Penicillin amidase

About: Penicillin amidase is a research topic. Over the lifetime, 576 publications have been published within this topic receiving 15563 citations. The topic is also known as: penicillin amidohydrolase & ampicillin acylase.

Strategies in the design of an enzymatic process for the synthesis of ampicillin: A whole cell E. coli recombinant penicillin amidase biocatalyst

Abstract: A recombinant immobilized E. coli cell biocatalyst was used to design an enzymatic process for the synthesis of ampicillin. Reduction of water activity did not increase conversion yield. Inhibition of PGME hydrolysis by methanol and pH control allowed synthesis reactions with 75% yields.

Penicillin amidase and penicillinase production in nitrogen- and sulfur-limited chemostats

Abstract: The production of penicillin amidase fromBacillus megaterium ATCC 14945 was examined in a sulfur-limited chemostat. Below 50% of the maximum growth rate, enzyme production was independent of dilution rate; above this point production decreased as growth rate increased. A simple and defined medium forBacillus licheniformis 749/C was developed, and extracellular penicillinase production was studied in a chemostat under sulfur- and nitrogenlimited conditions. While the yield of this enzyme differed little between these growth-limiting conditions, the specific activity of the penicillinase per unit weight of extracellular protein was increased 50% under nitrogenlimited conditions. Furthermore, the proportion of extracellular enzyme was greater in continuous, than in batch culture.

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.

Purification of Penicillin Amidohydrolase, an Enzyme for Semisynthetic Procedures

Abstract: Penicillin amidohydrolase (EC 35111) is one of the few enzymes used successfully for deprotection of primary amino groups of semisynthetic peptides The available material is usually contamined by endo- and exopeptidases We managed to prepare the enzyme devoid of trypsin- and chymotrypsin-like activities using affinity chromatography with specific ligands: Gly-D-Phe-Phe-Tyr-Thr-Pro-Lys-Thr (the fF peptide) and Leu-Gly-Val-D-Arg-Arg-Gly-Phe (the rR peptide) For further purification of the enzyme affinity chromatography with N-phenylacetyl-D-tert-Leu as a ligand was used