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.     

β-Lactamase-Free Penicillin Amidase from Alcaligenes sp.: Isolation Strategy, Strain Characteristics, and Enzyme Immobilization

Isolation and characterization of a β-lactamase (EC 3.5.2.6)-free, penicillin amidase (penicillin amidohydrolase, EC 3.5.1.11)-producing organism is reported. The test strain was isolated by an enrichment technique with a substrate other than penicillins. The isolated strain belongs to the genus Alcaligenes. Phenylacetic acid was found to be the inducer of penicillin amidase. The amidase has a broad substrate spectrum. It is very active against penicillin G and semisynthetic cephalosporins, whereas penicillin V and semisynthetic penicillins acted moderately as a substrate. Immobilized cells of Alcaligenes sp. were shown to act as a reversible enzyme. Received: 28 April 1999 / Accepted: 18 May 1999     

Decreased production ofpara-hydroxypenicillin V in penicillin V fermentations

Summary Penicillin V (phenoxymethyl penicillin) is produced by industrial strains ofPenicillium chrysogenum in the presence of phenoxyacetic acid (POAc), a side-chain precursor for the penicillin V molecule. The wild-type strain ofP. chrysogenum produces an undesirable penicillin byproduct,para-hydroxypenicillin V (p-OH penicillin V), in addition to penicillin V, viapara-hydroxylation of POAc and subsequent incorporation of thep-OH phenoxyacetic acid into the penicillin molecule. Most of thep-OH penicillin V is produced late in cycle when the POAc concentration in the medium is nearly depleted. The level ofp-OH penicillin V produced by the control strain ranges up to 10–15% of the total penicillins produced. 3-Phenoxypropionic acid andp-bromophenylacetic acid partially inhibit the formation ofp-OH penicillin V with a minimal effect on penicillin V productivity. Mutants deficient in their ability to hydroxylate POAc were found to produce lower levels ofp-OH penicillin V. Multi-step mutation and screening, starting with the wild-type strain, have culminated in isolation of mutants which producep-OH penicillin V as 1% of the total penicillins with no adverse effect on penicillin V productivity.     

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.     

Regulation and compartmentalization of β‐lactam biosynthesis

Penicillins and cephalosporins are β-lactam antibiotics widely used in human medicine. The biosynthesis of these compounds starts by the condensation of the amino acids l -α-aminoadipic acid, l -cysteine and l -valine to form the tripeptide δ-l -α-aminoadipyl-l -cysteinyl-d -valine catalysed by the non-ribosomal peptide ‘ACV synthetase’. Subsequently, this tripeptide is cyclized to isopenicillin N that in Penicillium is converted to hydrophobic penicillins, e.g. benzylpenicillin. In Acremonium and in streptomycetes, isopenicillin N is later isomerized to penicillin N and finally converted to cephalosporin. Expression of genes of the penicillin (pcbAB, pcbC, pendDE) and cephalosporin clusters (pcbAB, pcbC, cefD1, cefD2, cefEF, cefG) is controlled by pleitropic regulators including LaeA, a methylase involved in heterochromatin rearrangement. The enzymes catalysing the last two steps of penicillin biosynthesis (phenylacetyl-CoA ligase and isopenicillin N acyltransferase) are located in microbodies, as shown by immunoelectron microscopy and microbodies proteome analyses. Similarly, the Acremonium two-component CefD1–CefD2 epimerization system is also located in microbodies. This compartmentalization implies intracellular transport of isopenicillin N (in the penicillin pathway) or isopenicillin N and penicillin N in the cephalosporin route. Two transporters of the MFS family cefT and cefM are involved in transport of intermediates and/or secretion of cephalosporins. However, there is no known transporter of benzylpenicillin despite its large production in industrial strains.     

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     

Kinetic and microstructural characterization of immobilized penicillin acylase from Streptomyces lavendulae on Sepabeads EC-EP

Recombinant penicillin acylase from Streptomyces lavendulae was covalently bound to epoxy-activated Sepabeads EC-EP303®. Optimization of the immobilization process led to a homogeneous distribution of the enzyme on the support surface avoiding the attachment of enzyme aggregates, as shown by confocal electron microscopy. The optimal immobilized biocatalyst had a specific enzymatic activity of 26.2IUgwetcarrier^?1 in the hydrolysis of penicillin V at pH 8.0 and 40°C. This biocatalyst showed the highest activity at pH 8.5 and 65°C, 1.5 pH units lower and 5°C higher than its soluble counterpart. Substrate specificity of the derivative also showed its ability to efficiently hydrolyze other natural aliphatic penicillins such as penicillins K, F and dihydroF. The immobilized enzyme was highly stable at 40°C and pH 8.0 (t_1/2=625 h vs. t_1/2=397 h for the soluble enzyme), and it could be recycled for at least 30 consecutive batch reactions without loss of catalytic activity.     

Applications of Biocatalysis to Biotechnology

β-lactam antibiotics (e.g. penicillins, cephalosporins) are of major clinical importance and contribute to over 40% of the total antibiotic market. These compounds are produced as secondary metabolites by certain actinomycetes and filamentous fungi (e.g. Penicillium, Aspergillus and Acremonium species). The industrial producer of penicillin is the fungus Penicillium chrysogenum. The enzymes of the penicillin biosynthetic pathway are well characterized and most of them are encoded by genes that are organized in a cluster in the genome. Remarkably, the penicillin biosynthetic pathway is compartmentalized: the initial steps of penicillin biosynthesis are catalyzed by cytosolic enzymes, whereas the two final steps involve peroxisomal enzymes. Here, we describe the biochemical properties of the enzymes of β-lactam biosynthesis in P. chrysogenum and the role of peroxisomes in this process. An overview is given     

Factors affecting the synthesis of penicillins by the immobilized penicillin acylase from Escherichia coli

The effect of pH, temperature, reactant concentration, and reaction time has been investigated for the synthesis of N-benzhydryl-N′-acetamidopiperazyl-6-penicillanic acid and N-benzyl-N′-acetamidopiperazyl-6-penicillanic acid from 6-aminopenicillanic acid by the immobilized penicillin acylase from Escherichia coli. The synthesis of penicillins from carboxylic acids proceeds most rapidly at pH 5; with ethyl ester derivatives of carboxylic acids the pH optimum is higher (6–7). The most rapid synthesis of penicillins was obtained with ethyl ester derivatives of carboxylic acids. The optimum temperatures were 25–35°C.     

Engineering the Substrate Specificity of a Thermophilic Penicillin Acylase from Thermus thermophilus

A homologue of the Escherichia coli penicillin acylase is encoded in the genomes of several thermophiles, including in different Thermus thermophilus strains. Although the natural substrate of this enzyme is not known, this acylase shows a marked preference for penicillin K over penicillin G. Three-dimensional models were created in which the catalytic residues and the substrate binding pocket were identified. Through rational redesign, residues were replaced to mimic the aromatic binding site of the E. coli penicillin G acylase. A set of enzyme variants containing between one and four amino acid replacements was generated, with altered catalytic properties in the hydrolyses of penicillins K and G. The introduction of a single phenylalanine residue in position α188, α189, or β24 improved the K_m for penicillin G between 9- and 12-fold, and the catalytic efficiency of these variants for penicillin G was improved up to 6.6-fold. Structural models, as well as docking analyses, can predict the positioning of penicillins G and K for catalysis and can demonstrate how binding in a productive pose is compromised when more than one bulky phenylalanine residue is introduced into the active site.     

Comparison of penicillins produced by inter-species hybrids fromPenicillium chrysogenum

Summary From inter-species heterokaryons produced following induced protoplast fusion betweenPenicillium chrysogenum andP. stoloniferum, P. patulum orP. verrucosum var.cyclopium, hybrid progeny with different stability and morphology could be isolated.Analysis of penicillins excreted from the hybrid progeny showed that antibiotic production was influenced both quantitatively and qualitatively. In most instances, and except forP. chrysogenum+P. stoloniferum hybrids, penicillin yield was adversely affected after inter-species hybridization. Investigations of the different natural penicillins produced, including pentyl-, benzyl- and heptylpenicillin, indicated that the relative amount of pentylpenicillin was considerably decreased in favour of benzyl- and/or heptylpenicillin. In addition, as observed forP. chrysogenum+P. verrucosum var.cyclopium, genes from different species and coding for different biochemical properties could simultaneously be expressed in inter-species hybrids.     

Extracellular Accumulation of Phospholipids,UDP-N-Acetylhexosamine Derivatives and L-Glutamic Acid by Penicillin-treated Corynebacterium alkanolyticum

The addition of penicillin to cells of Corynebacterium alkanolyticum No. 314 growing on n-paraffins medium caused the simultaneous excretion of phospholipids, UDP-N-acetylhexosamine derivatives and L-glutamic acid.

Among many antibiotics which inhibit cell wall synthesis, only the inhibitors of peptideglycan transpeptidase such as penicillin G and cephaloridine were effective for inducing the excretion of phospholipids, UDP-N-acetylhexosamine derivatives and L-glutamic acid, while the others promoted only the excretion of UDP-N-acetylhexosamine derivatives.

From the close relationship between the excretion of L-glutamic acid and the excretion of phospholipids, it was suggested that the action of penicillins and cephalosporins on the cell membrane resulted in the excretion of L-glutamic acid.     

Amoxycillin: A new Semi-synthetic Penicillin

Amoxycillin (α-amino-p-hydroxybenzylpenicillin) is a new semi-synthetic penicillin with a broad spectrum of antibacterial activity similar to that of ampicillin. Penicillin-sensitive strains of staphylococci, streptococci, and pneumococci were sensitive to concentrations of 0·1 μg or less of amoxycillin/ml. Strains of Haemophilus influenzae were inhibited by a level of 0·5 μg/ml, and most strains of Escherichia coli, Proteus mirabilis, Shigella sonnei, Salmonella species, and Streptococcus faecalis were sensitive to a concentration of 5 μg or less of amoxycillin/ml. Penicillinase-producing strains of Staphylococcus aureus and strains of Pseudomonas aeruginosa, indole-positive Proteus, Klebsiella, and Enterobacter were insensitive to amoxycillin. The new penicillin was bactericidal in activity, as with other penicillins, and its antibacterial activity was not reduced in the presence of serum. After oral administration to volunteer subjects amoxycillin produced serum concentrations twice as high as those obtained with similar doses of ampicillin, and the penicillin was recovered unchanged in high concentrations in the urine. The absorption of amoxycillin was not greatly influenced by food, and administration of probenecid resulted in increased and more prolonged concentrations of amoxycillin in serum.     

A novel penicillin acylase from the environmental gene pool with improved synthetic properties

A new penicillin acylase was isolated by cloning and functional screening of DNA isolated from a sand soil enrichment culture. Sequence analysis of this enzyme, PAS2, revealed homology to a group of prominent penicillin G acylases, including the intensively studied enzyme of Escherichia coli ATCC 11105. Accordingly, PAS2 was found to be an Ntn-hydrolase with an N-terminal serine as the catalytic nucleophile, located on its 61.9 kDa β-subunit. The α-subunit was shown to have a molecular mass of 25.5 kDa.To evaluate the biocatalytic performance of the new enzyme, the complex kinetic parameters α, β₀, and γ were determined for the kinetically controlled synthesis of a number of important semi-synthetic penicillins and cephalosporins. While α is a measure for the relative affinity of the enzyme for the activated acyl donor (AD), β₀ and γ quantify the efficiency of acyl-transfer to the β-lactam nucleophile. Compared to the penicillin acylase of E. coli, PAS2 showed superior potential for the synthesis of 6-aminopenicillanic acid (6-APA)-derived antibiotics, allowing the accumulation of up to 2.3-fold more target product at significantly higher conversion rates. In the synthesis of amoxicillin, for instance, 1.6-fold more antibiotic was formed using the new enzyme, making PAS2 an interesting candidate for biocatalytic application.     

Radical‐scavenging activity of penicillin G,ampicillin, oxacillin,and dicloxacillin

The aim of this study was to characterize the antioxidant activity of penicillin G (PG), ampicillin (AMP), oxacillin (OX) and dicloxacillin (DOX) through their reactivity towards reactive oxygen species (superoxide anion radical, ; hydroxyl radical, HO^?; peroxyl radical, ROO^?; hydrogen peroxide, H₂O₂; DPPH^?) using various in vitro antioxidant assays with chemiluminescence (CL) and spectrophotometry as measurement techniques. In hydroxyl radical assays , PG, OX and AMP were found to inhibit the CL signal arising from the Fenton‐like reaction in a dose‐dependent manner with IC₅₀ = 0.480 ± 0.020 mM, IC₅₀ = 0.569 ± 0.021 mM, and IC₅₀ = 0.630 ± 0.019 mM, respectively. The highest reactivity of PG among the tested penicillins towards the HO radical was confirmed in the deoxyribose degradation assay. In the ABAP‐derived ROO radical assay, the radical‐scavenging ability of the test penicillins was in the following order: AMP > PG > DOX > OX. The number of reduced DPPH radicals by the drugs tested was <1 being the biggest for PG. The weak antioxidant capacity of the test penicillins was confirmed in the trolox antioxidant capacity assay (0.075 ± 0.004; 0.093 ± 0.006; 0.123 ± 0.005; 0.126 ± 0.004) for OX, AMP, DOX, PG, respectively. Use of luminol as a CL probe for estimation of penicillin reactivity towards H₂O₂ showed that only AMP was able to quench light emission; the remaining antibiotics demonstrated a strong enhancing effect. All the examined compounds showed a weak antioxidant potential when estimated using the ferric‐ferrozine assay. This study is the first to report the evaluation of test penicillins as antioxidants under the same reaction conditions.     

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     

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     

Streptococcus pneumoniae R6 interspecies transformation: genetic analysis of penicillin resistance determinants and genome‐wide recombination events

Interspecies gene transfer has been implicated as the major driving force for the evolution of penicillin resistance in Streptococcus pneumoniae. Genomic alterations of S. pneumoniae R6 introduced during four successive transformations with DNA of the high‐level penicillin‐resistant Streptococcus mitis B6 with beta‐lactam selection have now been determined and the contribution of genes to high resistance levels was analysed genetically. Essential for high level resistance to penicillins of the transformant CCCB was the combination of murM_B6 and the 3′ region of pbp2b_B6. Sequences of both genes were detected in clinical isolates of S. pneumoniae, confirming the participation of S. mitis in the global gene pool of beta‐lactam resistance determinants. The S. mitis PBP1b gene which contains an authentic stop codon within the transpeptidase domain is now shown to contribute only marginal to resistance, but it is possible that the presence of its transglycosylase domain is important in the context of cognate PBPs. The genome sequence of CCCB revealed 36 recombination events, including deletion and acquisition of genes and repeat elements. A total of 78 genes were affected representing 67 kb or 3.3% of the genome, documenting extensive alterations scattered throughout the genome.     

Penicillinase (β-lactamase) formation by blue-green algae

-Lactamase (penicillinase) activity was found in a number of strains of blue-green algae. In some cases, this enzyme permitted algae to overcome the inhibitory effects of penicillin. Production and localization of -lactamase were studied in a unicellular species, Coccochloris elabens (strain 7003), and in a filamentous, nitrogen-fixing Anabaena species (strain 7120). When cells were grown in a neutral medium with NaNO₃ as N source, the pH rose during growth; at a pH of about 10, most of the enzyme was extracellular and all the cell-bound enzyme was expressed equally well in intact or disrupted cells. If the pH was kept near neutrality during growth by gassing with CO₂ in N₂ or by growth under conditions of N₂ fixation, the enzyme remained cell-bound and cryptic for most of the growth phase, being measurable only after cells were disrupted. The enzymes from strains 7003 and 7120 had greater activity on benzyl penicillin and other penicillins than on cephalo-sporins. Some differences were observed in the substrate profiles of penicillinases from the two strains against different penicillins.A preliminary account of this work was presented at the 1974 meetings of the American Society for Microbiology in Chicago (Abstracts of Meetings, M37)     

Sensitivity of Yersinia enterocolitica to antibiotics]

Sensitivity of 33 foreign and 10 native strains of Y. enterocolitica to tetracycline, aminoglycosides, penicillins, levomycetin and polymyxin was studied. All the strains proved to be resistant to penicillin, oxacillin and ampicillin: they produced penicillinase. The level of resistance to penicillin did not always correlate with penicillin activity. The ability of the native strains to acquire R-factor in vitro from Coli bacteria was shown.