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BACKGROUND The oxy‐functionalization of non‐activated carbon bonds by the bacterial cytochrome P450 BM3 from Bacillus megaterium, presents a promising field in biosynthesis and it has gained much interest in recent decades. Nevertheless, the need for the expensive cofactor NADPH, together with low operational stability of the enzyme have made the implementation of this biocatalyst unfeasible in most cases for industry. RESULTS P450 BM3 and glucose dehydrogenase (GDH), as a cofactor regeneration enzyme, were successfully co‐immobilized obtaining a bi‐functional self‐sufficient oxidative biocatalyst. First, a broad screening of 13 different supports was carried out. Five selected agaroses with three different functionalities (epoxy, amine and aldehyde) were studied and their immobilization processes optimized. Finally, P450 BM3 and GDH were co‐immobilized on those supports showing the best performance for P450 BM3 immobilization: epoxy‐agarose (epoxy‐agarose‐UAB) presenting 83% and 20% retained activities respectively; AMINO‐agarose presenting 28% and 25%, and Lentikats® with which both enzymes retained 100% of the initial activity. Furthermore, the re‐utilization of the self‐sufficient immobilized derivatives was tested in five repeated cycles. CONCLUSIONS P450 BM3 and GDH have been successfully immobilized on three supports and their re‐usability has been tested in a model reaction. It represents a step forward for future P450 BM3 industrial implementations. © 2018 Society of Chemical Industry

Optimization and scale up of the Baeyer-Villiger oxidation of 3,3,5-trimethyl-cyclohexanone to trimethyl-ε-caprolactones (CHL) was studied in order to demonstrate this technology on 100 L pilot plant scale. The reaction was catalyzed by a cyclohexanone monooxygenase from Thermocrispum municipale (TmCHMO) that utilizes the costly redox cofactor NADPH, which was regenerated by a glucose dehydrogenase (GDH). As a first stage, different cyclohexanone monooxygenase formulations were tested: cell free extract, whole cells, fermentation broth and sonicated fermentation broth. Using broth resulted in the highest yield (63%) and required the least biocatalyst preparation effort. Two commercial glucose dehydrogenases (GDH-105 and GDH-01) were evaluated resulting in similar performances. Substrate dosing rate and biocatalyst loadings were optimized. At 30 mL scale, the best conditions were found when 30 mM h-1 dosing rate, 10% (v/v) cyclohexanone monooxygenase broth and 0.05% (v/v) of glucose dehydrogenase (GDH-01) liquid enzyme formulation were applied. These same conditions (with oxygen instead of air) were applied at 1 liter scale with 92% conversion, achieving a specific activity of 13.3 U g-1 cell wet weight (cww), a space time yield of 3.4 g CHL L-1 h-1 and a biocatalyst yield of 0.83 g CHL g-1 cww. A final 100 liter demonstration was performed in a pilot plant facility. After 9 hours, the reaction reached 85% conversion, 12.8 U g-1 cww, a space time yield of 2.7 g L-1 h-1 and a biocatalyst yield of 0.60 g CHL g-1 cww. The extraction of product resulted in 2.58 kg isolated final product. The overall isolated CHL yield was 76% (distal lactone 47% and proximal lactone 53%).

The monoterpenoid α-isophorone is sourced from the available and renewable plant dry matter, as well as a waste recovery operation from acetone. This compound, can be hydroxylated to 4-hydroxy-isophorone which is the main precursor for the synthesis of ketoisophorone. On its turn, ketoisophorone is a key intermediate for the production of carotenoids and Vitamin E. Here, the enzymatic oxidation of 4-hydroxy-isophorone to ketoisophorone is demonstrated employing an alcohol dehydrogenase (ADHaa) from Artemisia annua and a NADPH oxidase (NOX), as a cofactor regeneration enzyme. After 24 h of reaction and an initial substrate concentration of 50 mM, 95.7 % yield and a space time yield of 6.52 g L À 1 day À 1 could be obtained. Furthermore, the immobilization of the alcohol dehydrogenase was studied on 17 different supports. An epoxy-functionalized agarose resulted in the highest metrics, 100 � 0% immobilization yield and 58.2 � 3.5 % retained activity. Finally, the immobilized ADHaa was successfully implemented in 4 reaction cycles (96 h operation) presenting a biocatalyst yield of 23.4 g product g À 1 of enzyme. It represents a 2.5-fold increase compared with the reaction with soluble enzymes.

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