Improved thermostability of AEH by combining B-FIT analysis and structure-guided consensus method.

α-Amino ester hydrolases (AEH, E.C. 3.1.1.43) catalyze the synthesis and hydrolysis of α-amino ß-lactam antibiotics. The AEH enzymes have been shown to feature excellent synthetic capability but suffer from poor thermostability. AEH from Xanthomonas campestris exhibits an optimal activity temperature of 25 °C, an observed half-life of 5 min at 30 °C, and a "T-50" value, the temperature at which the half-life is 30 min, of 27 °C. To improve the thermostability of AEH, a modified structure-guided consensus model of seven homologous enzymes was generated along with analysis of the B-values from the available crystal structures of AEH from Xanthomonas citri. A family of stabilized variants was created including a consensus-driven triple variant, A275P/N186D/V622I. Independent NNK saturation of two high B-factor sites, K34 and E143, on the triple variant resulted in our best variant, the quadruple mutant E143H/A275P/N186D/V622I, with a "T-50" value of 34 °C (7 °C improvement) and 1.3-fold activity compared to wild-type.

Improving the diastereoselectivity of penicillin G acylase for ampicillin synthesis from racemic substrates.

Semi-synthetic ß-lactam antibiotics are synthesized enzymatically with the use of penicillin G acylase (PGA). Currently, PGA only exhibits weak diastereoselectivity with respect to the alpha amino group of rac-phenylglycine methyl ester (rac-PGME) when it is coupled with 6-aminopenicillanic acid to synthesize ampicillin. Therefore, we sought to improve the diastereoselectivity of PGA by targeting residues for site-saturation based on the proximity to the substrate's chiral center. Four variants with improved selectivity for (R)-ampicillin synthesis were identified, all resulting from a mutation at the ß24 position. ßPhe24Ala, while not identified from our library screening, was obtained with site-directed mutagenesis because it has been previously shown to be selective for (R)-enantiomers with substituents other than an amino group. The diastereomeric excess (d.e.(R)) value of 37% for the wild-type enzyme was improved to a d.e.(R) value of 98% for our most selective mutant, ßPhe24Ala. Also, four mutations at the α146 position that resulted in (S)-selective PGA variants were identified. ßPhe24 and αPhe146 are on opposite sides of the alpha carbon of the substrate, and we have shown that altering these residues results in enhanced selectivity in opposite directions. All variants that showed selectivity for (S)-ampicillin synthesis showed decreased synthetic activity for pure substrates and a decreased synthesis-to-hydrolysis ratio. In contrast, the mutants that were selective for (R)-ampicillin showed significantly decreased primary and secondary hydrolysis when synthesizing ampicillin from pure (R)-PGME, resulting in up to 4-fold decrease in the synthesis to hydrolysis ratio and up to 2-fold increase in the yield achieved. Finally, it was discovered that the selective PGA variants have racemase or epimerase activity, a fascinating phenomenon that has never been reported.

Status of protein engineering for biocatalysts: how to design an industrially useful biocatalyst.

Recent advances in the development of both experimental and computational protein engineering tools have enabled a number of further successes in the development of biocatalysts ready for large-scale applications. Key tools are first, the targeting of libraries, leading to far smaller but more useful libraries than in the past, second, the combination of structural, mechanistic, and sequence-based knowledge often based on prior successful cases, and third, the advent of structurally based algorithms allowing the design of novel functions. Based on these tools, a number of improved biocatalysts for pharmaceutical applications have been presented, such as an (R)-transaminase for the synthesis of active pharmaceutical ingredients (APIs) of sitagliptin (Januvia®) and ketoreductases, glucose dehydrogenases, and haloalkane dehalogenases for the API synthesis toward atorvastatin (Lipitor®) and montelukast (Singulair®).

Ampicillin Synthesis Using a Two-Enzyme Cascade with Both α-Amino Ester Hydrolase and Penicillin G Acylase.

The current enzymatic production of semisynthetic ß-lactam antibiotics requires isolation and purification of the intermediate 6-aminopenicillanic acid which adds cost and complexity to the manufacturing process. In this work, we took advantage of the unique substrate specificity of a-amino ester hydrolases to perform a purely aqueous one-pot production of ampicillin from penicillin G and D-phenylglycine methyl ester, catalyzed by α-amino ester hydrolase and penicillin G acylase. The synthesis was performed in both a one-pot, one-step synthesis resulting in a maximum conversion of 39%, and a one-pot, two-step process resulting in a maximum conversion of 47%. The two-enzyme cascade reported in this paper is a promising alternative to the current enzymatic two-step, two-pot manufacturing process for semisynthetic ß-lactam antibiotics which requires intermittent isolation of 6-aminopenicillanic acid.

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 ß-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 ß-lactam antibiotics including (R)-ampicillin and cephalexin, with optimal ampicillin hydrolytic activity at 25 °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 α, ß(o), and γ for ampicillin, determined here first for this class of proteins, are α = 0.25, ß(o) = 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.