Evolution of an acylase active on cephalosporin C

Abstract

Semisynthetic cephalosporins are synthesized from 7-amino cephalosporanic acid, which is produced by chemical deacylation or by a two-step enzymatic process of the natural antibiotic cephalosporin C. The known acylases take glutaryl-7-amino cephalosporanic acid as a primary substrate, and their specificity and activity are too low for cephalosporin C. Starting from a known glutaryl-7-amino cephalosporanic acid acylase as the protein scaffold, an acylase gene optimized for expression in Escherichia coli and for molecular biology manipulations was designed. Subsequently we used error-prone PCR mutagenesis, a molecular modeling approach combined with site-saturation mutagenesis, and site-directed mutagenesis to produce enzymes with a cephalosporin C/glutaryl-7-amino cephalosporanic acid catalytic efficiency that was increased up to 100-fold, and with a significant and higher maximal activity on cephalosporin C as compared to glutaryl-7-amino cephalosporanic acid (e.g., 3.8 vs. 2.7 U/mg protein, respectively, for the A215Y-H296S-H309S mutant). Our data in a bioreactor indicate an ,90% conversion of cephalosporin C to 7-aminocephalosporanic acid in a single deacylation step. The evolved acylase variants we produced are enzymes with a new substrate specificity, not found in nature, and represent a hallmark for industrial production of 7-amino cephalosporanic acid.Keywords: cephalosporin C; 7-amino cephalosporanic acid; protein engineering; directed evolution; site-saturation mutagenesis; enzymes; active sites; structure/function studies; protein sequencing; modification; mass spectrometry; protein structure prediction; kinetics Semisynthetic cephalosporins are the most widely used antibiotics and are primarily synthesized from 7-amino cephalosporanic acid (7-ACA), which is usually obtained by chemical deacylation of the natural antibiotic cephalosporin C (CephC). The chemical route includes, however, several expensive steps and requires treatment of toxic wastes. A two-step enzymatic route can also be used that, in two separate reactors, uses D-amino acid oxidase and glutaryl-7-amino cephalosporanic acid (gl-7ACA) acylase activity (see Scheme 1, below): Although this route solves the environmental safety problems, it is also expensive and not entirely satisfactory for industrial production. Therefore, enzymatic conversion of CephC to 7-ACA is of great interest to cephalosporin antibiotics manufacturers (an annual worldwide market of ,400 million US dollars has been estimated) (Pilone and Pollegioni 2002). The greatest hindrance to enzymatic industrial production is that acylase takes glutaryl-7-ACA (gl-7ACA) as a primary substrate, and its specificity (and activity) is too low for CephC. Glutaryl-7-ACA acylases are members of the N-terminal hydrolases (Ntn) class of hydrolytic enzymes. The gene structure of the open reading frame (ORF) of the members of this class consists of a signal peptide, followed by an a-subunit, a spacer sequence (which is not present in the acylase under investigation), and a b-subunit...

Site-Directed Mutagenesis of Cephalosporin C Acylase and Enzymatic Conversion of Cephalosporin C to 7-Aminocephalosporanic Acid

“…But the 7-ACA, intermediate and byproduct probably inhibited the catalysis with the extension reaction time. Pollegioni found that the mutant H296S/H309S of cephalosporin C acylase from N176 had the improved product inhibition [5]. In this research, after 15 minutes, A12 perhaps was inhibited by the 7-ACA or byproduct produced numerously due to the mutation of S471β.…”

“…Researchers tried to separate cephalosporin C acylase from the micro-organisms that could convert CPC into 7-ACA directly [4]. One-step enzymatic method achieved an annual product value 400 million U.S dollars in the global market [5]. It was the biggest obstacle that the cephalosporin C acylase catalyzed the CPC to 7-ACA directly in a very low efficiency.…”

Novel biosensors based on optimized glycine oxidase

“…The PCR products (full-length plasmids) were used to transform JM109 E. coli cells (the cloning host strain); the plasmid DNA was then pooled and transferred to BL21(DE3)pLysS E. coli cells (the expression host strain); recombinant cells were used for the screening procedure as detailed below. For each position,~280 clones were screened (Table 5), a number that gives a probability of 96% that every single amino acid is introduced at the desired position [30], and 10 clones were sequenced to verify the mutagenesis efficiency. The introduction of the mutations was confirmed by automated DNA sequencing.…”

J1 acylase, a glutaryl‐7‐aminocephalosporanic acid acylase from Bacillus laterosporus J1, is a member of the α/β‐hydrolase fold superfamily

“…The activity of J1 acylase was determined by a spectrophotometric method as described in [19,20], with slight modifications. Equal volumes (125 ll) of enzyme and substrate (GL-7-ACA) were allowed to react in a Thermomixer (Eppendrof) at 37°C for a certain period of time before the reaction was terminated by the addition of 1 ml mixture of 20% acetic acid and 0.1 M sodium hydroxide.…”