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We report herein the screening, optimisation and scale up to 100 g of a bioreduction process that employs an in situ product removal (ISPR) technique to overcome the inherent equilibrium problem associated with the coupled-substrate approach to biocatalytic carbonyl reduction. This technique allowed the valuable chiral alcohol, (S)-2-bromo-2-cyclohexen-1-ol, to be isolated in 88% yield and 99.8% ee without the need for further purification, validating the general applicability of this experimental setup.

The use of ene-reductase (ERED) enzymes for the asymmetric reduction of olefins offers a green, renewable alternative to metal-catalysed asymmetric reduction. We report herein the first example of an ERED-catalysed enantiospecific reduction carried out at large scale using a carbonyl reductase (CRED) enzyme in the cofactor recycle. This reaction has been paired with a hydrolase-mediated regioselective ester hydrolysis to generate a valuable chiral building block using a straightforward one-pot process.

The LOLAC assay is the first reported enzymatic method that accurately quantitates lactose in lactose-free samples. © 2018 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

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Carbonyl reductase (CRED) technology has been shown to be an important tool for the rapid and efficient preparation of cis-and trans-4-tert-butylcyclohexanol, the precursors to the high volume fragrance ingredients known as Woody Acetate. ' INTRODUCTIONBoth cis and trans forms of 4-tert-butylcyclohexyl acetate, 1 and 2, respectively ( Figure 1), are widely used as a perfume for cosmetics including soaps. The fragrance of its cis-isomer is more favorable than that of its trans-isomer, having a creamy, woody, and sweet odor. 1 cis-4-tert-Butylcyclohexyl acetate 1 is obtained by acetylation of the corresponding alcohol 3 (Scheme 1).Current methods for Woody Acetate production include the hydrogenation of 4-tert-butylphenol in the presence of a catalyst supported on a carrier under a superatmospheric pressure of hydrogen and at an elevated temperature. Sumitomo patented a hydrogenation process based on rhodium on carbon with HCl or sulfuric acid 2 that yields isomer ratios of approximately 80:20 to 90:10 cis/trans. More recently Abdur-Rashid et al. have submitted a patent application 3 for the stereoselective reduction of 4-tertbutylcyclohexanone with ruthenium-aminophosphine complexes in the presence of hydrogen gas and base. Ratios of 96:4 cis/trans have been achieved. Whilst the cis/trans ratios obtained are acceptable, these processes suffer from a number of drawbacks, namely, the use of expensive metal catalysts and/or corrosive reagents. With these facts in mind, the investigation of a biocatalytic route to generate cis-4-tert-butylcyclohexyl acetate becomes increasingly attractive.The application of biocatalysts to the synthesis of important enantiomerically pure compounds by the fine chemical and pharmaceutical industries has increased exponentially over the past decade. 4 The asymmetric reduction of prochiral ketones using carbonyl reductase (CRED) biocatalysts is becoming a straightforward approach for the synthesis of chiral alcohols. 5 CRED enzymes have received much attention by both academic and industrial groups, and many cloned reductases have been reported. 6 These biocatalysts have now received acceptance in the chemist's tool-box due to their high specificity and enantioselectivity under physiological conditions. For these systems to be used at scale, CRED enzymes require cofactor recycling, and several methods have been employed at multikilogram scale including glucose dehydrogenase (GDH) 7 and isopropyl alcohol (IPA) (Scheme 2). 8 The use of IPA as the cofactor recycle system has the added advantage that pH control is not required and can also be used as cosolvent.Due to their versatility, CRED enzymes must now be considered as an accepted tool in organic synthesis for the preparation of chiral alcohol intermediates. ' RESULTS AND DISCUSSIONAlmac's CESK 5000 carbonyl reductase enzyme kit was screened for the reduction of 4-tert-butylcyclohexanone. A selection of the screening results is shown in Table 1.The screening results show that both the cis-and trans-isomers are available in high yield and...

Colourimetric assays were used to measure the activities of six key hydrolases endogenous to barley: β‐glucanase, xylanase, cellulase, α‐amylase, beta‐amylase and limit dextrinase. The analysed barley malt samples were previously characterised by 27 conventional malt quality descriptors. Correlations between enzymatic activities and brewing parameters such as extract yield, fermentability, viscosity and filterability were investigated. A single extraction protocol for all six hydrolases was optimised and used for multi‐enzyme analysis using fully automatable assay formats. A regression analysis between malt parameters was undertaken to produce a relationship matrix linking enzyme activities and conventional malt quality descriptors. This regression analysis was used to inform a multi‐linear regression approach to create predictive models for extract yield, apparent attenuation limit, viscosity and filterability using the Small‐scale Wort rapId Filtration Test (SWIFT) and two different mashing protocols – Congress and a modified infusion mash at 65°C (MIM 65°C). It was observed that malt enzyme activities displayed significant correlations with the analysed brewing parameters. Both starch hydrolases and cell wall hydrolase activities together with modification parameters (i.e. Kolbach index) were found to be highly correlated with extract yield and apparent attenuation limit. Interestingly, it was observed that xylanase activity in malts was an important predictor for wort viscosity and filterability. It is envisaged that the automatable measurement of enzyme activity could find use in plant breeding progeny selection and for routine assessment of the functional brewing performance of malt batches. This analytical approach would also contribute to brewing process consistency, product quality and reduced processing times. © 2019 The Institute of Brewing & Distilling