Search Results (22)

Due to their remarkable biological and pharmacological activities such as antibacterial, antifungal, anticoagulant, antioxidant, anticancer, and anti-inflammatory properties, synthesis of coumarins and their derivatives has attracted considerable attention in research and development among both organic and medicinal chemists. In this paper, we demonstrated for the first time a two-step tandem enzymatic synthesis of coumarin bioamide derivatives through sustainable continuous-flow technology. Salicylaldehyde and dimethyl malonate were firstly reacted to obtain coumarin carboxylate methyl derivatives, which were then reacted with various biogenic amines at 50 °C for about 40 min under the catalysis of lipase TL IM from Thermomyces lanuginosus to obtain coumarin bioamide derivatives in continuous-flow reactors. Reaction parameters such as reaction solvent, reaction catalyst type, reactant ratio, residence time, reaction temperature and comparative experiments with traditional batch process were studied. Ideal product yields (62.7–87.1%) were obtained. Environmentally friendly methanol was applied as the reaction medium. Substantially shorter reaction times as well as a significant increase in the product yield were obtained as compared to the batch process. This innovative approach provides a promising green, efficient and rapid synthesis strategy for pharmaceutical synthesis and further research on novel coumarin bioamide derivatives. Full article

The enhanced and direct immobilization of the enzyme horseradish peroxidase on poly(methyl methacrylate) (PMMA) microchannel surfaces to create a miniaturized enzymatic reactor for the biocatalytic oxidation of phenols is demonstrated. Enzyme immobilization occurs by physical adsorption after oxygen plasma treatment, which micro-nanotextures the PMMA surfaces. A five-fold enhancement in immobilized enzyme activity was observed, attributed to the increased surface area and, therefore, to a higher quantity of immobilized enzymes compared to an untreated PMMA surface. The enzymatic reaction yield reached 75% using a flow rate of 2.0 μL/min for the reaction mixture. Additionally, the developed microreactor was reused more than 16 times without affecting the enzymatic conversion yield. These results demonstrate the potential of microchannels with plasma micro/nanotextured surfaces for the rapid and facile fabrication of microfluidic enzymatic microreactors with enhanced catalytic activity and stability. Full article

Microfluidic devices have attracted much attention in the current day owing to the unique advantages they provide. However, their application for industrial use is limited due to manufacturing limitations and high cost. Moreover, the scaling-up process of the microreactor has proven to be difficult. Three-dimensional (3D) printing technology is a promising solution for the above obstacles due to its ability to fabricate complex structures quickly and at a relatively low cost. Hence, combining the advantages of the microscale with 3D printing technology could enhance the applicability of microfluidic devices in the industrial sector. In the present work, a 3D-printed single-channel immobilized enzyme microreactor with a volume capacity of 30 μL was designed and created in one step via the fused deposition modeling (FDM) printing technique, using polylactic acid (PLA) as the printing material. The microreactor underwent surface modification with chitosan, and β-glucosidase from Thermotoga maritima was covalently immobilized. The immobilized biocatalyst retained almost 100% of its initial activity after incubation at different temperatures, while it could be effectively reused for up to 10 successful reaction cycles. Moreover, a multi-channel parallel microreactor incorporating 36 channels was developed, resulting in a significant increase in enzymatic productivity. Full article

Microreactors have become an efficient tool for many enzymatic reactions because the laminar fluid flow within the microchannel enables precise process control, rapid mixing, and short residence time. This paper provides a systematic overview of the application of reaction kinetics and the mathematical modeling of enzymatic processes in microreactors. Rapid heat and mass transfer and a high surface-to-volume ratio are usually the reasons why reactions in microchannels proceed faster and with higher yields and productivity compared to conventional macroreactors. Since there are no radial diffusion limitations, microreactors are also an effective tool for determining the kinetic parameters of enzyme-catalyzed reactions. By eliminating the mass transfer effect on the reaction rate, the kinetics estimated in the microreactor are closer to the intrinsic kinetics of the reaction. In this review, the advantages and disadvantages of using microreactors are highlighted and the potential of their application is discussed. Advances in microreactors result in process intensification and more efficient biocatalytic processes in line with the advantages offered by the application of microreactors, such as (i) higher yields, (ii) a cleaner and improved product profile, (iii) scale-independent synthesis, (iv) increased safety, and (v) the constant quality of the output product through (vi) accelerated process development. Furthermore, microreactors are an excellent tool for kinetic studies under specified mass transfer conditions, enhancing the capabilities of other methods. Full article

Benzimidazole scaffolds became an attractive subject due to their broad spectrum of pharmacological activities. In this work, a methodology was developed for the synthesis of N-substituted benzimidazole derivatives from benzimidazoles and α, β-unsaturated compounds (acrylonitriles, acrylate esters, phenyl vinyl sulfone) catalyzed by lipase TL IM from Thermomyces lanuginosus in continuous-flow microreactors. Investigations were conducted on reaction parameters such as solvent, substrate ratio, reaction temperature, reactant donor/acceptor structures, and reaction time. The transformation is promoted by inexpensive and readily available lipase in methanol at 45 °C for 35 min. A wide range of β-amino sulfone, β-amino nitrile, and β-amino carbonyl compounds were efficiently and selectively synthesized in high yields (76–97%). All in all, a microfluidic biocatalysis system was applied to the synthesis of N-substituted benzimidazole derivatives, and could serve as a promising fast synthesis strategy for further research to develop novel and highly potent active drugs. Full article

In this study, 3D printing technology was exploited for the development of immobilized enzyme microreactors that could be used for biocatalytic processes in Deep Eutectic Solvent (DES)-based media. 3D-printed polylactic acid (PLA) microwell plates or tubular microfluidic reactors were modified with polyethylenimine (PEI) and lipase from Candida antarctica (CALB) was covalently immobilized in the interior of each structure. DESs were found to have a negligible effect on the activity and stability of CALB, and the system proved highly stable and reusable in the presence of DESs for the hydrolysis of p-nitrophenyl butyrate (p-NPB). A kinetic study under flow conditions revealed an enhancement of substrate accessibility in the presence of Betaine: Glycerol (Bet:Gly) DES, while the system was not severely affected by diffusion limitations. Incubation of microreactors in 100% Bet:Gly preserved the enzyme activity by 53% for 30 days of storage at 60 °C, while the buffer-stored sample had already been deactivated. The microfluidic enzyme reactor was efficiently used for the trans-esterification of ethyl ferulate (EF) with glycerol towards the production of glyceryl ferulate (GF), known for its antioxidant potential. The biocatalytic process under continuous flow conditions exhibited 23 times higher productivity than the batch reaction system. This study featured an effective and robust biocatalytic system with immobilized lipase that can be used both in hydrolytic and synthetic applications, while further optimization is expected to upgrade the microreactor system performance. Full article

While a few derivatives of cinnamamides exhibited anti-inflammatory and/ or analgesic activity, in this study, we developed a highly efficient method for the synthesis of cinnamamides from methyl cinnamates and phenylethylamines catalyzed by Lipozyme^® TL IM in continuous-flow microreactors. The reaction parameters and broad substrate range of the new method was studied. Maximum conversion (91.3%) was obtained under the optimal condition of substrate molar ratio of 1:2 (methyl 4-chlorocinnamate: phenylethylamine) at 45 $°\mathrm{C}$ for about 40 min. The remarkable features of this work include short residence time, mild reaction conditions, easy control of the reaction process, and that the catalyst can be recycled or reused, which provide a rapid and economical strategy for the synthesis and design of cinnamamide derivatives for further research on drug activity. Full article

Oceans possess tremendous diversity in microbial life. The enzymatic machinery that marine bacteria present is the result of extensive evolution to assist cell survival under the harsh and continuously changing conditions found in the marine environment. Several bacterial cells and enzymes are already used at an industrial scale, but novel biocatalysts are still needed for sustainable industrial applications, with benefits for both public health and the environment. Metagenomic techniques have enabled the discovery of novel biocatalysts, biosynthetic pathways, and microbial identification without their cultivation. However, a key stage for application of novel biocatalysts is the need for rapid evaluation of the feasibility of the bioprocess. Cultivation of not-yet-cultured bacteria is challenging and requires new methodologies to enable growth of the bacteria present in collected environmental samples, but, once a bacterium is isolated, its enzyme activities are easily measured. High-throughput screening techniques have also been used successfully, and innovative in vitro screening platforms to rapidly identify relevant enzymatic activities continue to improve. Small-scale approaches and process integration could improve the study and development of new bioprocesses to produce commercially interesting products. In this work, the latest studies related to (i) the growth of marine bacteria under laboratorial conditions, (ii) screening techniques for bioprospecting, and (iii) bioprocess development using microreactors and miniaturized systems are reviewed and discussed. Full article

Coumarin carboxamide derivatives are important building blocks for organic synthesis and chemical biology due to their excellent biopharmaceutical properties. In this paper, we demonstrate for the first time a two-step enzymatic synthesis of coumarin carboxamide derivatives. Salicylaldehyde and dimethyl malonate were reacted to obtain coumarin carboxylate methyl derivatives, which were then reacted with various amines under the catalysis of lipase TL IM from Thermomyces lanuginosus to obtain coumarin carboxamide derivatives in continuous flow reactors. We studied various reaction parameters on the yields. The important features of this method include mild reaction conditions, a short reaction time (40 min), reduced environmental pollution, higher productivity (STY = 31.2941 g L⁻¹ h⁻¹) and enzymes being relatively easy to obtain. Full article

The enzymatic conversion of biomass-derived compounds represents a key step in the biorefinery flowsheet, allowing low-temperature high-efficiency reactions. β-Glucosidases are able to hydrolyze cellobiose into glucose. Wrinkled silica nanoparticles (WSNs) were demonstrated to be a good support for the immobilization of β-glucosidases, showing better performance than free enzymes in batch reaction; on the other hand, immobilized enzyme microreactors (IEMs) are receiving significant attention, because small quantities of reagents can be used, and favorable heat and mass transfer can be achieved with respect to conventional batch systems. In this work, we prepared, characterized, and tested structured enzymatic reactor compounds by a honeycomb monolith, a WSN washcoat, and β-glucosidases as the active phase. Powder and structured materials were characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), N₂ physisorption, thermogravimetric analysis (TGA), and Fourier-transform infrared spectroscopy (FT-IR). Structured catalysts were tested under both batch and continuous flow reaction conditions and compared to powder catalysts (batch reaction). The WSN washcoat was attached well onto the monolith walls, as suggested by the negligible weight loss after ultrasound treatment; the WSNs preserved their shape, porosity, and individual nature when deposited onto the monolith walls. The immobilized enzyme microreactors proved to be very efficient in hydrolysis of cellobiose to glucose, showing a complete conversion under continuous flow reaction at a batch-equivalent contact time equal to 120 min vs. 24 h obtained in the batch experiments. The apparent K_M value showed a 20-fold decrease with respect to the batch process, due to the absence of external diffusive transport limitations. Full article

Soybean peroxidase is an enzyme extracted from soybean seed hulls. In the presence of hydrogen peroxide, the enzyme has the potential to catalyze the biodegradation of toxic substances like chlorophenols. For this reason, its use in wastewater treatment processes is environmentally friendly since the enzyme can be obtained from a renewable and abundant raw material. In this work, enzymatic biodegradation of 2,4,6-trichlorophenol performed by soybean peroxidase in a microreactor was studied experimentally and theoretically. The experimental data set was obtained with a volume of 250 μL by using different soybean peroxidase concentrations and different reaction times. The fluid dynamics of the microreactor was modeled as well, using ANSYS CFX. The simulations exhibited secondary flows, which enhanced mixing. Although the laminar flow was developed, it can be assumed to be a well-mixed medium. The kinetic data were evaluated through a mechanistic model, the modified bi-bi ping-pong model, which is adequate to represent the enzymatic degradation using peroxidases. The model was composed of an initial value problem for ordinary differential equations that were solved using MATLAB. Some kinetic constants were estimated using the least square function. The results of the model fit well the experimental data. Full article

The interest in large-scale integrated (LSI) microfluidic systems that perform high-throughput biological and chemical laboratory investigations on a single chip is steadily growing. Such highly integrated Labs-on-a-Chip (LoC) provide fast analysis, high functionality, outstanding reproducibility at low cost per sample, and small demand of reagents. One LoC platform technology capable of LSI relies on specific intrinsically active polymers, the so-called stimuli-responsive hydrogels. Analogous to microelectronics, the active components of the chips can be realized by photolithographic micro-patterning of functional layers. The miniaturization potential and the integration degree of the microfluidic circuits depend on the capability of the photolithographic process to pattern hydrogel layers with high resolution, and they typically require expensive cleanroom equipment. Here, we propose, compare, and discuss a cost-efficient do-it-yourself (DIY) photolithographic set-up suitable to micro-pattern hydrogel-layers with a resolution as needed for very large-scale integrated (VLSI) microfluidics. The achievable structure dimensions are in the lower micrometer scale, down to a feature size of 20 µm with aspect ratios of 1:5 and maximum integration densities of 20,000 hydrogel patterns per cm². Furthermore, we demonstrate the effects of miniaturization on the efficiency of a hydrogel-based microreactor system by increasing the surface area to volume (SA:V) ratio of integrated bioactive hydrogels. We then determine and discuss a correlation between ultraviolet (UV) exposure time, cross-linking density of polymers, and the degree of immobilization of bioactive components. Full article

Biodiesel, which was earlier used only as an alternative fuel, is now an indispensable component of commercial diesel. Conventional production processes are unable to cope with the increasing demand for biodiesel, and therefore more and more work is being done to intensify the existing processes. The intensification of the biodiesel production process, taking into account the environmental and economic factors, is based on increasing productivity. One way to achieve that is by reducing the volume of production units. The application of the enzymatic reaction path, while reducing the volume of process equipment to the micro-level, has significantly magnified the productivity of the biodiesel production process, which is primarily due to better mass transfer in microsystems. Additional breakthrough is the use of deep eutectic solvents (DES) instead of buffers for enzyme stabilization. In this study, a lipase from Thermomyces lanuginosus (TlL) (both commercial and produced by solid-state fermentation) was used as a catalyst for biodiesel production. Edible and waste sunflower oil, as well as methanol, were used as substrates. The reaction mediums were buffer and DES. The transesterification reaction was carried out in a batch reactor and the emphasis was made on different microreactor configurations. The highest yield of 32% for residence time of only τ = 30 min was obtained in the microreactor system with an emulsion of waste oil and a commercial enzyme suspended in a buffer. This indicates that enzymatic transesterification could be a valuable reaction path for dealing with waste oils. Furthermore, biodiesel synthesis in DES showed somewhat lower yields, but by increasing the water content in the system, the reaction could prove much better results. In the end, the effects of reaction conditions on the volumetric productivity of the process were analyzed. Full article

A continuous-flow procedure for the synthesis of β-amino acid esters has been developed via lipase-catalyzed Michael reaction of various aromatic amines with acrylates. Lipase TL IM from Thermomyces lanuginosus was first used to catalyze Michael addition reaction of aromatic amines. Compared with other methods, the salient features of this work include green reaction conditions (methanol as reaction medium), short residence time (30 min), readily available catalyst and a reaction process that is easy to control. This enzymatic synthesis of β-amino acid esters performed in continuous-flow microreactors is an innovation that provides a new strategy for the fast biotransformation of β-amino acid esters. Full article

Although enzymatic microbioreactors have recently gained lots of attention, reports on the use of whole cells as biocatalysts in microreactors have been rather modest. In this work, an efficient microreactor with permeabilized Saccharomyces cerevisiae cells was developed and used for continuous biotransformation of fumaric into industrially relevant L-malic acid. The immobilization of yeast cells was achieved by entrapment in a porous structure of various hydrogels. Copolymers based on different ratios of sodium alginate (SA) and polyvinyl alcohol (PVA) were used for hydrogel formation, while calcium chloride and boric or phenylboronic acid were tested as crosslinking agents for SA and PVA, respectively. The influence of hydrogel composition on physico-chemical properties of hydrogels prepared in the form of thin films was evaluated. Immobilization of permeabilized S. cerevisiae cells in the selected copolymeric hydrogel resulted in up to 72% retained fumarase activity. The continuous biotransformation process using two layers of hydrogels integrated into a two-plate microreactor revealed high space time yield of 2.86 g/(L·h) while no activity loss was recorded during 7 days of continuous operation. Full article