Showing papers on "Penicillin amidase published in 2008"
TL;DR: The advancements made in PGA biotechnology are described and its simulation for production of β-lactam antibiotics is advocated.
117 citations
TL;DR: The design and synthesis of novel self-immolative spacer systems aiming at the release of phenol-containing compounds and their utility was illustrated by the preparation of original fluorogenic substrates of penicillin amidase whose strong fluorescence is unveiled through enzyme-initiated domino reactions.
61 citations
TL;DR: In this article, a conjugation of penicillin acylase (PA) to poly-N-isopropylacrylamide (polyNIPAM) was studied as a way to prepare a thermosensitive biocatalyst for industrial applications to antibiotic synthesis.
Abstract: Conjugation of penicillin acylase (PA) to poly-N-isopropylacrylamide (polyNIPAM) was studied as a way to prepare a thermosensitive biocatalyst for industrial applications to antibiotic synthesis. Condensation of PA with the copolymer of NIPAM containing active ester groups resulted in higher coupling yields of the enzyme (37%) compared to its chemical modification and copolymerization with the monomer (9% coupling yield) at the same NIPAM:enzyme weight ratio of ca. 35. A 10-fold increase of the enzyme loading on the copolymer resulted in 24% coupling yield and increased by 4-fold the specific PA activity of the conjugate. Two molecular forms of the conjugate were found by gel filtration on Sepharose CL 4B: the lower molecular weight fraction of ca. 106 and, presumably, cross-linked protein-polymer aggregates of MW > 107. Michaelis constant for 5-nitro-3-phenylacetamidobenzoic acid hydrolysis by the PA conjugate (20 M) was found to be slightly higher than that of the free enzyme (12 M), and evaluation of Vmax testifies to the high catalytic efficiency of the conjugated enzyme. PolyNIPAM-cross-linked PA retained its capacity to synthesize cephalexin from D-phenylglycin amide and 7-aminodeacetoxycephalosporanic acid. The synthesis-hydrolysis ratios of free and polyNIPAM-cross-linked enzyme in cephalexin synthesis were 7.46 and 7.49, respectively. Thus, diffusional limitation, which is a problem in the industrial production of -lactam antibiotics, can be successfully eliminated by cross-linking penicillin acylase to a smart polymer (i.e., polyNIPAM). (Less)
51 citations
TL;DR: The present support could be a good candidate for the immobilization of industrial enzymes rich in amino groups, especially the thermophilic ones.
Abstract: κ-Carrageenan hydrogel crosslinked with protonated polyethyleneimine (PEI+) and glutaraldehyde (GA) was prepared and evaluated as a novel biocatalytic support for covalent immobilization of penicillin G acylase (PGA). The method of modification of the carrageenan biopolymer is clearly illustrated using a schematic diagram and was verified by FTIR, elemental analysis, DSC, and INSTRON using the compression mode. Results showed that the gels' mechanical strength was greatly enhanced from 3.9 kg/cm2 to 16.8 kg/cm2 with an outstanding improvement in the gels thermal stability. It was proven that, the control gels were completely dissolved at 35°C, whereas the modified gels remained intact at 90°C. The DSC thermogram revealed a shift in the endothermic band of water from 62 to 93°C showing more gel-crosslinking. FTIR revealed the presence of the new functionality, aldehydic carbonyl group, at 1710 cm−1 for covalent PGA immobilization. PGA was successfully immobilized as a model industrial enzyme retaining 71% of its activity. The enzyme loading increased from 2.2 U/g (control gel) to 10 U/g using the covalent technique. The operational stability showed no loss of activity after 20 cycles. The present support could be a good candidate for the immobilization of industrial enzymes rich in amino groups, especially the thermophilic ones. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008
37 citations
TL;DR: The results showed that the relative activity of the immobilized PA was increased to 145% of that of free enzyme and the stability of PA was ameliorated greatly after immobilization, and the proposed covalent immobilizing technique would rank among the potential strategies for efficient immobilization of PA.
Abstract: To improve the performance of covalently immobilized penicillin acylase (PA), the immobilization was carried out in mesocellular silica foams (MCFs) using p-benzoquinone as cross linker. The characterizations of the immobilized enzyme were studied carefully. The results showed that the relative activity of the immobilized PA was increased to 145% of that of free enzyme. The activity was 3.7 folds of that of PA on the silica nanoparticles. The enzyme in MCFs presented a turnover equal to that of free enzyme. It was also found that the optimum pH of the immobilized PA shifted to pH 7.5 and the optimum reaction temperature rose from 45 to 50 °C. Furthermore, the stability of PA was ameliorated greatly after immobilization. Fourier transform infrared spectroscopy showed no major secondary structural change for PA confined in MCFs. The proposed covalent immobilizing technique would rank among the potential strategies for efficient immobilization of PA.
36 citations
TL;DR: Analysis of the crystal structure of PA has shown that residues alphaR145 and alphaF146 undergo extensive repositioning upon binding of large ligands to the active site, suggesting that these residues may be good targets for mutagenesis aimed at improving the catalytic performance of PA.
31 citations
TL;DR: Microwave irradiation improved the adsorption and immobilization of enzymes in mesocellular siliceous foams and immobilized papain and penicillin acylase in MCFs.
24 citations
TL;DR: Intra-particle diffusion limitation was investigated using biocatalysts prepared with different enzyme loads and agarose with different mean pore diameters, and it was showed that when more than 90 IU of enzyme activity were used per mL of support, the system was influenced by intra-particles mass transfer.
Abstract: Mass transfer effects were investigated for the synthesis of ampicillin and amoxicillin, at pH 6.5 and 25 °C, catalyzed by penicillin G acylase immobilized on agarose. The influence of external mass transfer was analysed using different stirring rates, ranging form 200 to 800 rpm. Above 400 rpm, the film resistance may be neglected. Intra-particle diffusion limitation was investigated using biocatalysts prepared with different enzyme loads and agarose with different mean pore diameters. When agarose with 6, 8 and 10% of crosslinking were used, for the same enzyme load, substrates and products concentration profiles presented no expressive differences, suggesting pore diameter is not important parameter. An increase on enzyme load showed that when more than 90 IU of enzyme activity were used per mL of support, the system was influenced by intra-particle mass transfer. A reactive-diffusive model was used to estimate effective diffusivities of substrates and products.
20 citations
TL;DR: A dodecane/thermosensitive polymer/water three-liquid-phase system was introduced for enzymatic hydrolysis of penicillin G (Pen G) for 6-aminopenicillanic acid (6-APA) to achieve reaction equilibrium towards the product’s side with 6- APA productivity of 80% at 42°C, pH 5.5.
Abstract: A dodecane/thermosensitive polymer/water three-liquid-phase system was introduced for enzymatic hydrolysis of penicillin G (Pen G) for 6-aminopenicillanic acid (6-APA). The enzyme was covalently attached to the terminal of PEO-PPO-PEO polymer (L63), which would be transferred into a polymer coacervate phase at high temperature above its "cloud point". 6-APA was primarily resided in the aqueous phase due to its zwitterionic nature. More than 70% phenylacetic acid (PAA) was transferred into the organic phase using trioctylmethylammonium hydroxide and trihexyl-(tetradecyl)phosphonium bis 2,4,4-trimethylpentylphosphinate ionic liquids (Cyphos IL-104) mixture at pH 5.5, while most of Pen G resided in water. As a result, high operational pH was permitted in three-liquid-phase system, which leads to higher enzymatic activity (120 IU at 40 degrees C) and stability (enzymatic half-time up to 55 h at 60 degrees C) in comparison with the value in butyl acetate/water two-phase system. On the other hand, two products in three-liquid-phase system might be automatically separated from the enzyme sphere into different phases at the same time, which facilitated the reaction equilibrium towards the product's side with 6-APA productivity of 80% at 42 degrees C, pH 5.5.
16 citations
TL;DR: Different carrier-free and carrier-bound penicillin acylases were evaluated in the thermodynamically controlled enzymatic synthesis in organic medium of deacetoxycephalosporin G using phenylacetic acid and 7-amino-deacetoxycefalosporanic acid used as a model reaction system.
14 citations
TL;DR: In this article, two feed-forward neural networks were trained and used to forecast the rates of amoxicillin and p-hydroxyphenylglycine (POHPG) net production.
Abstract: This study focuses in the mathematical modelling of the enzymic synthesis of amoxicillin by the reaction of p-hydroxyphenylglycine methyl ester and 6-aminopenicillanic acid (6APA), catalyzed by penicillin G acylase (PGA) immobilized on glutaraldehyde-chitosan, at 25°C and pH 6.5. Previous work on the kinetics and mechanism of reaction showed that the use of neural networks seems to be an interesting alternative to simulate experimental data of antibiotic production. Therefore, two feedforward neural networks, with one hidden layer, were trained and used to forecast the rates of amoxicillin and p-hydroxyphenylglycine (POHPG) net production. First of all, some parameters that affect the network performed were investigated, such as the number of nodes between the input and hidden layers and the number of interactions during the learning phase. Afterwards, hybrid models that coupled artificial neural networks to mass-balance equations were used to reproduce the performance of batch reactors for the production of amoxicillin. This approach provided accurate results, within the range of substrate concentration studied.
TL;DR: A simple method was developed to release periplasmic penicillin G acylase from Escherichia coli BL21(DE3) during the fermentation process and this method was efficient and would facilitate further investigation of peniillin G Acylase for industrial applications.
Abstract: 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.