This study implements a convenient microreactor for biocatalysis with enzymes immobilized on magnetic nanoparticles (MNPs). The enzyme immobilized onto MNPs by adsorption or by covalent bonds was lipase B from Candida antarctica (CaLB). The MNPs for adsorption were obtained by covering the magnetite core with a silica shell and later with hexadecyltrimethoxysilane, while for covalent immobilization, the silica-covered MNPs were functionalized by a layer forming from mixtures of hexadecyl- and 3-(2-aminoethylamino)propyldimethoxymethylsilanes in 16:1 molar ratio, which was further activated with neopentyl glycol diglycidyl ether (NGDE). The resulting CaLB-MNPs were tested in a convenient continuous flow system, created by 3D printing to hold six adjustable permanent magnets beneath a polytetrafluoroethylene tube (PTFE) to anchor the MNP biocatalyst inside the tube reactor. The anchored CaLB-MNPs formed reaction chambers in the tube for passing the fluid through and above the MNP biocatalysts, thus increasing the mixing during the fluid flow and resulting in enhanced activity of CaLB on MNPs. The enantiomer selective acylation of 4-(morpholin-4-yl)butan-2-ol (±)-1, being the chiral alcohol constituent of the mucolytic drug Fedrilate, was carried out by CaLB-MNPs in the U-shape reactor. The CaLB-MNPs in the U-shape reactor were compared in batch reactions to the lyophilized CaLB and to the CaLB-MNPs using the same reaction composition, and the same amounts of CaLB showed similar or higher activity in flow mode and superior activity as compared to the lyophilized powder form. The U-shape permanent magnet design represents a general and easy-to-access implementation of MNP-based flow microreactors, being useful for many biotransformations and reducing costly and time-consuming downstream processes.
Activity of diisopropyl malonate (2) as a novel acylating agent was investigated in kinetic resolution (KR) of various racemic amines [(±)-1a-d] catalyzed by lipase B from Candida antarctica. Diisopropyl malonate (2) proved to be effective acylating agent with four racemic amines [(±)-2-aminoheptane, (±)-1-methoxy-2-propylamine, (±)-1-phenylethylamine and (±)-4-phenylbutan-2-amine; (±)-1a-d, respectively] selected for this study. The lipase-catalyzed acylation of the amines (±)-1a-d with 2 proceeded with good conversions (44.9–52.1%) and provided the expected (R)-amides [(R)-3a-d] in moderate to excellent yields (51–98%) with high enantiomeric excess (ee(R)-3a-d 92.0–99.9%) after 4 h reaction time under mild reaction conditions in batch mode. The best conversion (50%) combined with high enantiomeric purity (ee(R)-2d > 99%ee) was achieved in the KR from racemic 2-aminoheptane (±)-1a. The four novel (R)-amides [(R)-3a-d] were isolated and properly characterized.
The application of enzyme-based therapies has received significant attention in modern drug development. Lipases are one of the most versatile enzymes that can be used as therapeutic agents in basic skin care and medical treatment related to excessive sebum production, acne, and inflammation. The traditional formulations available for skin treatment, such as creams, ointments or gels, are widely applied; however, their use is not always accompanied by good drug penetration properties, stability, or patient adherence. Nanoformulated drugs offer the possibility of combining enzymatic and small molecule formulations, making them a new and exciting alternative in this field. In this study polymeric nanofibrous matrices made of polyvinylpyrrolidone and polylactic acid were developed, entrapping lipases from Candida rugosa and Rizomucor miehei and antibiotic compound nadifloxacin. The effect of the type of polymers and lipases were investigated, and the nanofiber formation process was optimized to provide a promising alternative in topical treatment. Our experiments have shown that entrapment by electrospinning induced two orders of magnitude increase in the specific enzyme activity of lipases. Permeability investigations indicated that all lipase-loaded nanofibrous masks were capable of delivering nadifloxacin to the human epidermis, confirming the viability of electrospinning as a formulation method for topical skin medications.
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