Amino ester hydrolase from Xanthomonas campestris pv. campestris, ATCC 33913 for enzymatic synthesis of ampicillin

α-Amino ester hydrolases (AEH) are a small class of proteins, which are highly specific for hydrolysis or synthesis of α-amino containing amides and esters including β-lactam antibiotics such as ampicillin, amoxicillin, and cephalexin. A BLAST search revealed the sequence of a putative glutaryl 7-aminocephalosporanic acid (GL-7-ACA) acylase 93% identical to a known AEH from Xanthomonas citri. The gene, termed gaa, was cloned from the genomic DNA of Xanthomonas campestris pv. campestris sp. strain ATCC 33913 and the corresponding protein was expressed into Escherichia coli. The purified protein was able to perform both hydrolysis and synthesis of a variety of α-amino β-lactam antibiotics including (R)-ampicillin and cephalexin, with optimal ampicillin hydrolytic activity at 25 °C and pH 6.8, with kinetic parameters of kcat of 72.5 s−1 and KM of 1.1 mM. The synthesis parameters α, βo, and γ for ampicillin, determined here first for this class of proteins, are α = 0.25, βo = 42.8 M−1, and γ = 0.23, and demonstrate the excellent synthetic potential of these enzymes. An extensive study of site-directed mutations around the binding pocket of X. campestris pv. campestris AEH strongly suggests that mutation of almost any first-shell amino acid residues around the active site leads to inactive enzyme, including Y82, Y175, D207, D208, W209, Y222, and E309, in addition to those residues forming the catalytic triad, S174, H340, and D307.

The plot of ampicillin synthesized over (R)-phenylglycine generated was fit with the common model for beta-lactam synthesis and demonstrates the great potential of amino ester hydrolase for beta-lactam synthesis (α = 0.25, βo = 42.8 M−1, and γ = 0.23).Figure optionsDownload as PowerPoint slideResearch highlights▶ A BLAST search against two existing amino ester hydrolases yielded the sequence of a putative glutaryl 7-aminocephalosporanic acid (GL-7-ACA) acylase from Xanthomonas campestris pv. campestris sp. strain ATCC 33913. ▶ The protein was able to perform both hydrolysis and synthesis of a variety of α-amino β-lactam antibiotics including (R)-ampicillin and cephalexin, with optimal ampicillin hydrolytic activity at 25 °C and pH 6.8, with kinetic parameters of kcat of 72.5 s−1 and KM of 1.1 mM. ▶ The synthesis parameters α, βo, and γ for ampicillin, determined here first for this class of proteins, are α = 0.25, βo = 42.8 M−1, and γ = 0.23, and demonstrate the excellent synthetic potential of these enzymes. ▶ Mutation of almost any first-shell amino acid residues around the active site leads to inactive enzyme.