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Showing papers on "Penicillin amidase published in 2013"


Journal ArticleDOI
TL;DR: 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.
Abstract: 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 66-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

12 citations


Journal ArticleDOI
TL;DR: The cloning of the pac gene encoding KcPGA and the preparation of a slow-processing mutant precursor are reported, and the purification, crystallization and preliminary X-ray analysis of crystals of this precursor protein are described.
Abstract: Kluyvera citrophila penicillin G acylase (KcPGA) has recently attracted increased attention relative to the well studied and commonly used Escherichia coli PGA (EcPGA) because KcPGA is more resilient to harsh conditions and is easier to immobilize for the industrial hydrolysis of natural penicillins to generate the 6-aminopenicillin (6-APA) nucleus, which is the starting material for semi-synthetic antibiotic production Like other penicillin acylases, KcPGA is synthesized as a single-chain inactive pro-PGA, which upon autocatalytic processing becomes an active heterodimer of α and β chains Here, the cloning of the pac gene encoding KcPGA and the preparation of a slow-processing mutant precursor are reported The purification, crystallization and preliminary X-ray analysis of crystals of this precursor protein are described The protein crystallized in two different space groups, P1, with unit-cell parameters a = 540, b = 1246, c = 1351 A, α = 1041, β = 1014, γ = 965°, and C2, with unit-cell parameters a = 2651, b = 540, c = 2492 A, β = 1044°, using the sitting-drop vapour-diffusion method Diffraction data were collected at 100 K and the phases were determined using the molecular-replacement method The initial maps revealed electron density for the spacer peptide

10 citations


Patent
12 Jun 2013
TL;DR: In this article, a self-induced medium composed of 2-10g/L of arabinose, 0.5-2.5g of glucose, 5-25g of glycerol, 8-12g of peptone, 1.1-1.4g of phosphate and NH4Cl was used to induce the expression of an interest protein.
Abstract: The invention discloses a self-induced medium and an application thereof. The self-induced medium is composed of 2-10g/L of arabinose, 0.5-2.5g/L of glucose, 5-25g/L of glycerol, 8-12g/L of peptone, 6.5-7.4g/L of phosphate, 1.1-1.3g/L of sulfate, 2.6-2.7g/L of NH4Cl and an appropriate amount of trace element solution. The self-induced medium is used in producing penicillin amidase. Compared with the prior art, the self-induced medium disclosed by the invention has the advantages of strong regulation performance and high protein expression index by utilizing the arabinose as a substrate to induce the expression of an interest protein. In one specific embodiment, the enzyme activity of the penicillin amidase reaches 6U/mL, which is much higher than the protein expression index induced by lactose, and thus providing an important guiding significance for a new process of developing and producing penicillin amidase.

3 citations


23 Apr 2013
TL;DR: The study revealed the potential of agricultural wastes’ capability to produce amylase by B.megaterium at high temperature which could be an indication of P.amidase produced as thermostable.
Abstract: Temple puja wastes are released in water bodies or land creating severe environmental pollution and health hazards. The temple wastes extract was used to isolate the Bacillus megaterium for the production of enzyme penicillin amidase as it recorded the largest zone of activity. The result obtained showed that on nutrient agar medium the grown colonies were confirmed by gram staining as Bacillus megaterium in temple wastes. P.amidase activity was determined using DNSA method. Highest yield of enzyme activity by B.megaterium was obtained after 48 hrs. of incubation. The optimum temperature and pH for the enzyme activity was obtained at 60 o C (6.0 + 0.55) and pH 7.0 (2.04 ± 0.49). The results shown that B.megaterium is a good producer of extracellular P.amidase at high temperature which could be an indication of P.amidase produced as thermostable. The study revealed the potential of agricultural wastes’ capability to produce amylase by B.megaterium .

2 citations