Engineering of a borneol dehydrogenase from P. putida for the enzymatic resolution of camphor

Several thousand different terpenoid structures are known so far, and many of them are interesting for applications as pharmaceuticals, flavors, fragrances, biofuels, insecticides, or fine chemical intermediates. One prominent example is camphor, which has been utilized since ancient times in medical applications. Especially (−)-camphor is gaining more and more interest for pharmaceutical applications. Hence, a commercial reliable source is needed. The natural sources for (−)-camphor are limited, and the oxidation of precious (−)-borneol would be too costly. Hence, synthesis of (−)-camphor from renewable alpha-pinene would be an inexpensive alternative. As the currently used route for the conversion of alpha-pinene to camphor produces a mixture of both enantiomers, preferably catalytic methods for the separation of this racemate are demanded to yield enantiopure camphor. Enzymatic kinetic resolution is a sustainable way to solve this challenge but requires suitable enzymes. In this study, the first borneol dehydrogenase from Pseudomonas sp. ATCC 17453, capable of catalyzing the stereoselective reduction of camphor, was examined. By using a targeted enzyme engineering approach, enantioselective enzyme variants were created with E-values > 100. The best variant was used for the enzymatic kinetic resolution of camphor racemate, yielding 79% of (−)-camphor with an ee of > 99%. Key points • Characterization of a novel borneol dehydrogenase (BDH) from P. putida. • Development of enantioselective BDH variants for the reduction of camphor. • Enzymatic kinetic resolution of camphor with borneol dehydrogenase. Graphical abstract

Overview: Enzyme-catalyzed Enantioselective Biotransformation of Chiral Active Compounds Used in Hypertension Treatment

Enzymatic kinetic resolution is one of the methods which allows for the synthesis of enantiomerically pure various active pharmaceutical ingredients. In contrast to chemical routes, enzymatic reactions have characteristics, including mild reaction conditions, a few byproducts, and relatively high activity of the used enzymes. β-adrenolytic drugs are widely used in the treatment of hypertension and cardiovascular disorders. Due to the fact that β- blockers possess an asymmetric carbon atom in their structure, they are presented in two enantiomeric forms. It was reported by many studies that only the (S)-enantiomers of these drugs possess the desired therapeutic effect, whereas the administration of the racemate may cause dangerous side effects, such as bronchoconstriction or diabetes. Nevertheless, β- blockers are still commercially available drugs mainly used in medicine as racemates, whereas there are several methods that are widely used in order to obtain enantiomerically pure compounds.

The production of enantiomerically pure 1-phenylethanol by enzymatic kinetic resolution method using response surface methodology

As the enantiomers of 1-phenylethanol are valuable intermediates in several industries, the lipase catalyzed kinetic resolution of (R,S) -1-phenylethanol is a relevant research topic. In this study, the goal was to determine the optimum reaction parameters to produce enantiomerically pure 1-phenylethanol by lipase (Novozyme 435) catalyzed kinetic resolution using response surface methodology (RSM). Reactions were performed with 40–400 mM (R,S)-1-phenylethanol, 120–1200 mM vinyl acetate and 2–22 mg/ mL biocatalyst concentrations (BCL), at 20–60 °C and with a stirring rate of 50–400 rpm for 5–120 min. The samples were analyzed using high performance liquid chromatography (HPLC) with a Chiralcel OB column. Optimum reaction parameters to reach 100% enantiomeric excess for the substrate (ees) were determined as follows: substrate concentration (Cs): 240 mM, BCL: 11 mg/mL, at 42 °C with a reaction time of 75 min. Model validation was performed using these conditions and ees was calculated as 100%, which indicates the predicted model was efficient and accurate. When compared to the literature, it was observed that the reaction time decreased significantly. This is an important result considering the industrial scale perspective.