Isolation of Penicillinase-deficient Mutants of Kluyvera citrophila KY 3641

Penicillinase-deficient mutants were isolated from Kluyvera citrophila KY 3641 capable of carrying out enzymatic synthesis of 6-aminopenicillanic acid (6-APA) or d(–)-α-amino-benzylpenicillin (APc). Acridine orange treatment was found effective for the isolation of such mutants, some of which could produce larger amounts of 6-APA or APc than the parent strain. Particularly during an earlier stage of growth, the penicillin acylase activity of these penicillinase-deficient mutants did emerge due to the absence of penicillinase, while that of the parent strain did not appear because of its penicillinase activity.     

Selection of Pseudomonas melanvgenum KY 3987 as a New Ampicillin-producing Bacteria

Further selection for a better strain capable of producing D(?)-α-aminobenzylpenicillin (APc) from 6-aminopenicillanic acid (6–APA) was carried out. Pseudomonas melanogenum KY 3987 was consequently selected as a new strain possessing an APc-specific penicillin acylase.

The acylase could synthesize APc in good yields from 6–APA and phenylglycine ester and form 6–APA only from APc, not from other common penicillins. Since the Pseudomonas acylase was found incapable of forming penicillin G (Pc–G) from 6–APA and phenylacetic acid, in contrast with E. coli and Kluyvera citrophila enzymes, the enzymatic hydrolysate of Pc–G, for example by K. citrophila cells, which contained 6–APA and phenylacetate, became employed as a source of 6–APA instead of purified 6–APA to synthesize APc by the cells of P. melanogenum.     

Some Problems Involved in Ampicillin Formation by Kluyvera’s Penicillin Acylase

The cell growth of Kluyvera citrophila KY3641, capable of producing α-aminobenzyl-penicillin (APc) from 6-aminopenicillanic acid (6-APA) and phenylglycine, was stimulated by glutamic acid, serine or proline, or by pH control with tartaric acid or fumaric acid.

Penicillinase produced in an early stage of growth or pH-controlled culture was inactivated by alkaline treatment (incubation of cells at 40°C for 5 to 24 hr in pH 7.5 to 9.5) without inactivation of penicillin acylase. Surface active agents enhanced APc production. On the other hand, phenylalanine and some inorganic compounds inhibited this production.

This bacterium formed APc from penicillin G, but amounts of APc formed were only 9 μg from 20 mg of penicillin G.     

Production of 6-Aminopenicillanic Acid by Kluyvera citrophila KY 3641

Kluyvera citrophila KY 3641 cultivated aerobically more than 48 hr produced penicillin acylase. Sucrose, monosodium glutamate and aspartic acid stimulated the growth of the cells, whereas glucose, fructose, maltose and lactose inhibited the growth and acylase production. Enzymic reaction was carried out with whole broth, too, instead of using separated intact cells. The cells maintained its acylase activity more than one month and could be used repeatedly. Acetone-dried or freeze-dried cell was also available for enzymic reaction. Identification of 6-APA was also described.     

Search for Microorganisms Producing Cephalosporin Acylase and Enzymatic Synthesis of Cephalosporins

Microorganisms were tested for production of cephalosporin acylase. Some bacteria showed strong acylase activity for all of cephalexin, cephaloridine, cephalotin, penicillin G and ampicillin. Some showed a rather specific activity for cephalexin. Pseudomonas melanogenum KY 3987 showed specific activity only for cephalexin and ampicillin which contain a side chain of d-phenylglycine. Most of these acylase-producing bacteria had the ability to synthesize cephalexin and other cephalosporins from 7-aminocephem compounds and organic acid esters. Among them, Ktuyvera citrophila KY 7844 was one of the most promising organisms for enzymatic synthesis of cephalosporins. This organism had the ability to catalyze N-acylation of 7-aminocephem compound not only with α-amino acid ester, but also with such acid esters as 1-(1 H)-tetrazolylacetate methylester which has no α-amino group.     

Production of d(—)-α-Aminobenzylpenicillin by Kluyvera citrophila KY 3641

Intact cells of Kluyvera citrophila KY 3641 produced enzymaticaily d(—)-α-aminobenzyl-penicillin from 6-arainopenicillanic acid and phenylglycine derivatives. The optimum pH of the acylase was 6.5. Among various phenylglycine derivatives examined as substrates, d-phenylglycine methylester HC1 was the best compound giving the yields of about 10.7 mg/ml of d(—)-α-aminobenzy]penicillin in the enzymic reaction mixture. The product was isolated in a crystalline form and identified as d(—)-α-aminobenzylpenicillin.     

Formation of 6-Aminopenicillanic Acid, Penicillins, and Penicillin Acylase by Various Fungi

Several penicillin-producing fungi were examined for ability to produce 6-aminopenicillanic acid (6-APA) and penicillin acylase. 6-APA was found in corn steep liquor fermentations of Trichophyton mentagrophytes, Aspergillus ochraceous, and three strains of Penicillium sp. 6-APA was not detected in fermentations of Epidermophyton floccosum although penicillins were produced. 6-APA formed a large part of the total antibiotic production of T. mentagrophytes. The types of penicillins produced by various fungi were identified by paper chromatography, and it was found that all cultures produced benzylpenicillin. T. mentagrophytes and A. ochraceous showed increased yields of benzylpenicillin and the formation of phenoxymethylpenicillin in response to the addition to the fermentation medium of phenylacetic acid and phenoxyacetic acid, respectively. Washed mycelia of the three Penicillium spp. and two high penicillin-yielding strains of P. chrysogenum possessed penicillin acylase activity against phenoxymethylpenicillin. A. ochraceous, T. mentagrophytes, E. floccosum, and Cephalosporium sp. also had penicillin acylase activity against phenoxymethylpenicillin. Only two of the above fungi, T. mentagrophytes and E. floccosum, showed significant penicillin acylase activity against benzylpenicillin; in both cases it was very low. The acylase activity of A. ochraceous was considerably increased by culturing in the presence of phenoxyacetic acid. It is concluded that 6-APA frequently but not invariably accompanies the formation of penicillin, and that penicillin acylase activity against phenoxymethylpenicillin is present in all penicillin-producing fungi.     

Purification and Properties of Penicillin Acylase from Kluyvera citrophila

Penicillin acylase (EC 3.5.1.11) of Kluyvera citrophila KY7844 was purified approximately 120-fold by DEAE-cellulose chromatography, hydroxyapatite chromatography and isoelectro-focusing fractionation. The purified enzyme, with an approximate molecular weight of 63,000, appeared to be homogeneous in disc electrophoretic analysis, and showed isoelectric point (Ip) 8.12 and 13.0 units/mg of specific activity for cephalexin hydrolysis. The Michaelis constant (Km) for cephalexin and for 7-[1-(1H)-tetrazolylacetamido]-desacetoxycephalosporanic acid ((1H) T-7ADCA) was 1.4 mM and 3.6 mM, respectively. This enzyme was capable of producing (1H) T-7ADCA in 80% yield from 1-(1H)-tetrazolylacetate methylester and 7-aminodesacetoxycephalosporanic acid.     

An improved method to clone penicillin acylase genes: Cloning and expression in Escherichia coli of penicillin G acylase from Kluyvera citrophila

A simple and versatile procedure to clone penicillin acylase genes has been developed. It involves the construction of a plasmid library in a host presenting an amino acid auxotrophy. Recombinant clones carrying the acylase gene were selected on a minimal medium containing instead of the required amino acid its phenylacetyl derivative. Penicillin acylase genes from Escherichia coli ATCC 11105 and Kluyvera citrophila ATCC 21285 have been cloned in E. coli using this technique. The restriction map of the region containing the E. coli penicillin acylase gene was found to be similar to that described by H. Mayer et al. (in: Plasmids of Medical, Environmental and Commercial Importance (Timmis, K.M. and Paler, A., eds.), pp. 459–470, Elsevier, Amsterdam 1979). K. citrophila acylase gene was located within a 3.0 kb Hind III-PvuI fragment. Some differences were observed between the partial restriction maps of both genes. In addition, the production of those clones carrying the E. coli acylase was more sensitive to the growth temperature than that of the clones containing the K. citrophila gene. Bacteria harbouring plasmids containing the K. citrophila acylase sequence were able to produce about 30 fold more enzyme than the parental strain. A 60 000 dalton polypeptide corresponding to the K. citrophila acylase has been detected in a maxicell system. The industrial applications of the procedure are discussed.     

One-pot, two-step enzymatic synthesis of amoxicillin by complexing with Zn2+

A one-pot, two-step enzymatic synthesis of amoxicillin from penicillin G, using penicillin acylase, is presented. Immobilized penicillin acylase from Kluyvera citrophila was selected as the biocatalyst for its good pH stability and selectivity. Hydrolysis of penicillin G and synthesis of amoxicillin from the 6-aminopenicillanic acid formed and d-p-hydroxyphenylglycine methyl ester were catalyzed in situ by a single enzyme. Zinc ions can react with amoxicillin to form complexes, and the yield of 76.5% was obtained after optimization. In the combined one-pot synthesis process, zinc sulfate was added to remove produced amoxicillin as complex for shifting the equilibrium to the product in the second step. By controlling the conditions in two separated steps, the conversion of the first and second step was 93.8% and 76.2%, respectively. With one-pot continuous procedure, a 71.5% amoxicillin yield using penicillin G was obtained.     

Changing glycine 21 for glutamic acid in the β-subunit of penicillin G acylase from Kluyvera citrophila prevents protein maturation

Summary Active penicillin acylase from Kluyvera citrophila strain ATCC 21 285 consists of two different -and -subunits derived from a single precursor by post-translational processing. Using the chemical mutagen hydroxylamine we have treated plasmid pYKD59 containing the active penicillin acylase gene (pga) from K. citrophila and have generated different point mutant penicillin acylase genes, one producing a muturation deficient precursor. This point mutation has changed the gylcine 310 residue of the precursor for a glutamic acid (residue number 21 of the mature -subunit). The introduction of a charged residue in this position did not prevent translocation of the precursor to the periplasm but the resultant molecule was not able to undergo subsequent post-translational modification to yield the active protein.     

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.     

Enzymatic conversion in aqueous two-phase systems: deacylation of benzylpenicillin to 6-aminopenicillanic acid with penicillin acylase

The conversion of benzylpenicillin (BP) to 6-aminopenicillanic acid (6-APA) using penicillin acylase (penicillin amidohydrolase, EC 3.5.1.11) has been studied in aqueous two-phase systems. In a system composed of 8.9% (w/w) PEG 20000/7.6% (w/w) potassium phosphate the enzyme was almost completely partitioned to the bottom phase (K < 0.01), which allowed repeated batch conversions, recirculating the enzyme several times. The initial specific productivities were 0.31–1.47 μmol 6-APA mg protein^?1 min^?1 in repeated conversions over five steps. The yield obtained from the top phase was 0.47–0.71 mol 6-APA mol BP^?1. The results are discussed in relation to recirculating the enzyme by immobilizing it to a solid matrix. Despite the high phosphate concentration in the bottom phase the system needs to be titrated in order for the reaction to proceed. Titration of the top phase alone protected the enzyme from denaturation by strong alkali used for the titration.     

Monitoring bioreactors using principal component analysis: production of penicillin G acylase as a case study

The complexity of biological processes often makes impractical the development of detailed, structured phenomenological models of the cultivation of microorganisms in bioreactors. In this context, data pre-treatment techniques are useful for bioprocess control and fault detection. Among them, principal component analysis (PCA) plays an important role. This work presents a case study of the application of this technique during real experiments, where the enzyme penicillin G acylase (PGA) was produced by Bacillus megaterium ATCC 14945. PGA hydrolyzes penicillin G to yield 6-aminopenicilanic acid (6-APA) and phenyl acetic acid. 6-APA is used to produce semi-synthetic β-lactam antibiotics. A static PCA algorithm was implemented for on-line detection of deviations from the desired process behavior. The experiments were carried out in a 2-L bioreactor. Hotteling’s T ² was the discrimination criterion employed in this multivariable problem and the method showed a high sensibility for fault detection in all real cases that were studied.     

Kinetically controlled acylation of 6-APA catalyzed by penicillin acylase from Streptomyces lavendulae: effect of reaction conditions in the enzymatic synthesis of penicillin V

Abstract

Enzymatic synthesis of penicillin V (penV) by acylation of 6-aminopenicillanic acid (6-APA) was carried out using methyl phenoxyacetate (MPOA) as activated acyl donor and soluble penicillin acylase from Streptomyces lavendulae (SlPVA) as biocatalyst. The effect of different reaction conditions on penV synthesis was investigated, such as enzyme concentration, pH, molar ratio of 6-APA to MPOA, as well as presence of DMSO as water-miscible co-solvent at different concentrations. Time-course profiles of all reactions followed the typical pattern of kinetically controlled synthesis (KCS) of β-lactam antibiotics: penV concentration reached a maximum (highest yield or Y_max) and then decreased gradually. Such maximum was higher at pH 7.0, observing that final penV concentration was abruptly reduced when basic pH values were employed in the reaction. Under the selected conditions (100?mM Tris/HCl buffer pH 7.0, 30?°C, 2.7% (v/v) DMSO, 20?mM MPOA, 0.3 UI/ml of SlPVA), Y_max was enhanced by increasing the substrate molar ratio (6-APA to MPOA) up to 5, reaching a maximum of 94.5% and a S/H value of 16.4 (ratio of synthetic activity to hydrolytic activity). As a consequence, the use of an excess of 6-APA as nucleophile has allowed us to obtain some of the highest Y_max and S/H values among those reported in literature for KCS of β-lactam antibiotics. Although many penicillin G acylases (PGAs) have been described in kinetically controlled acylations, SlPVA should be considered as a different enzyme in the biocatalytic tool-box for novel potential synthetic processes, mainly due to its different substrate specificity compared to PGAs.     

Penicillin acylase mutants with altered site-directed activity from Kluyvera citrophila

Summary Oligonucleotide-directed mutagenesis has been used to obtain specific changes in the penicillin acylase gene from Kluyvera citrophila. Wild-type and mutant proteins were purified and the kinetic constants for different substrates were determined. Mutations in Met168 highly decreased the specificity constant of the enzyme for penicillin G, penicillin V and phenylacetyl-4-aminobenzoic acid and the catalytic constant k _cat for phenylacetyl-4-aminobenzoic acid. Likewise, the phenylmethylsulphonyl-fluoride sensitivity was significantly decreased. It is concluded that the 168 residue is involved in binding by interaction with the acid moiety of the substrate. A putative penicillin-binding domain was located in penicillin acylase by sequence homology with other penicillin-recognizing enzymes. Lys374 and His481, the conserved amino acid residues that are essential for catalysis in these enzymes, can be changed in penicillin acylase with no changes to the k _cat and phenylmethylsulphonyl fluoride reactivity, but change the K _m.The likelihood of the existence of this proposed penicillin binding site is discussed. The reported results might be used to alter the substrate specificity of penicillin acylase in order to hydrolyse substrates of industrial significance other than penicillins. Offprint requests to: I. Prieto     

Biotransformation of penicillin V to 6-aminopenicillanic acid using immobilized whole cells of E. coli expressing a highly active penicillin V acylase

The production of 6-aminopenicillanic acid (6-APA) is a key step in the manufacture of semisynthetic antibiotics in the pharmaceutical industry. The penicillin G acylase from Escherichia coli has long been utilized for this purpose. However, the use of penicillin V acylases (PVA) presents some advantages including better stability and higher conversion rates. The industrial application of PVAs has so far been limited due to the nonavailability of suitable bacterial strains and cost issues. In this study, whole-cell immobilization of a recombinant PVA enzyme from Pectobacterium atrosepticum expressed in E. coli was performed. Membrane permeabilization with detergent was used to enhance the cell-bound PVA activity, and the cells were encapsulated in calcium alginate beads and cross-linked with glutaraldehyde. Optimization of parameters for the biotransformation by immobilized cells showed that full conversion of pen V to 6-APA could be achieved within 1?hr at pH 5.0 and 35°C, till 4% (w/v) concentration of the substrate. The beads could be stored for 28 days at 4°C with minimal loss in activity and were reusable up to 10?cycles with 1-hr hardening in CaCl₂ between each cycle. The high enzyme productivity of the PVA enzyme system makes a promising case for its application for 6-APA production in the industry.     

ENHANCED PRODUCTION OF 6-AMINOPENICILLANIC ACID IN AQUEOUS METHYL ISOBUTYL KETONE SYSTEM WITH IMMOBILIZED PENICILLIN G ACYLASE

Enzymatic hydrolysis of penicillin G for production of 6-amino-penicillanic-acid (6-APA) was achieved by using penicillin G acylase as catalyst in an aqueous-methylisobutyl ketone (MIBK) system. The optimization was carried out and it was found that the best conversion was improved 10% more than the aqueous system, which was obtained at the conditions: initial pH 8.0, 5.0% (W/V) substrate (penicillin G), and temperature at 35°C, and the ratio of aqueous and organic phase was 3:1. The stability of the biocatalyst was studied at the operational conditions. After 5 cycles of semi-batch reactions, the residual activity of penicillin G acylase was 69.2% of the initial activity. There was no apparent loss of the yield of product. This process has a potential application in the industrial scale production of 6-APA because it simplifies the process effectively.     

Penicillin G acylase fromKluyvera citrophila new choice as industrial enzyme

Summary We propose a new and integrated method for the evaluation of industrial enzymes. The application of this method to the enzyme penicillin G acylase fromKlyvera citrophila shows very interesting industrial propects. This acylase presents a much better stability agains heat, pH or organic cosovents as compared with the more popular enzyme fromEscherichia coli. In addition, this enzyme is very easy to immobilize through its amine groups and to stabilize through multipoint covalent attachment on activated pre-existing supports.