Blum_2010_J.Mol.Catal.B.Enzym_67_21

Reference

Title : Amino ester hydrolase from Xanthomonas campestris pv. campestris, ATCC 33913 for enzymatic synthesis of ampicillin - Blum_2010_J.Mol.Catal.B.Enzym_67_21
Author(s) : Blum JK , Bommarius AS
Ref : J Mol Catal B Enzym , 67 :21 , 2010
Abstract : 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.
ESTHER : Blum_2010_J.Mol.Catal.B.Enzym_67_21
PubMedSearch : Blum_2010_J.Mol.Catal.B.Enzym_67_21
PubMedID: 22087071
Gene_locus related to this paper: xanax-GAA

Related information

Gene_locus related to this paper: xanax-GAA

Citations formats

Blum JK, Bommarius AS (2010)
Amino ester hydrolase from Xanthomonas campestris pv. campestris, ATCC 33913 for enzymatic synthesis of ampicillin
J Mol Catal B Enzym 67 :21

Blum JK, Bommarius AS (2010)
J Mol Catal B Enzym 67 :21