Solvent-free System Research Articles

Chemically vs Enzymatically Synthesized Polyglycerol-Based Esters: A Comparison between Their Surfactancy.

Polyglycerol fatty acid esters (PGFAEs) are gaining interest in several industrial sectors due to their excellent surfactant properties and their wide range of hydrophilic-lipophilic balance (HLB) values. Moreover, they can be prepared from renewable resources, i.e., fatty acids and glycerol. In this study, polyglycerol-2 stearic acid esters (PG2SAEs) were synthesized by the enzymatic esterification of polyglycerol-2 (PG2) and stearic acid (SA) using the immobilized lipase Novozym 435 as a biocatalyst in a solvent-free system. Reaction conditions, i.e., temperature (80 °C), reactant ratio (1:1.8), and enzyme loading (2.7% w/w), were finely optimized; furthermore, biocatalyst recycling was studied by assessing the residual activity of the lipase after each reaction cycle, up to 20 times. The composition of the enzymatically synthesized products (E) was roughly evaluated by chromatographic methods and mass spectrometry and compared with that of the esters obtained by acid-catalyzed esterification (C). Then, the surfactant properties of the prepared polyglycerol-based surfactants were investigated by interfacial tension studies. Specifically, the emulsifying capacity and stability and the rheological behavior of O/W emulsions prepared in the presence of E were deeply investigated in comparison with those of the chemically synthesized and commercially available product C.

Green strategies using solvent-free biodegradable membranes in microfluidic devices. Liquid phase microextraction and electromembrane extraction

In this work, a novel solvent-free microfluidic method based liquid phase microextraction has been proposed for the first time. A comprehensive study of liquid phase microextraction (LPME) and electromembrane extraction (EME) implemented in microfluidic formats has been carried out to investigate the efficiency of biodegradable membranes (such as agarose) without organic solvent to develop fully environmental microfluidic methods. For this study, non-polar and polar basic compounds (five) were selected as model analytes and different agarose membrane compositions were synthesized and tested with and without organic solvent (solvent-free). Under optimal experimental conditions, the extraction efficiencies obtained using solvent-free LPME-chip devices were similar to the ones obtained using solvent-free EME-chip devices at very low voltages (0.25 V), however, LPME microfluidic format was selected due to its simplicity. The proposed green microfluidic device was successfully applied in urine samples with recoveries between 80 and 93% for all analytes and relative standard deviation below 7% for all analytes. Results were compared with experiments previously conducted using conventional (polypropylene) membranes, observing that solvent-free microfluidic systems based on biodegradable solid support materials have proven to be an attractive alternative and offered the same advantages in terms of membrane stability allowing consecutive extractions compared to supported liquid membranes (SLM) microfluidic methods.

Microwave-assisted enzymatic synthesis of geraniol esters in solvent-free systems: optimization of the reaction parameters, purification and characterization of the products, and biocatalyst reuse

Various geraniol esters act as insect pheromones and display pharmacological activities, especially as neuroprotective agents. Therefore, the search for synthetic strategies alternative to traditional chemical synthesis could help designing ecofriendly routes for the preparation of such bioactive compounds. Hence, this work aims at the microwave-assisted enzymatic synthesis of geranyl esters in solvent-free systems. The process variables were optimized for the synthesis of geranyl acetoacetate, achieving 85% conversion after 60 min using a 1:5 substrates molar ratio (ester to geraniol), 80 °C and 8.4% of Lipozyme 435 lipase without removal of the co-produced methanol. On the other hand, a 95% conversion was reached after 30 min using 1:6 substrates molar ratio, 70 °C and 7% lipase in the presence of 5Å molecular sieves for the methanol capture. In addition, the lipase showed good reusability, maintaining the same activity for five reaction cycles. Finally, under the above optimized conditions, other geraniol esters were successfully synthetized such as the geranyl butyrate (98%), geranyl hexanoate (99%), geranyl octanoate (98%), and geranyl (R)-3-hydroxybutyrate (56%). These results demonstrate the microwave-assisted lipase-catalyzed transesterification in a solvent-free system as an excellent and sustainable catalytic methodology to produce geraniol esters.Graphical

β-Sitosterol Oleate Synthesis by Candida rugosa Lipase in a Solvent-Free Mini Reactor System: Free and Immobilized on Chitosan-Alginate Beads

Candida rugosa lipase (CRL) was immobilized by the ionic gelling technique using alginate and chitosan as encapsulating agents. An immobilization yield of 99% and an immobilization efficiency of 51% were obtained. Maximum hydrolytic activity for free and immobilized CRL was detected at 40 °C and for synthesis activity at 35 °C. The optimum pH for immobilized and free CRL hydrolysis activity was 8.0. The Vmax obtained for the hydrolysis reaction was higher for free CRL (4121.4 μmol/min/g) compared to immobilized CRL (2359.13 μmol/min/g). A Vmax of 2.24 μmol/min/g was detected for the synthetic activity of free CRL. The Km obtained for the hydrolysis reaction was higher (660.02 μmol/L) for immobilized CRL than for free CRL (403.06 μmol/L). For the synthetic activity, a Km of 234.44 μmol/L was calculated. The conversion of β-sitosterol oleate ranged from 80.85 to 96.84% for free CRL, higher than the maximum found for immobilized CRL (32%). The scale-up (scale factor: 50) with the free CRL was successfully performed, achieving a high conversion value (92%) in a 500 mL bioreactor. This conversion value was within the range predicted by the mathematical model obtained using mini reactors. These mini reactors are good models to test several conditions of enzyme reactions that are intended for large scales.

Immobilized KDN Lipase on Macroporous Resin for Isopropyl Myristate Synthesis

Free enzymes often face economic problems because of their non-repeatability and variability, which limit their application in industrial production. In this study, KDN lipase was immobilized with the macroporous resin LXTE-1000 and glutaraldehyde. The optimal conditions of enzyme immobilization were defined by a single factor experiment and response surface methodology (RSM). The concentration of the cross-linking agent glutaraldehyde was 0.46% (v/v), the cross-linking temperature was 25.0 °C, and the cross-linking time was 157 min. The enzyme activity of the immobilized KDN lipase after adsorption/cross-linking was 291.36 U/g, and the recovery of the enzyme activity was 9.90%. The optimal conditions for the synthesis of isopropyl myristate were catalyzed by the immobilized KDN lipase in a solvent-free system: immobilized enzyme 53 mg, reaction temperature 36.1 °C, myristic acid 228.4 mg, isopropanol 114 µL, and reaction time 18 h. The yield of isopropyl myristate was 66.62%. After ten cycles, the activity of the immobilized KDN lipase preserved more than 46.87% of its initial enzyme activity, and it demonstrated high tolerance to solvents compared to free KDN lipase.

Green biosynthesis of rare DHA-phospholipids by lipase-catalyzed transesterification with edible algal oil in solvent-free system and catalytic mechanism study.

Docosahexaenoic acid (DHA)-enriched phosphatidylcholine (PC) has received significant scientific attention due to the health benefits in food and pharmaceutical products. In this work, the edible algal oil rich in DHA-triacylglycerol (DHA-TAG) without pretreatment was first used as the DHA donor for the transesterification of phospholipids (PLs) to prepare three kinds of rare PLs, including DHA-PC, DHA-phosphatidylethanolamine (DHA-PE), and DHA-phosphatidylserine (DHA-PS). Here, 153 protein structures of triacylglycerol lipase (EC 3.1.1.3) were virtually screened and evaluated by transesterification. PLA1 was the best candidate due to a higher DHA incorporation. Results showed that the transesterification of PC with DHA-TAG at 45°C and 0.7% water content (without additional water addition) could produce DHA-PC with 39.1% DHA incorporation at 30min. The different DHA donors, including forms of fatty acid, methyl ester, and triglycerides, were compared. Molecular dynamics (MD) was used to illustrate the catalytic mechanism at the molecular level containing the diffusions of substrates, the structure-activity relationship of PLA1, and the effect of water content.

Continuous synthesis of cinnamate ester using membrane reactor in a solvent-free system

Cinnamic acid is a phenolic compound with the potential to act as a natural antioxidant. Cinnamic acid esterification can be performed by adding a long-chain alcohol to one of its hydroxyl groups to improve its antioxidant activity. In this study, cinnamic esters were synthesized in an enzymatic membrane reactor (EMR) and were carried out continuously. This study aimed to determine the optimum conditions for the synthesis of cinnamic esters using various types of alcohols (e.g., butanol, hexanol, and octanol) as alkyl group donors and the impact of the esterification on the antioxidant activity. The continuous synthesis of cinnamic esters with TL IM lipozyme as the biocatalyst had the highest conversion (20.91%) with 1-butanol as the alkyl donor, in a synthesis strategy of a 12-h batch followed by continuous operation with a residence time of 9 h. The resulting ester increased the antioxidant activities by 2.18 and 3.85-fold, respectively based on the DPPH and FRAP assays.

Design of a New Chemoenzymatic Process for Producing Epoxidized Monoalkyl Esters from Used Soybean Cooking Oil and Fusel Oil

The aim of this study was to produce epoxidized monoalkyl esters (EMAE), a valuable class of oleochemicals used in a wide range of products and industries, from used soybean cooking oil (USCO) and fusel oil via a three-step chemoenzymatic process. This process consists of a first enzymatic hydrolysis of USCO to produce free fatty acids (FFA). Here, five microbial lipases with different specificities were tested as biocatalysts. Full hydrolysis of USCO was obtained after a 180 min reaction time under vigorous stirring (1500 rpm) using a non-specific lipase from Candida rugosa (CRL). Then, monoalkyl esters (MAE) were produced via the esterification of FFA and fusel oil in a solvent-free system using the lipase Eversa® Transform 2.0 (ET2.0) immobilized via physical adsorption on poly(styrenene-divinylbenzene) (PSty-DVB) beads as a biocatalyst. Different water removal strategies (closed and open reactors in the presence or absence of molecular sieves at 5% m.m−1) on the reaction were evaluated. Maximum FFA conversions of 64.3 ± 2.3% (open reactor after a 30 min reaction time) and 73.5 ± 0.4% (closed reactor after a 45 min reaction time) were observed at 40 °C, using a stoichiometric FFA:fusel oil molar ratio (1:1), without molecular sieves, and 5 mg of immobilized protein per gram of reaction mixture. Under these conditions, maximum FFA conversion was only 30.2 ± 2.7% after a 210 min reaction time in a closed reactor using soluble lipase. Reusability tests showed better retention of the original activity of immobilized ET2.0 (around 82%) after eight successive batches of esterification reactions conducted in an open reactor. Finally, the produced MAE was epoxidized via the Prilezhaev reaction, a classical chemical epoxidation process, using hydrogen peroxide and formic acid as a homogeneous catalyst. The products were characterized by standard methods and identified using proton nuclear magnetic resonance (1H NMR). Maximum unsaturated bond conversions into epoxy groups were at approximately 33%, with the experimental epoxy oxygen content (OOCexp.) at 1.75–1.78%, and selectivity (S) at 0.81, using both MAEs produced (open or closed reactors). These results show that this new process is a promising approach for value-added oleochemical production from low-cost and renewable raw materials.

Post-synthesis modification of metal–organic framework boosts solvent-free enzymatic esterifications

Metal-organic frameworks (MOFs) have been widely used as carriers in bio-catalysis. However, the micropores of the MOFs usually decrease the activity of bio-catalysts in the solvent-free and viscous reaction system. Herein, we report a post-modification method with adjustable apertures to reduce the mass transfer resistance in the described reaction system. The defected lipase-MOF composites (CRL@dZIF-90), synthesized by the post-modification method, can efficiently catalyze the esterification reactions in the solvent-free systems to synthesize phytosterol esters (conversion to stigmasterol conjugated linoleate ester up to 95%). The CRL@dZIF-90 demonstrated comparable activity to the free CRL with more than 80% activity after 5 cycles and 85% activity after one-month storage, validating that post-modification of the MOFs is a potential way to boost the activity of enzyme@MOF bio-composite in the solvent-free system.

Cleaner Production of Isopropyl Laurate using Lipase Catalyzed Esterification: Optimization by Response Surface Methodology

This work aims to produce a common emollient ester isopropyl laurate (IPL) in a solvent-free system. Isopropyl alcohol (IPA) and lauric acid (LA) were esterified in a closed batch reactor, employing immobilized

Butyl-esters synthesis from palm fatty acid distillate catalyzed by immobilized lipases in solvent-free system – Optimization using a simplified method (SER)

Palm fatty acid distillate (PFAD) is a residual fatty acids rich-stream obtained in the palm oil refining process. Its potential as raw material for butyl esters syntheses using immobilized lipases (Novozym 435® and Lipozyme RM IM®) in solvent-free system have been explored. A simplified tool for optimizing reaction parameters (Substrate-Enzyme Relation, SER) conciliates the masses of reagents and biocatalysts to obtain valuable insights into the shift of chemical equilibrium and the probability of deleterious effects on immobilized lipases. This mathematical relation had been studied when a single acid and alcohol are involved, but here SER is adopted when a mixture of similar chain-length acids is present in the reaction media. Experimental assays have revealed a range of reaction conditions of molar ratio and biocatalyst loadings equivalent to SER − 42 to − 12, that achieved maximum conversions higher than 90 % using biocatalysts loading lower than 2.0 % (wt/wt of PFAD mass) and less than 10 % of n-butanol stoichiometric excess. Similar SER trends were observed when comparing single reactants conversions results and a mixture of acids (C16–C18) and one alcohol involved, indicating the applicability of SER as optimization tool for enzymatic esterifications using a complex matrix.

Enzymatic Synthesis of Thymol Octanoate, a Promising Hybrid Molecule

Interest in the synthesis and application of thymol esters has increased in recent years due to the numerous applications associated with its biological activities. The enzymatic synthesis of thymol octanoate by esterification of thymol and octanoic acid was explored using soluble lipases and immobilized lipase biocatalysts in solvent-free systems. Candida antarctica lipase B in its soluble form was the most active biocatalyst for this reaction. Different thymol and lipase feeding strategies were evaluated to maximize thymol octanoate production. The results suggest that there could be lipase inhibition by the ester product of the reaction. In this way, the optimal reaction condition was given using a thymol/acid molar ratio of 1:4 mol/mol. Under these conditions the conversion of thymol was close to 94% and the lipase maintained more than 90% of its initial activity after the reaction, showing the potential of the enzyme to be used in successive reaction cycles.

Performance evaluation studies of PEG esters as biolubricant base stocks derived from non-edible oil sources via enzymatic esterification

Polyethylene glycol esters (PEGEs) as, environment-friendly biolubricants were produced from different fatty acids derived from plant oils by enzymatic esterification reaction in a solvent-free system using commercially available immobilized Candida antarctica Lipase B (NS 435) biocatalyst. The raw materials used were a 1:1 ratio of oleic acid: linseed oil fatty acid, linseed oil fatty acid: ricinoleic acid, and oleic acid: ricinoleic acid as mixed fatty acids. Enzymatic esterification exhibited around 98.1%, 97.8%, and 98.5% maximum yield for three different PEGEs under the following optimum reaction conditions under vacuum: the molar ratio of mixed fatty acids to polyethylene glycol 200–2:1, reaction time - 4 h, catalyst loading - 10% (w/w), and reaction temperature - 60 °C. This study aimed to improve the thermo-oxidative stability and cold flow behavior of the esterified products. The synthesized esters were confirmed and characterized by acid value, Nuclear Magnetic Resonance Spectroscopy (NMR), and Fourier Transform Infrared Spectroscopy (FTIR). The fatty acid composition of PEGEs was determined by GC-FID (Gas Chromatography). Physicochemical properties, thermo-oxidative stability, and friction-reducing properties of the PEGEs were studied thoroughly and compared against conventional petroleum-based lubricants. Results revealed that the plant oil-based biolubricants had good lubricating properties like high flash point, low pour point, high viscosity index, high thermo-oxidative stability, and improved friction-reducing properties compared with petroleum-based hydraulic lube oil. Overall, the data indicated that enzymatically esterified biolubricants have significant potential as ISO VG-32, VG-46, and VG-68 grade biolubricant base stocks for various commercialized industrial applications.

Fabrication of dialysis membrane from cotton Giza 86 cellulose di-acetate prepared using Ac2O and NiCl2 as a new catalyst

This attempt has been made to synthesize the cellulose di-acetate in a solvent-free acetylation system of cotton Giza 86 cellulose with Ac2O (200 and 300 ml) in the presence of NiCl2.6HO (1.0, 1.5 and 2.0 g) as an effectively available and new catalyst by the conventional reflux and microwave irradiation methods. This study also illustrates the preparation of a dialysis membrane made from a cellulose di-acetate–dichloromethane-methanol-polyethylene glycol (MW: 200) casting solution. The microwave irradiation method for the synthesis of cellulose di-acetate showed excellent yields and short reaction time, which is an important feature of this method. The impact of the two methods on the cellulose di-acetate formation and its used in the dialysis membrane formulations was studied. The experimental degree of substitution of the prepared cellulose di-acetate values (DS = 2.00–2.7) showed an agreement with the calculated values by FTIR and 1H-NMR analysis methods. The formation of cellulose di-acetate with percentage yields varied from 62.85 to 89.85%. The applicability of the prepared membrane in dialysis operation was evaluated in terms of urea clearance, rejection of Bovine Serum Albumin (BSA) and flux of pure water. Characterization of cellulose di-acetate was achieved through 1H-NMR, FTIR, TGA, and BET analyses. The CA-PEG blend membrane was examined by contact angle measurement, porosity, and water uptake of the membrane. The cellulose acetate membrane surface morphology was determined using SEM. It is observable that the fabricated CA-PEG blend membrane from synthesized cellulose di-acetate by using Nickel chloride as a catalyst is showing remarkable rejection of BSA and urea clearance up to 100 and 67.2%, respectively. The present work is promising and applicable in dialysis membranes.

Structured lipids produced from palm-olein oil by interesterification: A controllable lipase-catalyzed approach in a solvent-free system

Palm olein (POL) was modified by enzymatic interesterification with different degrees of acyl migration in a solvent-free packed bed reactor. The fatty acid and acylglycerol composition, isomer content, thermodynamic behavior, and relationship between crystal polymorphism, solid fat content (SFC), crystal microstructure, and texture before and after modification were studied. We found that the increase in sn-2 saturation interesterification was not only due to the generated tripalmitin (PPP) but also caused by acyl migration, and the SFC profiles were changed accordingly. The emergence of high melting point acylglycerols was an important factor accelerating the crystallization rate, further shortening the crystallization induction time, leading to the formation of large crystal spherulites, thereby reducing the hardness. The transformation from the β' to the β form occurred during post-hardening during storage. The isomer content also affected the physicochemical properties of the modified POL.

Bench-stable oxidant sodium percarbonate for functional group transformation of arylboronic acids

Sodium percarbonate (2Na2CO3·3H2O2, sodium carbonate-hydrogen peroxide, SPC), an easily accessible and bench-stable hydrogen peroxide-solid adduct based on inorganic salt, was efficiently used as a solid hydroxylating reagent for the ipso-hydroxylation of arylboronic acids under mild conditions, affording the corresponding phenols in an excellent manner at a fast rate. Moreover, a solvent-free solid-state reaction system using SPC for the oxidative hydroxylation of arylboronic acids was also demonstrated. Furthermore, subsequent treatment with benzyl halide was successfully performed, providing an alternative synthetic route for preparing benzyl phenyl ethers in a one-pot tandem reaction system.

Green Synthesis of Liquid Metal-Doped Carbon Dots for Treating Multidrug-Resistant Gram-Negative Bacteria.

Global emergence of multidrug-resistant (MDR) gram-negative bacteria triggers severe infections that result in an epidemic. It is urgent to discover novel classes of antibacterial agents. Here, a green route for synthesizing polyethylene glycol-based carbon dots (PEG-CDs) doped with liquid metal (LM-Cdots) via a solvent-free system is presented. LM-Cdots synthesized via ultrasound exhibit great antibacterial activity against gram-negative bacteria (E. coli, P. aeruginosa, K. pneumoniae, and A. baumannii) and their multidrug-resistant (MDR) clinical isolates. In the in vitro antibacterial test with MDR K. pneumoniae, LM-Cdots show an extremely low minimum inhibition concentration (MIC) of 0.63µg mL-1 . Compared to naked PEG-CDs, the MIC is improved by 1000 times. In vivo results reveal that LM-Cdots can accelerate wound healing with low biotoxicity and inflammation.

Engineering a Bacillus subtilis esterase for selective hydrolysis of d, l-menthyl acetate in an organic solvent-free system.

Esterase/lipase-catalyzed selective hydrolysis of d, l-menthyl esters has become one of the promising approaches for producing l-menthol, one of the most important flavoring chemicals with extensive uses. However, the activity and l-enantioselectivity of the biocatalyst are not sufficient for meeting the industrial requirements. Herein, a highly active para-nitrobenzyl esterase from Bacillus subtilis 168 (pnbA-BS) was cloned and then engineered to enhance its l-enantioselectivity. On the basis of the strategy tailoring the steric exclusion effect and structural flexibility of the region adjacent to the substrate, the substitution of Ala400 to Pro caused a remarkable improvement in the E value from 1.0 to 466.6. The variant A400P was purified and further confirmed with strict l-enantioselectivity in the selective hydrolysis of d, l-menthyl acetate, whereas the improved l-enantioselectivity caused decreased activity. To develop an efficient, easy-to-use, and green methodology, organic solvent was omitted and substrate constant feeding was integrated into the whole-cell catalyzed system. During the catalytic process, the selective hydrolysis of 1.0 M d, l-menthyl acetate in 14 h offered a conversion of 48.9%, e.e.p value of >99%, and space-time yield of 160.52 g (l d)-1.

Improvement of amidase production with high specific acyltransferase activity using Bacillus smithii IITR6B2

This work introduces an approach to increase the productivity of the whole-cell amidase's acyltransferase activity of the Bacillus smithii IITR6b2 in terms of its specific activity. Amidase has a wide range of substrate specificities. An increased amidase with acyl transfer activity will help produce an abundance of different hydroxamates and acid hydrazides with only a small amount of biocatalyst. Unlike the chemical process, it involves a greener approach using an aqueous media and one-step biotransformation. The statistical optimization of amidase production was done through Plackett-Burman design and Response Surface Methodology. After the screening, the ANOVA showed a p-value of <0.001 with a determination coefficient (R2) of 0.994. The four critical factors selected from the Plackett-Burman design were yeast extract, sodium chloride, manganese chloride, and trisodium citrate. After the Central Composite Design, the ANOVA revealed a p-value of <0.001 and a determination coefficient (R2) of 0.9887. The overall enzyme activity increased from 18 IU/ml to 91.30 IU/ml (a 5-fold increase), and specific enzyme activity from 16.1 IU/mg to 47.66 IU/mg (a nearly 3-fold increase). After validation of the predicted model from Response Surface Methodology, yeast extract had the maximum influence on amidase production and activity in combination with manganese chloride. Further, the produced enzyme was explored for the synthesis of pyrazine-2-carboxylic acid hydrazide in a solvent-free reaction system.

Recent Advances in the Enzymatic Synthesis of Polyester.

Polyester is a kind of polymer composed of ester bond-linked polybasic acids and polyol. This type of polymer has a wide range of applications in various industries, such as automotive, furniture, coatings, packaging, and biomedical. The traditional process of synthesizing polyester mainly uses metal catalyst polymerization under high-temperature. This condition may have problems with metal residue and undesired side reactions. As an alternative, enzyme-catalyzed polymerization is evolving rapidly due to the metal-free residue, satisfactory biocompatibility, and mild reaction conditions. This article presented the reaction modes of enzyme-catalyzed ring-opening polymerization and enzyme-catalyzed polycondensation and their combinations, respectively. In addition, the article also summarized how lipase-catalyzed the polymerization of polyester, which includes (i) the distinctive features of lipase, (ii) the lipase-catalyzed polymerization and its mechanism, and (iii) the lipase stability under organic solvent and high-temperature conditions. In addition, this article also focused on the advantages and disadvantages of enzyme-catalyzed polyester synthesis under different solvent systems, including organic solvent systems, solvent-free systems, and green solvent systems. The challenges of enzyme optimization and process equipment innovation for further industrialization of enzyme-catalyzed polyester synthesis were also discussed in this article.