V acylase formation, while phenoxyacetic acid, the side-chain moiety of penicillin V, did not alter enzyme production significantly. The enzyme was stable between pH 6 and 11 and at temperatures from 20 °C to 55 °C. This extracellular enzyme was able to hydrolyse natural penicillins and unable to hydrolyse penicillin G. Received: 22 March 1999 / Received revision: 16 June 1999 / Accepted: 20 June 1999     

Molecular biology of β-lactam acylases

-Lactam acylases such as penicillin G acylases, penicillin V acylases and glutaryl 7-aminocephalosporanic acid acylases are used in the manufacture of 6-aminopenicillanic acid, 7-aminodesacetoxycephalosporanic acid and 7-aminocephalosporanic acid (7-ACA). Genetically-engineered strains producing 1050 U/g, 3200 U/g and 7000 to 10,000 U/I of penicillin G acylase, penicillin V acylase and glutaryl-7-ACA acylase, respectively, have been developed. The penicillin G acylase studied to date and the glutaryl-7-ACA acylase from Pseudomonas sp. share some common features: the active enzyme molecules are composed of two dissimilar subunits that are generated from respective precursor polypeptide; the proteolytic processing is a post-translational modification which is regulated by temperature; and the Ser residue at the N-terminus of the -sub-unit (Ser²⁹⁰; penicillin G acylase numbering) is implicated as the active site residue. Protein engineering, to generate penicillin G acylase molecules and their precursors with altered sequences, and the structure-function correlation of the engineered molecules are discussed.The authors are with Research and Development, Hindustan Antibiotics Ltd, Pimpri, Pune 411 018, India;     

Variation of penicillin acylase partition coefficient with phase volume ratio in poly(ethylene glycol)–sodium citrate aqueous two-phase systems

The influence of phase volume ratio on partition and purification of penicillin acylase from Escherichia coli on poly(ethylene glycol)–sodium citrate aqueous two-phase systems was studied. In PEG 1000 systems both partition coefficients of the enzyme and total protein increased with decreasing phase volume ratio. However, in PEG 3350 containing NaCl, penicillin acylase follows a reverse trend, while total protein behaves in the same way. Implications for protein purification designs are discussed.