Research involving alpha/beta hydrolases, including alpha-amino acid ester hydrolase and cocaine esterase, has been limited by the lack of an online high throughput screening assay. The development of a high throughput screening assay capable of detecting alpha/beta hydrolase activity toward specific substrates and/or chemical reactions (e.g., hydrolysis in lieu of amidase activity and/or synthesis instead of thioesterase activity) is of interest in a broad set of scientific questions and applications. Here we present a general framework for pH-based colorimetric assays, as well as the mathematical considerations necessary to estimate de novo the experimental response required to assign a 'hit' or a 'miss,' in the absence of experimental standard curves. This combination is valuable for screening the hydrolysis and synthesis activity of alpha/beta hydrolases on a variety of substrates, and produces data comparable to the current standard technique involving High Performance Liquid Chromatography (HPLC). In contrast to HPLC, this assay enables screening experiments to be performed with greater efficiency.
Title: Amino ester hydrolase from Xanthomonas campestris pv. campestris, ATCC 33913 for enzymatic synthesis of ampicillin Blum JK, Bommarius AS Ref: J Mol Catal B Enzym, 67:21, 2010 : PubMed
alpha-Amino ester hydrolases (AEH) are a small class of proteins, which are highly specific for hydrolysis or synthesis of alpha-amino containing amides and esters including beta-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 alpha-amino beta-lactam antibiotics including (R)-ampicillin and cephalexin, with optimal ampicillin hydrolytic activity at 25 degrees C and pH 6.8, with kinetic parameters of k(cat) of 72.5 s(-1) and K(M) of 1.1 mM. The synthesis parameters alpha, beta(o), and gamma for ampicillin, determined here first for this class of proteins, are alpha = 0.25, beta(o) = 42.8 M(-1), and gamma = 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.
