Leakage of recombinant penicillin G acylase from Escherichia coli by adding chloroform under fermentation conditions

A simple method was developed to release periplasmic penicillin G acylase from Escherichia coli BL21(DE3) during the fermentation process. More than 80% of the total penicillin G acylase was released into the broth when 3% (v/v) chloroform was added at 3 h after induction. The activity of extracellular penicillin G acylase reached 20699 U/l. This method was efficient and would facilitate further investigation of penicillin G acylase for industrial applications.     

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     

Beijerinckia indica var.penicillanicum penicillin V acylase: enhanced enzyme production by catabolite repression-resistant mutant and effect of solvents on enzyme activity

Summary Beijerinckia indica var.penicillanicum mutant UREMS-5, producing 168% more penicillin V acylase, was obtained by successive treatment with UV, -irradiation and ethylmethane sulfonate. Penicillin V acylase production by the mutant strain was resistant to catabolite repression by glucose. Incorporation of glucose, sodium glutamate and vegetable oils in the medium enhanced enzyme production. The maximum specific production of penicillin V acylase was 244 IU/g dry weight of cells. Effect of solvents on hydrolysis of penicillin V by soluble penicillin V acylase and whole cells was studied. Methylene chloride, chloroform and carbon tetrachloride significantly stimulated the rate of penicillin V hydrolysis by whole cells.     

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.     

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;     

Cloning and expression of penicillin acylase genes from overproducing strains of Escherichia coli and Bacillus megaterium

Summary Penicillin acylase genes from Escherichia coli 194, of an overproducing mutant (194-3) of this strain, and of a similar overproducing mutant of Bacillus megaterium UN1 were cloned in E. coli DH1 on the plasmid vector pACYC184. The sizes of chromosomal DNA fragments essential for penicillin acylase production were found by Tn1000 mutagenesis and in vitro deletions to be between 2.2 and 2.5 kb in the case of both E. coli genes and between 2.3 and 2.7 kb in the case of the mutant Bacillus gene. Restriction mapping indicated substantial sequence differences between the E. coli and B. megaterium penicillin acylase genes. Enzyme production in E. coli recombinants from both overproducing mutants was found to be constitutive and higher than in the original strains. The Bacillus penicillin acylase was produced intracellularly in E. coli recombinants, which is in contrast to the normal extracellular production of this enzyme in B. megaterium. Recombinant plasmids containing penicillin acylase genes from either source were found to be unstable in the absence of selection pressure for retention of the vector.     

Cloning of E. coli penicillin G acylase gene with mini-Mu containing a plasmid replicon

Summary An in vivo cloning system based on mini-Mu derivatives was used for cloning of E. coli penicillin G acylase gene (pac). We have constructed several recombinant clones producing penicillin G acylase and some of them exhibit approximately two times higher activity than original strains.     

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.     

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.     

Mutagenic Activity of Autoxidized Linolenic and Linoleic Acid

Isolation of mutants with an enhanced productivity of 7β-(4-carboxybutanamido)-cephalosporanic acid acylase (penicillin amidohydrolase, EC 3.5.1.11) was attempted. A mutant, Ci-36, isolated by a method using glutarylamlide, produced approximately 5-times more acylase than did the parental strain. However, this acylase formation was still dependent on glutaric acid which was previously found to be essential in the case of the wild strain, Pseudomonas SY-77-1. The inducible-acylase formation was found to be firmly associated with the process of cell multiplication. Subsequently, a mutant, GK-16 was derived from Ci-36, which was shown to produce the acylase at maximum level without the addition of glutaric acid. The productivity of GK-16 was 2.4-times higher than that of Ci-36.     

Rapid continuous colorimetric enzyme assay for penicillin G acylase

A rapid, continuous, colorimetric enzyme assay for penicillin G acylase has been developed. The assay measures the formation of the acidic products of penicillin G hydrolysis by following the decrease in pH using Phenol Red as an indicator. The activity measured is directly proportional to the amount of enzyme added to the assay, having a linear relationship with an R ² value of 0.9994.     

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     

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.     

Immobilization of <Emphasis Type="Italic">Alcaligenes faecalis</Emphasis> penicillin G acylase on epoxy-type supports

Alcaligenes faecalis penicillin G acylase has several desired features over other penicillin G acylases and its use in industry requires immobilization. In this work, two novel supports ZH-EP (epoxy type) and ZH-HA (epoxy-amino type) were used to immobilize Alcaligenes faecalis penicillin G acylase (AfPGA) with Eupergit C as reference. The saturation of immobilized protein on ZH-EP (269 mg/g, 116 h) and ZH-HA (296 mg/g, 15 h) was obtained more rapidly than Eupergit C (197 mg/g, 260 h). And the activity of immobilized AfPGA on ZH-EP (520 U/g) and ZH-HA (2200 U/g) was higher than that on Eupergit C (310 U/g). The properties of three immobilized enzymes were compared and no obvious difference was observed, which indicated that ZH-EP and ZH-HA were promised in industry.     

Crystal structure of penicillin G acylase from the Bro1 mutant strain of Providencia rettgeri.

Penicillin G acylase is an important enzyme in the commercial production of semisynthetic penicillins used to combat bacterial infections. Mutant strains of Providencia rettgeri were generated from wild-type cultures subjected to nutritional selective pressure. One such mutant, Bro1, was able to use 6-bromohexanamide as its sole nitrogen source. Penicillin acylase from the Bro1 strain exhibited an altered substrate specificity consistent with the ability of the mutant to process 6-bromohexanamide. The X-ray structure determination of this enzyme was undertaken to understand its altered specificity and to help in the design of site-directed mutants with desired specificities. In this paper, the structure of the Bro1 penicillin G acylase has been solved at 2.5 A resolution by molecular replacement. The R-factor after refinement is 0.154 and R-free is 0.165. Of the 758 residues in the Bro1 penicillin acylase heterodimer (alpha-subunit, 205; beta-subunit, 553), all but the eight C-terminal residues of the alpha-subunit have been modeled based on a partial Bro1 sequence and the complete wild-type P. rettgeri sequence. A tightly bound calcium ion coordinated by one residue from the alpha-subunit and five residues from the beta-subunit has been identified. This enzyme belongs to the superfamily of Ntn hydrolases and uses Ogamma of Ser beta1 as the characteristic N-terminal nucleophile. A mutation of the wild-type Met alpha140 to Leu in the Bro1 acylase hydrophobic specificity pocket is evident from the electron density and is consistent with the observed specificity change for Bro1 acylase. The electron density for the N-terminal Gln of the alpha-subunit is best modeled by the cyclized pyroglutamate form. Examination of aligned penicillin acylase and cephalosporin acylase primary sequences, in conjunction with the P. rettgeri and Escherichia coli penicillin acylase crystal structures, suggests several mutations that could potentially allow penicillin acylase to accept charged beta-lactam R-groups and to function as a cephalosporin acylase and thus be used in the manufacture of semi-synthetic cephalosporins.     

Improved activity and pH stability of E. coli ATCC 11105 penicillin acylase by error-prone PCR

Penicillin G acylase is the key enzyme used in the industrial production of β-lactam antibiotics. This enzyme hydrolyzes penicillin G and related β-lactam antibiotics releasing 6-aminopenicillanic acid, which is an intermediate in the production of semisynthetic penicillins. To improve the enzymatic activity of Escherichia coli penicillin acylase, sequential rounds of error-prone polymerase chain reaction were applied to the E. coli pac gene. After the second round of evolution, the best mutant M2234 with enhanced activity was selected and analyzed. DNA sequence analyses of M2234 revealed that one amino acid residue (K297I), located far from the center of the catalytic pocket, was changed. This mutant (M2234) has a specific activity 4.0 times higher than the parent enzyme and also displayed higher stability at pH 10.     

Enhanced production of penicillin G acylase from a recombinant Escherichia coli

Penicillin G acylase (pac) gene was cloned into a stable asd ⁺ vector (pYA292) and expressed in Escherichia coli. This recombinant strain produced 1000 units penicillin G acylase g^–1 cell dry wt, which is 23-fold more than that produced by parental Escherichia coli ATCC11105. This enzyme was purified to 16 units mg^–1 protein by a novel two-step process.     

Biosynthesis of penicillin V acylase by Fusarium sp.: effect of culture conditions

Penicillin V acylase was produced, both intracellularly and extracellularly, by Fusarium sp. SKF 235 grown in submerged fermentation. When neopeptone was added to the medium, >95% of the penicillin V acylase was extracellular. In the absence of a complex organic nitrogen source, the fungus produced low levels of totally intracellular penicillin V acylase. MgSO₄ was essential for synthesis of the enzyme, which was induced by phenoxyacetic acid and penicillin V. The maximum yield of penicillin V acylase was 430 IU/g dry cell wt. The optimum pH value and temperature for the penicillin V acylase were 6.5 and 55°C, respectively.     

Selective transformations of penicillins and cephalosporins with pen G acylase

Summary Immobilised Penicillin G acylase from E. coli hydrolyses penicillin and cephalosporin derivatives protected at the carboxy group as the phenylacetoxymethylene esters. The corresponding hydrolysis of penicillin V retains the phenoxyacetyl moiety. Kinetic data of the hydrolysis are reported.