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Epimerization of cholic and chenodeoxycholic acid (CA and CDCA, respectively) is a notable conversion for the production of ursodeoxycholic acid (UDCA). Two enantiocomplementary hydroxysteroid dehydrogenases (7α‐ and 7β‐HSDHs) can carry out this transformation fully selectively by specific oxidation of the 7α‐OH group of the substrate and subsequent reduction of the keto intermediate to the final product (7β‐OH). With a view to developing robust and active biocatalysts, novel NADH‐active 7β‐HSDH species are necessary to enable a solely NAD+‐dependent redox‐neutral cascade for UDCA production. A wild‐type NADH‐dependent 7β‐HSDH from Lactobacillus spicheri (Ls7β‐HSDH) was identified, recombinantly expressed, purified, and biochemically characterized. Using this novel NAD+‐dependent 7β‐HSDH enzyme in combination with 7α‐HSDH from Stenotrophomonas maltophilia permitted the biotransformations of CA and CDCA in the presence of catalytic amounts of NAD+, resulting in high yields (>90 %) of UCA and UDCA. [ABSTRACT FROM AUTHOR]

Ene-reductases (ERs) are widely applied for the asymmetric synthesis of relevant industrial chemicals. A novel ER OYERo2 was found within a set of 14 putative old yellow enzymes (OYEs) obtained by genome mining of the actinobacterium Rhodococcus opacus 1CP. Multiple sequence alignment suggested that the enzyme belongs to the group of 'thermophilic-like' OYEs. OYERo2 was produced in Escherichia coli and biochemically characterized. The enzyme is strongly NADPH dependent and uses noncovalently bound FMNH2 for the reduction of activated α,β-unsaturated alkenes. In the active form OYERo2 is a dimer. Optimal catalysis occurs at pH 7.3 and 37℃. OYERo2 showed highest specific activities (45-50 U mg-1) on maleimides, which are efficiently converted to the corresponding succinimides. The OYERo2-mediated reduction of prochiral alkenes afforded the (R)-products with excellent optical purity (ee > 99%). OYERo2 is not as thermo-resistant as related OYEs. Introduction of a characteristic intermolecular salt bridge by site-specific mutagenesis raised the half-life of enzyme inactivation at 32℃ from 28 to 87 min and improved the tolerance toward organic co-solvents. The suitability of OYERo2 for application in industrial biocatalysis is discussed. [ABSTRACT FROM AUTHOR]

The enantioselective Michael addition using water as both nucleophile and solvent has to date proved beyond the ability of synthetic chemists. Herein, the direct, enantioselective Michael addition of water in water to prepare important β-hydroxy carbonyl compounds using whole cells of Rhodococcus strains is described. Good yields and excellent enantioselectivities were achieved with this method. Deuterium labeling studies demonstrate that a Michael hydratase catalyzes the water addition exclusively with anti-stereochemistry. [ABSTRACT FROM AUTHOR]

Lipase-catalyzed synthesis of estolides starting from different saturated (C16 16OH, C18 12OH) and unsaturated (C18:1 9 cis 12-OH) hydroxy-fatty acids was investigated. For this reason, the catalytic efficiency of several native and immobilized lipases in different organic reaction media at temperatures up to 75 °C was studied. The formation of mono- and di-lactone as well as estolide's chain elongation depends on the type and source of lipase. The lipase from Pseudomonas stutzeri immobilized by cross-linking as cross-linked enzymes aggregates (CLEAs) was the best biocatalyst in terms of chain elongation. Estolides with polymerization degree up to 10 were obtained at substrate conversions higher than 80 %. [ABSTRACT FROM AUTHOR]

In industrial biocatalytic processes the class of lyases (EC 4) is underrepresented and only a few enzymes are used in industrial scale reactions. Hydro-lyases (EC 4.2.1) catalyze the non-hydrolytic and non-oxidative addition of water to a CC bond. Without a biocatalyst, this reaction proceeds under strong acidic conditions with no enantioselectivity. From a chemical point of view carotenoid 1,2-hydratase (CrtC) performs a very challenging addition of water to an isolated CC bond. This hydration reaction proceeds without assistance of electron withdrawing groups or transition metals, which makes it very interesting from both an industrial, as well as enzymatic point of view. Oleate hydratase (OHase) catalyzes the enantioselective conversion of oleic acid into 10-hydroxystearic acid. The crystal structure of OHase from Lactobacillus acidophilus was published recently, which contributes to the understanding of this enzyme. These enzymes could broaden the organic chemist's toolbox. [ABSTRACT FROM AUTHOR]