title: Optimization of glutaryl-7-aminocephalosporanic acid acylase expression in E. coli.
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 approximately 80- and 120-fold increase in specific and volumetric enzyme productivity, respectively. Under the best expression conditions, approximately 1380 U/g cell and 16,100 U/L of GLA were produced versus the approximately 18 U/g cell and the approximately 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.
authors.affiliation: Dipartimento di Biotecnologie e Scienze Molecolari, Università degli Studi dell'Insubria, via J.H. Dunant 3, 21100 Varese, VA, Italy.
authors.forename: Federica
authors.initials: F
authors.lastname: Volontè
authors.forename: Flavia
authors.initials: F
authors.lastname: Marinelli
authors.forename: Luciano
authors.initials: L
authors.lastname: Gastaldo
authors.forename: Silvia
authors.initials: S
authors.lastname: Sacchi
authors.forename: Mirella S
authors.initials: MS
authors.lastname: Pilone
authors.forename: Loredano
authors.initials: L
authors.lastname: Pollegioni
authors.forename: Gianluca
authors.initials: G
authors.lastname: Molla
mesh_terms: D000581:Amidohydrolases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D019149:Bioreactors; D003470:Culture Media / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004790:Enzyme Induction / Q000187:drug effects; D004926:Escherichia coli / Q000737:chemistry / Q000187:drug effects / Q000502:physiology*; D007218:Industrial Microbiology / Q000191:economics / Q000379:methods; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D010399:Penicillin Amidase / Q000096:biosynthesis* / Q000737:chemistry / Q000187:drug effects; D011549:Pseudomonas / Q000201:enzymology*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D012965:Sodium Chloride / Q000494:pharmacology; D013997:Time Factors
chemical_list: D003470:Culture Media; D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside; D012965:Sodium Chloride; D000581:Amidohydrolases; D010399:Penicillin Amidase; C046586:glutarylamidocephalosporanic acid acylase