title: Strategic manipulation of an industrial biocatalyst--evolution of a cephalosporin C acylase.
abstract: Semi-synthetic cephalosporins are synthesized from the 7-amino cephalosporanic acid (7-ACA) nucleus produced from the antibiotic cephalosporin C (CephC). In recent years, a single-step enzymatic process in which CephC is directly converted into 7-ACA by a cephalosporin C acylase (CA) has attracted industrial interest because of the prospects of simplifying the process and reducing costs. CAs are members of the glutaryl acylase family that specifically use CephC as their substrate; however, known natural glutaryl acylases show very low activity on the antibiotic. We previously enhanced the catalytic efficiency on CephC of a glutaryl acylase from Pseudomonas N176 (named VAC) by a protein engineering approach, and solved the structures of the VAC, thus providing insight into the substrate binding and catalytic activity of CAs. However, the properties of such enzymes are not sufficient to encourage 7-ACA manufacturers to shift to single-step enzymatic conversion of CephC. Here, we combine structural knowledge, semi-rational design, computational approaches and evolution analysis to isolate VAC variants with altered substrate specificity (i.e. with a > 11,000-fold increase in specificity constant for CephC versus glutaryl-7-amino cephalosporanic acid, compared to wild-type) and with the highest kinetic efficiency so far obtained for a CA. Indeed, the H57βS-H70βS-L154βY VAC variant shows the highest conversion of CephC into 7-ACA under conditions resembling those used at industrial level because of its high kinetic efficiency and the absence of substrate or product inhibition effects, and may be suitable for industrial application of the mono-step process for CephC conversion.
authors.affiliation: Dipartimento di Biotecnologie e Scienze della Vita, Università degli studi dell'Insubria, Varese, Italy.
authors.forename: Gianluca
authors.initials: G
authors.lastname: Conti
authors.forename: Loredano
authors.initials: L
authors.lastname: Pollegioni
authors.forename: Gianluca
authors.initials: G
authors.lastname: Molla
authors.forename: Elena
authors.initials: E
authors.lastname: Rosini
mesh_terms: D019943:Amino Acid Substitution; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics; D002511:Cephalosporins / Q000096:biosynthesis*; D019020:Directed Molecular Evolution / Q000379:methods*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010399:Penicillin Amidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010802:Phylogeny; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods; D011549:Pseudomonas / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D002511:Cephalosporins; D011994:Recombinant Proteins; C025163:cephalosporin C; D010399:Penicillin Amidase; C046586:glutarylamidocephalosporanic acid acylase
keywords: acylase; biocatalysis; cephalosporin C; mono-step bioconversion; protein engineering