Biocatalytic oxidase processes can benefit from improved mixing and mass transfer, as can all multiphase processes. Flow reactors are established for their mixing capabilities, but there are practical challenges in terms of slurry and gas/ liquid handling. The oxidase of the DL-amino acid was studied and scaled up in flow in a Coflore system and compared to batch processing conditions. The improved mass transfer under flow conditions resulted in a reduction in reaction time, enzyme consumption, and pressure drop.

The epoxide hydrolase (EH) from Corynebacterium sp. C12, which grows on cyclohexene oxide as sole carbon source, has been purified to homogeneity in two steps, involving anion exchange followed by hydrophobic-interaction chromatography. The purified enzyme is multimeric (probably tetrameric) with a subunit size of 32 140 Da. The gene encoding Corynebacterium EH was located on a 3.5-kb BamHI fragment of C12 chromosomal DNA using a DNA probe generated by PCR using degenerate primers based on the N-terminal and an internal amino acid sequence. Sequencing and database comparison of the predicted amino acid sequence of Corynebacterium EH shows that it is similar to mammalian and plant soluble EH, and the recently published sequence of epichlorohydrin EH from Agrobacterium radiobacter AD1 [Rink, R., Fennema, M., Smids, M., Dehmel, U. & Janssen, D. B. (1997) J. Biol. Chem. 272, 14 650Ϫ14 657), particularly around the catalytic site. All of these proteins belong to the A/β-hydrolase-fold family of enzymes. Similarity to the mammalian microsomal EH is weaker.Keywords : epoxide hydrolase; Corynebacterium C12 ; cyclohexene oxide ; A/β hydrolase fold.Extensive studies on the mammalian soluble (cytosolic) and chemical studies of soluble EH have demonstrated the formation microsomal epoxide hydrolases (EH), enzymes with a central of intermediate aspartate adducts , and role in detoxification (Oesch, 1973;Hammock et al., 1997), single-turnover experiments with microsomal EH (Lacourciere have established that they belong to a family of proteins that and Armstrong, 1993) have confirmed that the hydroxyl group share regions of amino acid sequence similarity with the active transferred to the product derives from the enzyme and not disite of bacterial haloalkane dehalogenase . rectly from water. These experiments also demonstrated the laThe three-dimensional structure of the latter was characterised belling of a specific aspartate residue during turnover, by H 2 18 O. by Verschueren et al. and coworkers (1993) who demonstrated Site-directed-mutagenesis studies with soluble EH (Pinot et al., that the enzyme belongs to the A/β-hydrolase-fold class of en-1995; Arand et al., 1996) have also confirmed the essential role zymes and employs a two-step mechanism involving initial for-of a histidine and aspartate pair, predicted by sequence alignmation of an aspartate ester, which is hydrolysed subsequently ment with haloalkane dehalogenase and other A/β hydrolase fold by a water molecule activated by a histidine-aspartate pair. enzymes, to be involved in promoting the hydrolytic step. While the core structure of the A/β hydrolase family, formed by Genes encoding the soluble EH have been cloned from a sheet of parallel β strands sandwiched by A helices (Ollis et mammals and plants (Beetham et al., 1995), and microsomal EH al., 1992), supports the catalytic triads of hydrolytic enzymes genes have been cloned from mammals and an insect source such as lipase and serine carboxypeptidase, a distinguishing fea- (Wojtasek and Prestwich, 1996). W...

No abstract

No abstract

No abstract

No abstract

No abstract

Unnatural amino acids are a growing class of intermediates required for pharmaceuticals, agrochemicals and other industrial products. However, no single method has proven sufficiently versatile to prepare these compounds broadly at scale. To address this need, we have developed a general chemoenzymatic process to prepare enantiomerically pure l-and d-amino acids in high yield by deracemization of racemic starting materials. This method involves the concerted action of an enantioselective oxidase biocatalyst and a nonselective chemical reducing agent to effect the stereoinversion of one enantiomer and can result in an enantiomeric excess of >99% from the starting racemate, and product yields of over 90%. This approach compares very favourably with resolution processes, which have a maximum single-pass yield of 50%. We have developed efficient methods to adapt the process towards new target compounds and to optimize key factors that influence process efficiency and offer competitive economics at scale. C 2006 Biochemical Society 287