Crosslinked Aggregates of Fusion Enzymes in Microaqueous Organic Media

Abstract

Baeyer-Villiger monooxygenases (BVMOs) are attractive for selectively oxidizing various ketones using oxygen into valuable esters and lactones. However, the application of BVMOs is restrained by cofactor dependency and enzyme instability combined with water-related downsides such as low substrate loading, low oxygen capacity, and water-induced side reactions. Herein, we described a redox-neutral linear cascade with in-situ cofactor regeneration catalyzed by fused alcohol dehydrogenase and cyclohexanone monooxygenase in aqueous and microaqueous organic media. The cascade conditions have been optimized regarding substrate concentrations as well as the amounts of enzymes and cofactors with the Design of Experiments (DoE). The carrier-free immobilization technique, crosslinked enzyme aggregates (CLEAs), was applied to fusion enzymes. The resultant fusion CLEAs were proven to function in microaqueous organic systems, in which the enzyme ratios, water contents (0.5-5 vol. %), and stability have been systematically studied. The fusion CLEAs showed promising operational (up to 5 cycles) and storage stability.

Metal Affinity Fusion Enzyme Immobilization: Batch Process Showcase for Cascading Alcohol Oxidation and Bayer–Villiger Oxidation in Microaqueous Media

“…Regarding the substrate concentration (Figure c), a lower concentration leads to a higher reaction rate, as expected, since it is well-known that most of the BVMO enzymes suffer from substrate inhibition. , High yields were obtained for the reactions with 30, 60, and 125 mM CHL, resulting in 23, 48, and 99 mM ECL with 77, 80, and 79% yields, respectively, revealing 60 mM as the best one. For the higher concentrations, the yields drop significantly to 40% (100 mM ECL) and 13% (63 mM ECL) when using 250 and 500 mM CHL.…”

“…The coimmobilization of single enzymes is not considered due to an uncontrollable competitive immobilization. For the linear cascade, the previous study by the authors reported the optimal reaction conditions regarding substrate and cofactor concentrations, and temperature, which was further used in this work. Since the oxidation of CHO by CHMO is the rate-limiting step of the linear cascade, all reactions were performed with the same units of Tm CHMO .…”

“…On all the basis, the combination of fusion enzymes, immobilization techniques, and microaqueous media represents a prospective strategy for process intensification. Recently, the fused enzyme of alcohol dehydrogenase from Thermoanaerobacter brockii ( Tb ADH, EC 1.1.1.2) and CHMO from Thermocrispum municipale ( Tm CHMO, EC 1.14.13.22), , namely, Tb–Tm, has been applied for the linear cascade in microaqueous media in the form of cross-linked enzyme aggregates (CLEAs), resulting in multiple reuses (up to five cycles) of Tb–Tm . However, the practicality of CLEA preparations’ handling was limited while running repetitive batches.…”

“…One of the most known guidelines for chemical process development is the 12 principles of Green Chemistry, most of which a typical enzymatic biotransformation can fulfill. , This is especially noticeable in oxidative reactions, in which the chemical processes tend to use toxic metals as catalysts under high temperatures or high pressures. , Alternatively, enzymes can catalyze selective oxidations under mild conditions, representing one of the most important (bio)­transformations in the synthesis of organic chemicals without generating massive amounts of waste. − In particular, cyclohexanone monooxygenases (CHMOs), one of the most studied enzymes belonging to the family of Bayer–Villiger monooxygenases (BVMOs), show a great potential for industrial uses. − For instance, they have been employed for the synthesis of ε-caprolactone (ECL), a nylon-6 precursor with a demand of more than a million tons/year for diverse industrial interests. − Although the biocatalytic reaction is well-known, there are still challenges to overcome to enable its feasibility on a technical scale. The most critical challenges include overall enzyme instability and cofactor dependence, as well as limited oxygen accessibility due to low oxygen solubility, substrate and product inhibition, and autohydrolysis of lactone products in aqueous media. , …”

Cyclohexene oxide CA, a derivative of zeylenone, exhibits anti-cancer activity in glioblastoma by inducing G0/G1 phase arrest through interference with EZH2

“…Cell lysates were harvested, washed, and lysed using RIPA buffer and then centrifuged at 13,000 rpm for 45 min at 4°C ( Vernet et al, 2023 ). The protein concentration was determined using the BCA Protein Assay Kit and diluted in loading buffer (4X).…”