Cloning, purification, crystallization and preliminary structural studies of penicillin V acylase from Bacillus subtilis

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Abstract

Penicillin acylase proteins are amidohydrolase enzymes that cleave penicillins at the amide bond connecting the side chain to their ?-lactam nucleus. An unannotated protein from Bacillus subtilis has been expressed in Escherichia coli, purified and confirmed to possess penicillin V acylase activity. The protein was crystallized using the hanging-drop vapour-diffusion method from a solution containing 4 M sodium formate in 100 mM Tris-HCl buffer pH 8.2. Diffraction data were collected under cryogenic conditions to a spacing of 2.5 ?. The crystals belonged to the orthorhombic space group C222 1, with unit-cell parameters a = 111.0, b = 308.0, c = 56.0 ?. The estimated Matthews coefficient was 3.23 ?3 Da-1, corresponding to 62% solvent content. The structure has been solved using molecular-replacement methods with B. sphaericus penicillin V acylase (PDB code) as the search model. ? 2005 International Union of Crystallography All rights reserved.

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Rathinaswamy, P., Pundle, A. V., Prabhune, A. A., SivaRaman, H., Brannigan, J. A., Dodson, G. G., & Suresh, C. G. (2005). Cloning, purification, crystallization and preliminary structural studies of penicillin V acylase from Bacillus subtilis. Acta Crystallographica Section F: Structural Biology and Crystallization Communications, 61(7), 680–683. https://doi.org/10.1107/S1744309105017987

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