Optimization of a pseudo-affinity process for penicillin acylase purification

被引:0
|
作者
L. P. Fonseca
J. M. S. Cabral
机构
[1] Laboratório de Engenharia Bioquímica,
[2] Centro de Engenharia Biológica e Química,undefined
[3] Instituto Superior Técnico,undefined
[4] Av. Rovisco Pais,undefined
[5] 1000 Lisboa Portugal,undefined
来源
Bioprocess Engineering | 1999年 / 20卷
关键词
Enzymatic Extract; Ammonium Sulphate; Enzyme Adsorption; Penicillin Acylase; Continuous Adsorption;
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学科分类号
摘要
A pseudo-affinity process for penicillin acylase (EC 3.5.1.11) purification using an affinity ligand (Ampicillin) attached on Sepharose 4B-CNBr was optimized. The enzyme adsorption on this affiant (Amp-Seph) is independent of pH between 5.5 and 8.8, in 100 mM phosphate containing 22% (w/v) ammonium sulphate. The desorption of the penicillin acylase from the affinity gels was carried out, the best desorption results being obtained through a non specific eluent, 100 mM phosphate pH 4.6 with 15% (w/v) ammonium sulphate. The best purification results were obtained with an enzymatic extract, produced through osmotic shock of Escherichia coli cells (3.7 IU/mg prot). With this extract and an affinity gel of Sepharose 4B-CNBr derivatized with ampicillin (3.8 μmol/cm3 gel), a maximum activity capacity adsorbed of 20 IU/cm3 gel was obtained for initial values of activity and protein concentration of 1.7 IU/cm3 and 0.4 mg prot/cm3, respectively. With the optimized eluent it was possible to obtain penicillin acylase in only one purification step with a desorption yield of enzyme activity higher than 90%. The penicillin acylase produced with this process was characterized by a maximum purity of 34 IU/mg prot, corresponding to a purification degree higher than 150 in relation to the lowest pure enzymatic extract. The enzyme purity of the eluted fractions was certified by SDS gel electrophoresis and liquid chromatography through a Mono Q column in a FPLC apparatus. The gel electrophoresis presented 4 main stained bands with 2 corresponding to α and β subunits of the penicillin acylase with equivalent molecular weights of 27 and 63 kDa. No external diffusion resistance on penicillin acylase and total protein adsorption on this affiant (Amp-Seph 3.8 μmol/cm3 gel) were observed for continuous adsorption processes performed at two different agitation speeds (120 and 400 rpm).
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页码:513 / 524
页数:11
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