72657-23-9

15484-46-5

72657-23-9

GENERAL PROCEDURE: The general procedure for the synthesis of (-)-methyl-D-BETA-hydroxyisobutyrate from methyl 2-(hydroxymethyl)acrylate is as follows: the Ene-reductase activity assay needs to be carried out in anaerobic conditions (Coy Laboratory, Grass Lake, MI) at ambient temperature. Preparation of reaction stock solutions consisted of dissolving each substrate (see below for details) in 50 mM Tris-HCl buffer (pH 7.5) supplemented with 200 μM NADP+, 100 mM glucose, and glucose dehydrogenase (GDH) from Thermoplasma acidophilum (2 units were used for the IVTT reaction and 5 (2 units for the IVTT reaction and 5 units for the purified enzyme reaction). Individual substrate concentrations were selected to ensure that vmax conditions or maximum solubility were achieved. At these substrate concentrations, adjust reaction times to achieve 10-50% substrate conversion. For OYE activity assays in IVTT experiments, 20 μL of reaction stock solution was mixed with 2-10 μL of IVTT reaction mixture and the total assay volume was adjusted to 30 μL with 50 mM Tris-HCl (pH 7.5).For assays of purified OYE1 variants, the enzyme (final concentration: 250 nM) was added to 500 μL of reaction stock solution. The reaction process was quenched by removing a 30 μL aliquot from the assay solution and mixing it thoroughly with 30 μL of ethyl acetate containing 1 mM cyclohexanone (as internal standard). Organic phase samples were collected and analyzed by gas chromatography (GC) (see below for specific protocol). Enantiomeric/diastereoisomeric excesses were calculated by integrating the peak areas of the products and substrates. The relative conversion rate of each substrate was calculated by dividing the measured conversion rate of the OYE1 variant by the corresponding rate of wild-type OYE1.