Optimization of glutaryl-7-aminocephalosporanic acid acylase expression in E. coli |
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Authors: | Volontè Federica Marinelli Flavia Gastaldo Luciano Sacchi Silvia Pilone Mirella S Pollegioni Loredano Molla Gianluca |
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Affiliation: | aDipartimento di Biotecnologie e Scienze Molecolari, Università degli Studi dell’Insubria, via J.H. Dunant 3, 21100 Varese, VA, Italy |
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Abstract: | A recombinant glutaryl-7-aminocephalosporanic acid acylase (GLA) from Pseudomonas N176 has been over-expressed in BL21(DE3)pLysS Escherichia coli cells. By alternating screenings of medium components and simplified factorial experimental designs, an improved microbial process was set up at shake-flask level (and then scaled up to 2L-fermentors) giving a 80- and 120-fold increase in specific and volumetric enzyme productivity, respectively. Under the best expression conditions, 1380 U/g cell and 16,100 U/L of GLA were produced versus the 18 U/g cell and the 140 U/L obtained in the initial standard conditions. Osmotic stress caused by the addition of NaCl, low cell growth rate linked to high biomass yield in the properly-designed rich medium, optimization of the time and the amount of inducer’s addition and decrease of temperature during recombinant protein production, represent the factors concurring to achieve the reported expression level. Notably, this expression level is significantly higher than any previously described production of GLAs. High volumetric production, cost reduction and the simple one-step chromatographic purification of the His-tagged recombinant enzyme, makes this GLA an economic tool to be used in the 7-ACA industrial production. |
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Keywords: | Acylase Cephalosporin C Protein expression Medium optimisation Plackett– Burman factorial design Recombinant E. coli |
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