Structural studies of penicillin acylase

Penicillin acylases are used in the pharmaceutical industry for the preparation of antibiotics. The 3-D structure of Penicillin Gacylase from Escherichia coli has been solved. Here, we present structural data that pertain to the unanswered questions that arose from the original strucutre. Specificity for the amide portion of substrate was probed by the structure determination of a range of complexes with substitutions around the phenylacetyl ring of the ligand. Altered substrate specificity mutations derived from an in vivo positive selection process have also been studied, revealing the structural consequences of mutation at position B71. Protein processing has been analyzed by the construction of site-directed mutants, which affect this reaction with two distinct phenotypes. Mutations that allow processing but yield inactive protein provide the structure of an ES complex with a true substrate, with implications for the enzymatic mechanism and stereospecificity of the reaction. Mutations that preclude processing have allowed the structure of the precursor, which includes the 54a mino acid linker region normally removed from between the A and B chains, to be visualized.     

Purification and stability of glutaryl-7-ACA acylase from pseudomonas sp.

The enzyme glutaryl-7-ACA acylase fromPseudomonas sp. NCIMB 40474, produced by a recombinantEscherichia coli host, was purified to homogeneity. The enzyme is a tetramer composed of two couples of asymmetric dimers, each of them constituted of two subunits of mol wt 18 and 52 kDa, respectively. It was found that glutaric acid, one of the products of the substrate hydrolysis, is an effective acylase inhibitor. Between pH 6.0 and pH 10.0, the enzymatic activity is almost constant, but below pH 6.0 it progressively declines. The acylase activity decreased sharply as a function of guanidine HC1 concentration. The loss is significant even at concentrations of denaturant lower than those causing unfolding, as suggested by UV spectroscopy and fluorescence emission studies. In these conditions (low denaturant concentration and low pH) the inactivation of the enzyme is caused by the tetramer dissociation into dimers. The lability of the quaternary structure of the enzyme is a key feature that must be taken into account for the improvement of the catalyst stability.     

Selective adsorption of poly-His tagged glutaryl acylase on tailor-made metal chelate supports.

A poly-His tag was fused in the glutaryl acylase (GA) from Acinetobacter sp. strain YS114 cloned in E. coli yielding a fully active enzyme. Biochemical analyses showed that the tag did not alter the maturation of the chimeric GA (poly-His GA) that undergoes a complex post-translational processing from an inactive monomeric precursor to the active heterodimeric enzyme. This enzyme has been used as a model to develop a novel and very simple procedure for one-step purification of poly-His proteins via immobilized metal-ion affinity chromatography on tailor-made supports. It was intended to improve the selectivity of adsorption of the target protein on tailor-made chelate supports instead of performing a selective desorption. The rate and extent of the adsorption of proteins from a crude extract from E. coli and of pure poly-His tagged GA on different metal chelate supports was studied. Up to 90% of proteins from E. coli were adsorbed on commercial chelate supports having a high density of ligands attached to the support through long spacer arms, while this adsorption becomes almost negligible when using low ligand densities, short spacer arms and Zn2+ or Co2+ as cations. On the contrary, poly-His GA adsorbs strongly enough on all supports. A strong affinity interaction between the poly-His tail and a single chelate moiety seems to be the responsible for the adsorption of poly-His GA. By contrast, multipoint weak interactions involving a number of chelate moieties seem to be mainly responsible for adsorption of natural proteins. By using tailor-made affinity supports, a very simple procedure for one-step purification of GA with minimal adsorption of host proteins could be performed. Up to 20 mg of GA were adsorbed on each ml of chelate support while most of accompanying proteins were hardly adsorbed on such supports. Following few washing steps, the target enzyme was finally recovered (80% yield) by elution with 50 mM imidazole with a very high increment of specific activity (up to a 120 purification factor).     

Immobilization-stabilization of Penicillin G acylase fromEscherichia coli

We have developed a strategy for immobilization-stabilization of penicillin G acylase from E. coli, PGA, by multipoint covalent attachment to agarose (aldehyde) gels. We hve studied the role of three main variables that control the intensity of these enzyme-support multiinteraction processes: 1. surface density of aldehyde groups in the activated support; 2. temperature; and 3. contact-time between the immobilized enzyme and the activated support prior to borohydride reduction of the derivatives. Different combinations of these three variables have been tested to prepare a number of PGA-agarose derivatives. All these derivatives preserve 100% of catalytic activity corresponding to the soluble enzyme that has been immobilized but they show very different stability. The less stable derivative has exactly the same thermal stability of soluble penicillin G acylase and the most stable one is approximately 1,400 fold more stable. A similar increase in the stability of the enzyme against the deleterious effect of organic solvents was also observed. On the other hand, the agarose aldehyde gels present a very great capacity to immobilize enzymes through multipoint covalent attachment. In this way, we have been able to prepare very active and very stable PGA derivatives containing up to 200 International Units of catalytic activity per mL. of derivative with 100% yields in the overall immobilization procedure.     

Supermacroporous hydrophobic affinity sorbents for penicillin acylase purification

Penicillin acylase (PA, EC 3.5.1.11) is used as a raw material in the production of semi-synthetic penicillins. Although there are many methods for PA purification, affinity chromatography is advantageous as it provides efficient one step purification. In this study, poly(2-hydroxyethyl methacrylate) based cryogel column containing hydrophobic N-methacryloyl-L-tryptophan (MATrp) functional monomer as a ligand was prepared. Interaction of MATrp with amino acids in PA structure is the basis of hydrophobic interaction chromatography in this study. PHEMA and PHEMATrp cryogel columns were characterized by surface area measurements, infrared spectroscopy, swelling tests, elemental analysis and scanning electron microscopy (SEM). Initial PA concentration, pH, effect of temperature, amount of ligand, flow rate, ionic strength and time on PA adsorption on PHEMATrp cryogel were investigated. Optimum pH was determined as 5.0 for PA adsorption and maximum adsorption capacity was obtained as 6.40 mg/g. It was observed that adsorption capacity increased with the increasing of temperature. Also, PA adsorption increased up to 0.25 M salt concentration and decreased in higher salt concentrations. Data obtained in this affinity system suggests that hydrophobic interactions are dominant. In the last stage of the study, PA was purified from Penicillium chrysogenum with 76.3% yield and 332.3 purification factor.     

Radiation-induced polymerization for the immobilization of penicillin acylase

The immobilization ofEscherichia coli penicillin acylase (EC 3.5.1.11) was investigated by radiation-induced polymerization of 2-hydroxyethyl methacrylate at low temperature. A leak-proof composite that does not swell in water was obtained by adding the crosslinking agent trimethylolpropane trimethacrylate to the monomer-aqueous enzyme mixture. Penicillin acylase, which was immobilized with greater than 70% yield, possessed a higherK _m value toward the substrate 6-nitro-3-phenylacetamidobenzoic acid than the free enzyme form (K _m = 1.7 × 10⁻⁵ and 1 × 10⁻⁵M, respectively). The structural stability of immobilized penicillin acylase, as assessed by heat, guanidinium chloride, and pH denaturation profiles, was very similar to that of the free-enzyme form, thus suggesting that penicillin acylase was entrapped in its native state into aqueous free spaces of the polymer matrix.     

Novel approaches to the purification of penicillin acylase

Penicillin acylase ofE. coli NCIM 2400 has been purified to homogeneity using a combination of hydrophobic interaction chromatography and DEAE-cellulose treatment. A variety of substituted matrices were synthesized using D- or DL-phenylglycine, norleucine, ampicillin, or amoxycillin as ligands, all of which retained penicillin acylase at high concentrations of ammonium sulfate or sodium sulfate. The enzyme could be eluted nonbiospecifically by buffer of lower ionic strength with over 95% recovery of the activity. Ammonium chloride, ammonium nitrate, sodium chloride, sodium nitrate, and potassium chloride were ineffective in either adsorption or elution of the enzyme on these columns. Further purification of this partially pure enzyme with DEAE-cellulose at pH 7.0–7.2 yielded an enzyme preparation of very high purity according to electrophoretic and ultracentrifugal analyses, its specific activity being as high as 37 U/mg protein. The purifiedf enzyme has a molecular weight of 67,000 a sedimentation coefficient of 4.0S, and resolves into two forms upon isoelectric focusing. Overall recoveries ranged between 75 and 85%. Ease of operation, high recoveries, high purity of the enzyme and prolonged reuse of the conjugates make the process economically feasible and possibly of great commercial importance.     

Immobilization of modified penicillin G acylase on Sepabeads carriers

An approach to stable covalent immobilization of chemically modified penicillin G acylase from Escherichia coli on Sepabeads® carriers with high retention of hydrolytic activity and thermal stability is presented. The two amino-activated polymethacrylate particulate polymers with different spacer lengths used in the study were Sepabeads® EC EA and Sepabeads® EC HA. The enzyme was first modified by cross-linking with polyaldehyde derivatives of starch in order to provide it with new useful functions. Such modified enzyme was then covalently immobilized on amino supports. The method seems to provide a possibility to couple the enzyme without risking a reaction at the active site which might cause the loss of activity. Performances of these immobilized biocatalysts were compared with those obtained by the conventional method with respect to activity and thermal stability. The thermal stability study shows that starch-PGA immobilized on Sepabeads EC-EA was almost 4.5-fold more stable than the conventionally immobilized one and 7-fold more stable than free non-modified PGA. Similarly, starch-PGA immobilized on Sepabeads EC-HA was around 1.5- fold more stable than the conventionally immobilized one and almost 9.5-fold more stable than free non-modified enzyme.     

Use of enantio-, chemo- and regioselectivity of acylase I. Resolution of polycarboxylic acid esters

Acylase I was used to catalyze the enantioselective butanolysis of trimethyl 2-[(carboxymethyl)oxy]succinate (E=30) and N-carboxymethylaspartate (E=9) exclusively at the most sterically hindered of the three ester groups (the position α to the asymmetric centre). Gram-scale resolution allowed the preparation of the less reactive trimethyl (S)-2-[(carboxymethyl)oxy]succinate (96% e.e.), that of the (R)-butyldimethyl regioisomer (78% e.e.) at 55% conversion and finally the preparation of the corresponding trisodium carboxylate by saponification. Acylase I was shown to transform (±)-methyl N-acetylmethionine and (±)-valine to the corresponding (S)-amino acids through ester hydrolysis-N-acetyl transfer sequence with absolute chemo- and enantioselectivity. Butanolysis of methyl N-acetylmethionine stopped in the formation of the butyl ester (E=12), the valine derivative being totally unreactive.     

Application of monolayer engineering for immobilization of penicillin G acylase

Present communication is concerned with the application of monolayer engineering, in particular of ‘protective plate’ technique, for the fabrication of alternate-layer assemblies based on enzyme penicillin G acylase. Several structures are compared with each other. The deposited films are tested to determine the values of enzymatic activity and the level of protein detachment in aqueous solutions. As the result, the deposition procedure is found, which enables to obtain biocatalytic media with enhanced performances. The biocatalyst efficiency is proved by three independent techniques including direct yield determination with HPLC. The advantage of applied method of enzyme immobilization with respect to other techniques is demonstrated.     

Isolation and characterization of an L-amino acid acylase fromAspergillus oryzae

A scheme of isolating a highly purified L-amino acylase fromAspergillus oryzae is described which excludes extraction of the enzyme from the preparation “Amilorizin,” fractionation with ethanol, chromatography on DEAE-cellulose, and gel filtration through Sephadex G-200 and Bio-Gel P-300. The enzyme, as purified 1240-fold, has a molecular weight of 118,000, apparently consists of two subunits with a molecular weight of 60,000, is stable in the pH range of 7–10 and has an optimum pH of 8.9 and a pI of 4.0. Its amino acid composition has been determined and its substrate specificity has been studied. The acylase is a metalloenzyme: Co^2+` ions in concentrations of 10^?4–5·10^?5 M increase the rate of hydrolysis of N-acetyl-L-amino acids three- to fourfold. It shows differences in its molecular and functional properties from acylase I obtained from porcine kidney.     

Immobilization of penicillin acylase in porous beads of polyacrylamide gel

A procedure is described for the immobilization of benzylpenicillin acylase from Escherichia coli within uniformly spherical, porous polyacrylamide gel beads. Aqueous solutions of the enzyme and sodium alginate and of acrylamide monomer, N,N'-methylene-bis-acrylamide, N,N,N,N'-tetramethylethylenediamine (TEMED) and sodium alginate are cooled separately, mixed, and dropped immediately into ice-cold, buffered calcium formate solution, pH 8.5, to give calcium alginate-coated beads. The beads are left for 30-60 min in the cold calcium formate solution for polyacrylamide gel formation. The beads are then treated with a solution of glutaraldehyde and the calcium alginate subsequently leached out with a solution of potassium phosphate. Modification of the native enzyme with glutaraldehyde results in a slight enhancement in the rate of hydrolysis of benzylpenicillin at pH 7.8 and 0.05M substrate concentration. The enzyme entrapped in porous polyacrylamide gel beads shows no measurable diffusional limitation in stirred reactors, catalyzing the hydrolysis of the substrate at a rate comparable to that of the glutaraldehyde-modified native enzyme. The immobilized enzyme preparation has been used in batch mode over 90 cycles without any apparent loss in hydrolytic activity.     

Single-step purification and immobilization of penicillin acylase using hydrophobic ligands

Five differenthydrophobic ligands immobilized on 4% (4XL) and 6% (6XL) crosslinked agarose were used to study the single-step purification of penicillin acylase from cell lysate. The 4XL gels showed relatively higher specific activity and recovery than the 6XL gels. In single-step purification, highly active enzyme (42 U/mg) was obtained using moderately hydrophobic ligand (octyl). The crude enzyme immobilized on octyl gel by adsorption showed significant operational stability over a period of 30 d at room temperature. Reactor studies demonstrated the feasibility of hydrophobic ligands as a medium for immobilization.     

Penicillin G acylase catalyzed Markovnikov addition of allopurinol to vinyl ester

A new enzymatic process is reported, in which penicillin G acylase from Escherichia coli displays a promiscuous activity in catalyzing the Markovnikov addition of allopurinol to vinyl ester.