Showing papers on "Penicillin amidase published in 2004"

A new, mild cross-linking methodology to prepare cross-linked enzyme aggregates.

Abstract: Cross-linked enzyme aggregates (CLEAs) were prepared from several enzymes (penicillin G acylase, hydroxynitrile lyase, alcohol dehydrogenase, and two different nitrilases) by precipitation and subsequent cross-linking using dextran polyaldehyde. In most cases, higher immobilization yields were obtained using the latter cross-linker as compared with the commonly used glutaraldehyde. Active site titration of penicillin acylase CLEAs showed that the higher activity originated from a significantly lower loss in active sites using dextran polyaldehyde as a cross-linking agent. It is proposed that macromolecular cross-linkers are too large to penetrate the protein active site and react with catalytically essential amino acid residues.

Construction, characterization, and use of small-insert gene banks of DNA isolated from soil and enrichment cultures for the recovery of novel amidases.

Abstract: Summary To obtain new amidases of biocatalytic relevance, we used microorganisms indigenous to different types of soil and sediment as a source of DNA for the construction of environmental gene banks, following two different strategies. In one case, DNA was isolated from soil without preceding cultivation to preserve a high degree of (phylo)genetic diversity. Alternatively, DNA samples were obtained from enrichment cultures, which is thought to reduce the number of clones required to find a target enzyme. To selectively sustain the growth of organisms exhibiting amidase activity, cultures were supplied with a single amide or a mixture of different aromatic and non-aromatic acetamide and glycine amide derivatives as the only nitrogen source. Metagenomic DNA was cloned into a high-copy plasmid vector and transferred to E. coli , and the resulting gene banks were searched for positives by growth selection. In this way, we isolated a number of recombinant E. coli strains with a stable phenotype, each expressing an amidase with a distinct substrate profile. One of these clones was found to produce a new and highly active penicillin amidase, a promising biocatalyst that may allow higher yields in the enzymatic synthesis of b -lactam antibiotics.

Stabilization of Penicillin G Acylase from Escherichia coli: Site-Directed Mutagenesis of the Protein Surface To Increase Multipoint Covalent Attachment

Abstract: Three mutations on the penicillin acylase surface (increasing the number of Lys in a defined area) were performed. They did not alter the enzyme's stability and kinetic properties; however, after immobilization on glyoxyl-agarose, the mutant enzyme showed improved stability under all tested conditions (e.g., pH 2.5 at 4 degrees C, pH 5 at 60 degrees C, pH 7 at 55 degrees C, or 60% dimethylformamide), with stabilization factors ranging from 4 to 11 compared with the native enzyme immobilized on glyoxyl-agarose.

A novel support of MCM-48 molecular sieve for immobilization of penicillin G acylase

Abstract: As a novel support of immobilizing penicillin G acylase (PGA), MCM-48 and Co-MCM-48 molecular sieves were synthesized and characterized by XRD, N2 adsorption, NH3-TPD, FT-IR and so on. The studies show that MCM-48 and Co-MCM-48 has well ordered long-range structure, narrow pore size distribution, larger surface area and higher concentration of the weakly acidic silanol groups on their surface. Penicillin G acylase was immobilized on MCM-48 or Co-MCM-48 by interacting silanol groups on the surface. The presence of cobalt in the framework of MCM-48 increases the amount of the weak acid sites. For the hydrolysis of penicillin G catalyzed by PGA/Co-MCM-48 (Co/Si=0.01), its specific activity reaches 1682 U/g. After used for six cycles, PGA/MCM-48(0.01) can keep 1375 U/g of the specific activity. If MCM-41 was used as the support, the activity of immobilized PGA is only 402 U/g.

Combinatorial formulation of biocatalyst preparations for increased activity in organic solvents: Salt activation of penicillin amidase

Abstract: A combinatorial experimental technique was used to identify salts and salt mixtures capable of activating penicillin amidase in organic solvents for the transesterification of phenoxyacetate methyl ester with 1-propanol. Penicillin amidase was lyophilized in the presence of various chloride and acetate salts within 96-deep-well plates and catalytic rates measured to determine lead candidates for highly salt-activated preparations. The kinetics of the most active formulations were then further evaluated. These studies revealed that a formulation consisting of 98% (w/w) of a 1:1 KAc:CsCl salt mixture, 1% (w/w) enzyme, and 1% (w/w) potassium phosphate buffer was approximately 35,000-fold more active than the salt-free formulation in hexane, as reflected in values of V(max)/K(m). This extraordinary activation could be extended to more polar solvents, including tert-amyl alcohol, and to formulations with lower total salt contents. A correlation was found between the kosmotropic/chaotropic behavior of the salts (as measured by the Jones-Dole B coefficients) and the observed activation. Strongly chaotropic cations combined with strongly kosmotropic anions yielded the greatest activation, and this is likely due to the influence of the ions on protein-water and protein-salt interactions.

Penicillin acylase-catalyzed synthesis of beta-lactam antibiotics in highly condensed aqueous systems: beneficial impact of kinetic substrate supersaturation.

Abstract: Advantages of performing penicillin acylase-catalyzed synthesis of new penicillins and cephalosporins by enzymatic acyl transfer to the beta-lactam antibiotic nuclei in the supersaturated solutions of substrates have been demonstrated. It has been shown that the effective nucleophile reactivity of 6-aminopenicillanic (6-APA) and 7-aminodesacetoxycephalosporanic (7-ADCA) acids in their supersaturated solutions continue to grow proportionally to the nucleophile concentration. As a result, synthesis/hydrolysis ratio in the enzymatic synthesis can be significantly (up to three times) increased due to the nucleophile supersaturation. In the antibiotic nuclei conversion to the target antibiotic the remarkable improvement (up to 14%) has been gained. Methods of obtaining relatively stable supersaturated solutions of 6-APA, 7-ADCA, and D-p-hydroxyphenylglycine amide (D-HPGA) have been developed and syntheses of ampicillin, amoxicillin, and cephalexin starting from the supersaturated homogeneous solutions of substrates were performed. Higher synthetic efficiency and increased productivity of these reactions compared to the heterogeneous "aqueous solution-precipitate" systems were observed. The suggested approach seems to be an effective solution for the aqueous synthesis of the most widely requested beta-lactam antibiotics (i.e., amoxicillin, cephalexin, cephadroxil, cephaclor, etc.).

Optimization of cephalexin synthesis with immobilized penicillin acylase in ethylene glycol medium at low temperatures

Abstract: Organic cosolvents, and among them, polyols, are suitable media to perform the enzymatic synthesis of β-lactam antibiotics with immobilized penicillin acylase, because they effectively reduce water activity, depressing hydrolytic reactions in favor of synthesis. Among polyols, ethylene glycol has proven to be particularly suited as reaction medium for their synthesis. Previous studies have shown that pH, temperature, and cosolvent concentration are the most relevant variables in the kinetically controlled synthesis of cephalexin from 7-amino-3-deacetoxy cephalosporanic acid and phenylglycine methyl ester, conversion yield increasing at low temperatures and high cosolvent concentrations. The objective of this work is the optimization of temperature, pH, and ethylene glycol concentration in the kinetically controlled synthesis of cephalexin with immobilized penicillin acylase at lower than ambient temperature in terms of substrate molar conversion yield. Phenylglycine was used as acyl donor and 7-amino-3-deacetoxy cephalosporanic acid was the limiting substrate at 30 mM. Optimization was performed using surface of response methodology, optimum conditions being 12 °C, pH 6.8, and 60% (v/v) ethylene glycol, at which cephalexin yield was close to stoichiometric with respect to the limiting nucleophile, which is unattainable in aqueous medium. Stability of the biocatalyst at optimum conditions for cephalexin synthesis was very high, with a projected half-life of 1500 h, making it a suitable catalyst for the large-scale production of cephalexin.

Activity and stability of immobilized penicillin amidase at low pH values

Abstract: Penicillin amidase is being applied widely in the production of semi-synthetic β-lactam antibiotics. Usually the processes are at pH 7–8, but for many new applications the range of pH 3–6 is of interest too. Therefore, we studied the activity of penicillin amidase at 25 °C in potassium phosphate buffer of pH 3.7–9, as well as its stability in potassium phosphate buffer of pH 3–6. At each pH, the enzyme was stable during at least 32 days. On the other hand, immobilized penicillin amidase incubated in butyl acetate lost its stability, showing after 32 days a decrease of 52% in relation to its initial enzymatic activity value. In phosphate buffer, the enzyme showed the highest activity at pH 8–9. A gradual decrease to about 20% of this activity occurred when the pH was decreased to 3.7. At even lower pH, the enzyme activity could not be determined with the assay that was used due to a very low stability of penicillin G (PenG). The course of penicillin G conversion and 6-aminopenicillanic acid (APA) production, during enzymatic hydrolysis at pH 4, could be quantitatively described by a simple model when the thermodynamic equilibrium of the hydrolysis was taken into account.

Evaluation of magnetic polymer micro-beads as carriers of immobilised biocatalysts for selective and stereoselective transformations

Abstract: The kinetic, selective and stereoselective properties of enzyme immobilised on magnetic polymer beads with diameters in the range 1 microm was studied with penicillin amidase from E. coli. The enzyme was immobilised on epoxy and glutaraldehyde-activated poly(vinyl alcohol), poly(methylmetacrylate) and poly(vinyl acetate-divinylbenzene) magnetic beads. The amount of covalently bound active protein was dependent on the chemical modification of the matrix and increased at higher ionic strength of the immobilisation buffer. The small size of the magnetic beads, that reduces mass transfer limitations, and the decreased charge density in the electric double layer resulted in lower apparent Km values and higher efficiency for benzylpenicillin hydrolysis, higher stereoselectivity in condensation of R-phenylglycine amide with S- and R-Phe and in hydrolysis of racemic phenylacetyl-Phe and higher selectivity in kinetically controlled synthesis of cephalexin compared to the enzyme immobilised on larger and porous carriers.

Production of 6-aminopenicillanic acid in aqueous two-phase systems by recombinant Escherichia coli with intracellular penicillin acylase

Abstract: Bioconversion of penicillin G in PEG 20000/dextran T 70 aqueous two-phase systems was achieved using the recombinant Escherichia coli A56 (ppA22) with an intracellular penicillin acylase as catalyst. The best conversion conditions were attained for: 7% (w/v) substrate (penicillin G), enzyme activity in bottom phase 52 U ml−1, pH 7.8, temperature 37 °C, reaction time 40 min. Five repeated batches could be performed in these conditions. Conversions ratios between 0.9–0.99 mol of 6-aminopenicillanic acid (6-APA) per mol of penicillin G, were obtained and volumetric productivity was 3.6–4.6 μmol min−1 ml−1. In addition the product 6-APA could be directly crystallized from the top phase with a purity of 96%.

Production of a fully functional, permuted single-chain penicillin G acylase

Abstract: Penicillin G acylase (PGA) is a heterodimeric enzyme synthesized as a single-polypeptide precursor that undergoes an autocatalytic processing to remove an internal spacer peptide to produce the active enzyme. We constructed a single-chain PGA not dependent on autoproteolytic processing. The mature sequence of the β-domain was expressed as the N terminus of a new polypeptide, connected by a random tetra-peptide to the α-domain, to afford a permuted protein. We found several active enzymes among variants differing in their linker peptides. Protein expression analysis showed that the functional single-chain variants were produced when using a Sec-dependent leader peptide, or when expressed inside the bacterial cytoplasm. Active-site titration experiments showed that the single-chain proteins displayed similar kcat values to the ones obtained with the wild-type enzyme. Interestingly, the single-chain proteins also displayed close to 100% of functional active sites compared to 40% to 70% functional yield usually obtained with the heterodimeric protein.

Total enzymatic synthesis of cholecystokinin CCK-5

Abstract: This paper describes the enzymatic synthesis of the C-terminal fragment H-Gly-Trp-Met-Asp-Phe-NH2 of cholecystokinin. Immobilized enzymes were used for the formation of all peptide bonds except thermolysin. Beginning the synthesis with phenylacetyl (PhAc) glycine carboxamidomethyl ester (OCam) and H-Trp-OMe by using immobilized papain as biocatalyst in buffered ethyl acetate, the dipeptide methyl ester was then coupled directly with Met-OEt.HCl by alpha-chymotrypsin/Celite 545 in a solvent free system. For the 3+2 coupling PhAc-Gly-Trp-Met-OEt had to be converted into its OCam ester. The other fragment H-Asp(OMe)-Phe-NH2 resulted from the coupling of Cbo-Asp(OMe)-OH with H-Phe-NH2.HCl and thermolysin as catalyst, followed by catalytic hydrogenation. Finally PhAc-Gly-Trp-Met-Asp-Phe-NH2 was obtained in a smooth reaction from PhAc-Gly-Trp-Met-OCam and H-Asp(OMe)-Phe-NH2 with alpha-chymotrypsin/Celite 545 in acetonitrile, followed by basic hydrolysis of the beta-methyl ester. The PhAc-group is removed with penicillin G amidase and CCK-5 is obtained in an overall isolated yield of 19.6%.

Enantioselective synthesis of phenylacetamides in the presence of high organic cosolvent concentrations catalyzed by stabilized penicillin G acylase. Effect of the acyl donor.

Abstract: Immobilization of penicillin G acylase on glyoxyl agarose and its further hydrophilization by physicochemical modification with ionic polymers has made it possible to perform the direct condensation between (+/-)-2-hydroxy-2-phenylethylamine [(+/-)-1] and different acyl donors in the presence of high concentrations of organic cosolvent (up to 90%) in the reaction medium. Using 50 mM phenyl acetic acid and these drastic reaction conditions, too harsh for any other PGA preparation, we have achieved an almost quantitative transformation (more than 99%) of 10 mM (+/-)-1 into the corresponding amide. Remarkably, the enantioselectivity of the enzyme immobilized on the amine was strongly dependent on the acyl donor employed. Thus, using phenylacetic acid (2), the enantioselectivity was almost negligible (1.3 favoring the S isomer), whereas using S-mandelic acid [(S)-4], the E factor reached a value of 21 (also favoring the S isomer). By using R-mandelic acid [(R)-4], we observed a different enantioselectivity (E was 3.6 favoring the R). At 4 degrees C, the E value reached a value higher than 100 when (S)-4 was used as the acyl donor. The reaction performed under these conditions allowed us to produce (2S,2'S)-N-2'-hydroxy-2'-phenyl)-2-hydroxyphenylacetamide [(2S,2'S)-7] with a diasteromeric excess higher than 98%.

One-step purification of poly-his tagged penicillin G acylase expressed in E. coli

Abstract: The inexpensive large-scale production of pure PGA (Penicillin G Acylase) has been a commercial goal. PGA has been used as a model enzyme in the development of simple one-step purification methods. In this study, the purification of poly-His tagged PGA protein secreted into the periplasmic space was carried out by using immobilized metal-ion affinity chromatography (IMAC). The PGA gene was obtained from E. coli ATCC 11105. Codons encoding histidines were fused at the C-terminus of the PGA gene by PCR. E. coli JM109 harboring pPGA-HIS6 vector produced active his-tagged acylases in the presence of lac promoter during cultivation at 26C. The maximum specific activity of the acylase purified by using one-step chromatography after osmotic shock was 38.5 U/mg and was recovered with the yield of 70%. Both 23 kDa (α) and 62 kDa (β) subunits were recovered by using IMAC with just C-terminus tagging of the β subunit. The purification of the periplasmic fraction by osmotic shock and that of purified acylase was increased by 2.6-fold and 19-fold, respectively, compared to the crude extract.

Purification, substrate specificity, and N-terminal amino acid sequence analysis of a β-lactamase-free penicillin amidase from Alcaligenes sp.

Abstract: A β-lactamase-free penicillin amidase from Alcaligenes sp. active against various β-lactams was purified to homogeneity. The enzyme can hydrolyze penicillin G to 6-amino penicillanic acid (6-APA) and furnish penicillin G from 6-APA and phenyl acetic acid by condensation. The penicillin amidase is a heterodimer of subunit masses of 63 kDa and 22 kDa, respectively. Its isoelectric point is at pH 8.5. Cephalothin was found to be the best substrate. This is a novel type II penicillin amidase which shares the properties of both type II and type III enzymes. It is thermostable and, unlike penicillin amidase from A. faecalis, its stability remains unperturbed even in presence of reductant. An inhibition study by 2-hydroxy-5-nitro benzylbromide indicated the involvement of tryptophan in catalysis by the enzyme.

Evidence for the involvement of arginyl residue at the active site of penicillin G acylase from Kluyvera citrophila.

Abstract: Penicillin G acylase (PGA) is used for the commercial production of semi-synthetic penicillins. It hydrolyses the amide bond in penicillin producing 6-aminopenicillanic acid and phenylacetate. 6-Aminopenicillanic acid, having the β-lactam nucleus, is the parent compound for all semi-synthetic penicillins. Penicillin G acylase from Kluyvera citrophila was purified and chemically modified to identify the role of arginine in catalysis. Modification with 20 mm phenylglyoxal and 50 mm 2,3-butanedione resulted in 82% and 78% inactivation, respectively. Inactivation was prevented by protection with benzylpenicillin or phenylacetate at 50 mm. The reaction followed psuedo-first order kinetics and the inactivation kinetics (Vmax, Km, and kcat) of native and modified enzyme indicates the essentiality of arginyl residue in catalysis.

Use of high acyl donor concentrations leads to penicillin acylase inactivation in the course of peptide synthesis

Abstract: Enzyme inactivation has been observed in the course of penicillin acylase-catalyzed hydrolysis and aminolysis of d- phenylglycine amide. Inactivation was very sensitive to the d- phenylglycine amide concentration: at pH 9.5, 25 °C and 400 mM substrate, penicillin acylase lost more than 90% of its initial catalytic activity in half an hour, in the presence of 100 mM substrate, 50% of the initial activity in two hours, whereas in the absence of substrate, no significant enzyme inactivation was observed in three hours. Observed enzyme inactivation limits use of high acyl donor concentrations at penicillin acylase-catalyzed peptide synthesis.

Mannan-penicillin G acylase neoglycoproteins and their potential applications in biotechnology

Abstract: Mannan-penicillin G acylase neoglycoproteins were prepared by the conjugation of Saccharomyces cerevisiae mannan with enzyme penicillin G acylase using the reductive amination method. Eight neoglycoproteins preparations were obtained after gel chromatography. The preparations contained from 42 to 67% (w/w) saccharides and their molar masses varied from 283 to over 1000 kDa. Significant biospecific interaction of separated fractions with the lectin concanavalin A was evaluated by the precipitation and sorption method (equilibrium constants) and further characterized using surface plasmon resonance to determine kinetic association and dissociation constants. K-D was determined over the range 10(-7) M. High-molar-mass preparations appeared to be more suitable for preparation of stable and active complexes with concanavalin A for prospective use as a penicillin G acylase biocatalyst in enzyme reactors. The enzyme stability of such complexes was significantly increased compared with the original neoglycoprotein. Lower-molar-mass preparations were more suitable for applications such as biocatalysts in bioanalytical devices. (Less)

Synthesis, modification and characterisation of magnetic micro-matrices for covalent immobilisation of biomolecules. Model investigations with penicillin amidase from E.coli

Abstract: The subject of this work was to develop and characterise magnetic micro-matrices for covalent immobilisation of enzymes. As a model biocatalyst penicillin amidase from E.coli - an enzyme of high industrial importance for the production of semi-synthetic b-lactam antibiotics, was used. Poly(vinylacetate) (PVAc) and poly(methylacrylate) (PMA) magnetic beads of sizes in a range 2-20 µm were synthesised by an oil-in-water suspension polymerisation method. Cross-linked poly(methyl methacrylate) (PMMA) beads of 6 µm diameter were converted into magnetic particles by a swell-deswell manufacturing procedure in a toluene-based magnetic fluid comprising of nano-crystals of magnetite covered by a bi-layer of surfactant. Amino-silanised magnetic particles of 1-2 µm diameter were produced by co-precipitation of Fe(II) and Fe(III) salts at high alkaline pH and covered by amino-silane and finally by poly(glutaraldehyde) layers. Poly(vinylalcohol) beads under the trade name M-PVA, of 1-3 µm diameter, were donated by chemagen Biopolymer Technologie AG and largely studied here. All of the magnetic matrices investigated had sizes in a range of 1-20 µm and showed superparamagnetic behaviour e.g. no magnetic memory effects once they were set out of an external magnetic field. Modification of the magnetic bead surfaces mainly with epoxy-, or amino- and subsequent aldehyde- spacers, was done in order to create sites, suitable for the covalent immobilisation of enzymes. Penicillin amidase from E.coli was covalently immobilised onto magnetic matrices investigated in this work via different spacers/reactions. For M-PVA commercial beads, with increasing of the spacer length an increase in the amount of the covalently bound active protein, of up to 22 mg enzyme per gram dry beads for the functionalised with hexamethylene diamine / glutardialdehyde (20.8 A length) spacer matrix at low (0.2 M) ionic strength of the immobilisation buffer was observed. For comparable immobilisation conditions, the same carrier with shorter (6.0 A length) epoxy-spacer immobilised only 4 mg enzyme per gram dry beads. Comparable results were obtained for subsequently modified amino-silanised magnetic particles with a different number of hexamethylene diamine / glutardialdehyde �spacer units� at the above-mentioned low ionic strength of the immobilisation buffer. For the last matrix an immobilisation maximum of 74 mg enzyme per gram dry beads at four spacer units (64.9 A length) was evaluated. This result was also the maximum enzyme loading obtained with the magnetic matrices studied here for the chosen biocatalyst and immobilisation conditions. For poly(vinylacetate) magnetic beads with increasing spacer length from 6.0 A (epoxy-spacer) to up to 21.0 A (hexamethylene diamine / glutardialdehyde spacer), the amount of the immobilised active penicillin amidase increased from 3 to up to 24 mg·g-1 dry beads. Furthermore, for M-PVA magnetic beads the influence of the immobilisation buffer ionic strength on the achievable enzyme loadings was more pronounced for the matrices modified with shorter epoxy- (epichlorohydrine) and imidazolyl-carbamate (N,N´-carbonyl diimidazole) spacers. For both spacers a significant increase - of almost three folds of the amount of the covalently bound enzyme, with rising ionic strength of up to 1 M, was observed. This can be due to minimising of un-desirable charge-charge interactions between the matrix surface (negative zeta potential at pH 7.5 determined) and the enzyme molecule (negative charge at pH 7.5) during the immobilisation process. It is logical, that the effect of the ionic strength was more significant in the case of the above-mentioned shorter spacers, which means shorter distances and stronger electrostatic repulsions between the matrix and the enzyme. Amino-modified and further glutardialdehyde-functionalised poly(methylacrylate) magnetic beads showed enzyme loading capacities comparable to those obtained with the commercial M-PVA matrices at similar immobilisation conditions. With a prolongation of the immobilisation reaction time from 24 h to 72 h, the amount of bound penicillin amidase increased from 35 to 54 mg per gram dry beads. The apparent kinetic constants Km and kcat of the penicillin amidase from E.coli covalently immobilised onto the studied magnetic matrices were investigated here for the hydrolysis of penicillin G. The evaluated apparent Km constants were several fold higher compared to the one for the free enzyme, but were one or two orders of magnitude lower compared to those for penicillin amidase bound onto larger (>100 µm) porous carriers. The selectivity of the enzyme immobilised onto M-PVA beads was investigated in kinetically controlled synthesis of cephalexin from R-phenylglycine amide and 7-aminodeacetoxycephalosporanic acid at 5 °C and an excess of nucleophile. The observed immobilised enzyme selectivity, calculated as nsyn/nhyd= 8 was comparable to this of the free penicillin amidase from E.coli e.g. nsyn/nhyd= 13. Enantioselectivity of the penicillin amidase from E.coli immobilised onto different magnetic matrices was studied for the equilibrium controlled hydrolysis of racemic phenylacetylphenylalanine and kinetically controlled condensation of R-phenylglycine amide with pure enantiomers or racemic mixture of phenylalanine. All studied enzymes, immobilised onto magnetic micro-particles, showed Ehyd,rac >100 for the investigated hydrolysis reaction.