David P. Pantaleone

Amine–boranes: effective reducing agents for the deracemisation of dl-amino acids using l-amino acid oxidase from Proteus myxofaciens

Abstract The deracemisation of dl -α-amino acids using l -amino acid oxidase from Proteus myxofaciens and amine-boranes as chemical reducing agents has been investigated. Amine-boranes were found to be of particular interest in terms of reactivity and chemoselectivity compared to sodium borohydride and cyanoborohydride. Starting from the racemate, a range of d -amino acids were obtained in yields of up to 90% and e.e. >99%.

Novel biosynthetic routes to non-proteinogenic amino acids as chiral pharmaceutical intermediates

Abstract Transaminases catalyse the reversible transfer of amino and keto groups between an amino acid and keto acid substrate pair. Many bacterial transaminases accept a wide array of keto acids as amino acceptors and are useful as commercial biocatalysts in the preparation of amino acids. Since the reaction equilibrium typically lies close to unity, several approaches have been described to improve upon the 50% product yield, using additional enzymes. The present work describes an efficient means to significantly increase product yield in transamination using the aromatic transaminase of Escherichia coli encoded by the tyrB gene, with l -aspartate as the amino donor. This is achieved by the introduction of the alsS gene encoding the acetolactate synthase of Bacillus subtilis, which eliminates pyruvate and alanine produced as a by-product of aspartate transamination. The biosynthesis of the non-proteinogenic amino acid l -2-aminobutyrate is described using a recombinant strain of E. coli containing the cloned tyrB and alsS genes. The strain additionally carries the cloned ilvA gene of E. coli encoding threonine deaminase to produce the substrate 2-ketobutyrate from l -threonine. An alternate coupled process uses lysine e-aminotransferase in concert with a transaminase using l -glutamate as the amino donor.

Engineering of a novel biochemical pathway for the biosynthesis of L-2-aminobutyric acid in Escherichia coli K12.

L-2-Aminobutyric acid was synthesised in a transamination reaction from L-threonine and L-aspartic acid as substrates in a whole cell biotransformation using recombinant Escherichia coli K12. The cells contained the cloned genes tyrB, ilvA and alsS which respectively encode tyrosine aminotransferase of E. coli, threonine deaminase of E. coli and alpha-acetolactate synthase of B. subtilis 168. The 2-aminobutyric acid was produced by the action of the aminotransferase on 2-ketobutyrate and L-aspartate. The 2-ketobutyrate is generated in situ from L-threonine by the action of the deaminase, and the pyruvate by-product is eliminated by the acetolactate synthase. The concerted action of the three enzymes offers significant yield and purity advantages over the process using the transaminase alone with an eight to tenfold increase in the ratio of product to the major impurity.

Purification and characterization of an L-amino acid deaminase used to prepare unnatural amino acids

Abstract l -Amino acid deaminase ( l -AAD) from Proteus myxofaciens was cloned and over-expressed in Escherichia coli K12. This enzyme has a broad substrate specificity, working on both natural and unnatural l -amino acids. Of the 20 naturally occurring l -amino acids, l -AAD prefers amino acid substrates that have aliphatic, aromatic or sulfur-containing side chains; those with charged side chains (–CO 2 − or –NH 3 + ) are poor or non-substrates. Enzyme activity was monitored using a microtiter-plate-based assay, which measures the formation of phenylpyruvic acid from l -phenylalanine. The reaction has an absolute requirement for O 2 , releases NH 3 and does not produce H 2 O 2 . Substrate comparisons were carried out by using an O 2 electrode to measure the O 2 utilization rates. Studies on partially purified enzyme show a pH optimum of 7.5 with a subunit molecular weight of approximately 51 kDa. Additional purification and characterization strategies will be presented. The use of whole cells containing l -AAD will be discussed to prepare chiral pharmaceutical intermediates.