Search Results (87)

This work aimed to explore novel polymeric material by synthesizing polyamide 6,5 via the direct enzymatic polycondensation of dimethyl glutarate and 1,6-Diaminohexane, using the lipase Novozym 435 as a biocatalyst. While maintaining a fixed monomer feed ratio, the effects of reaction temperature, duration, and enzyme concentrations on the molecular weight and yield of the resulting polyamide 6,5 were systematically investigated. The experimental results indicated that the optimal conditions for the Novozym 435-catalyzed synthesis were a reaction time of 3 days, a temperature of 90 °C, and enzyme concentrations of 20 wt%. The establishment of this enzymatic synthesis route for polyamide 6,5 not only provides a novel methodology for polymer synthesis but also offers a new perspective for the future green materials manufacturing industry. Full article

In this paper, batch stirred-tank alcoholysis reactions of used and refined sunflower oils were performed with n-octyl, myristyl, cetyl, oleyl, and stearyl alcohols using immobilized lipases Novozym 435 and Lipozyme IM as catalysts. Alcohol conversions ranged from 74% to 94%, with slight differences between used frying sunflower oil and refined sunflower oil. The resulting wax esters were purified via stepwise column chromatography. The different regioselectivity of the biocatalysts led to distinct reaction pathways, and Novozym 435 proved to be the most effective enzyme, providing higher conversions and no detectable by-products. This study demonstrates the valorization of waste frying oils into high-value wax esters through enzymatic alcoholysis, comparing two industrially relevant immobilized lipases and achieving high conversion across multiple long-chain alcohols. The results highlight a sustainable alternative to conventional chemical catalysis and extend biocatalytic applications beyond traditional biodiesel production. By incorporating waste lipids into value-added products, the overall sustainability and circularity of the system are improved, contributing to green and sustainable chemistry. Full article

The present study evaluates the impact of cavitation on the performance of the chemical refining of rapeseed oils and the enzymatic interesterification of fat blends using a powerful UP400S ultrasonicator (400 W, 20 kHz). Ultrasound-assisted alkali neutralization achieved efficiency comparable to that of the conventional 60 min process in only 7 min, with similar refining losses (5.04–6.80 wt.%), although slightly higher lipid peroxidation was observed. Performing the ultrasound cavitation under a protective nitrogen atmosphere minimized the formation of lipid peroxides and their breakdown products (i.e., hexanal, nonanal), partially protected tocopherols, and improved oxidative stability (IP at 120 °C = 3.9–4.4 h). Ultrasound-assisted enzymatic interesterification (EIE) of palm kernel fat and a palm stearin blend catalyzed by immobilized lipases (Lipozyme TL IM, Lipozyme RM IM, Novozyme 435) was carried out for the first time. Cavitation accelerated triacylglycerol rearrangement, reduced reaction time from 6 h (9.0·10⁻³ to 1.6·10⁻² min⁻¹) to only 1 h (5.5·10⁻² to 1.2·10⁻¹ min⁻¹), and significantly affected melting point stabilization and solid fat content profile. In summary, ultrasound cavitation substantially enhanced mass transfer and reaction kinetics, demonstrating strong potential for process intensification in the edible oil industry. Further optimization of reaction conditions is required before large-scale industrial implementation. Full article

Monoacylglycerols (MAGs) are significant intermediate byproducts in the hydrolysis of oils and fats. The accumulation of MAGs not only reduces the quality and purity of the final products in biodiesel production and edible oil refining but also poses challenges for downstream separation processes. Therefore, the development of efficient biocatalysts for the specific MAG conversion is of great industrial importance. The lipase from Aspergillus oryzae (AOL) has shown potential for lipid modification; however, the wild-type enzyme (WT) suffers from poor solubility, tendency to aggregate, and low specific activity towards MAGs in aqueous systems, which severely restricts its practical application. In this study, a combinatorial protein engineering strategy was employed to overcome these limitations. We integrated fusion protein technology with rational design to enhance both the functional expression and catalytic efficiency of AOL. Firstly, the superfolder green fluorescent protein (sfGFP) was fused to the N-terminus of AOL. The results indicated that the sfGFP fusion tag significantly improved the solubility and stability of the enzyme, preventing the formation of inclusion bodies. The fusion protein sfGFP-AOL exhibited a MAG conversion rate of approximately 65%, confirming the positive impact of the fusion tag on enzyme developability. To further boost catalytic performance, site-directed mutagenesis was performed based on structural analysis. Among the variants, the mutant sfGFP-Y92Q emerged as the most potent candidate. In the MAG conversion, sfGFP-Y92Q achieved a conversion rate of 98%, which was not only significantly higher than that of sfGFP-AOL but also outperformed the widely used commercial immobilized lipase, Novozym 435 (~54%). Structural modeling and docking analysis revealed that the Y92Q mutation optimized the geometry of the active site. The substitution of Tyrosine with Glutamine at position 92 likely enlarged the substrate-binding pocket and altered the local electrostatic environment, thereby relieving steric hindrance and facilitating the access of the bulky MAG substrate to the catalytic center. In conclusion, this work demonstrates that the synergistic application of sfGFP fusion and rational point mutation (Y92Q) can dramatically transform the catalytic properties of AOL. The engineered sfGFP-Y92Q variant serves as a robust and highly efficient biocatalyst for MAG degradation. Its superior performance compared to commercial standards suggests immense potential for cost-effective applications in the bio-manufacturing of high-purity fatty acids and biodiesel, offering a greener alternative to traditional chemical processes. Full article

Developing highly efficient and cost-effective immobilized biocatalysts is essential for optimizing diacylglycerol (DAG) production via biotransformation of natural oil. To address this, the 1,3-regiospecific MAS1-H108W lipase, derived from marine Streptomyces sp. strain W007, was produced through high-density fermentation (20 °C, pH 7.0, 132 h). This lipase was immobilized by XAD1180 resin adsorption, yielding an immobilized MAS1-H108W lipase with a lipase activity of 4943.5 U/g and a protein loading of 201.5 mg/g under selected conditions (lipase/support ratio 100 mg/g, initial buffer pH of 8.0). After immobilization, the lipase maintained its optimal temperature at 70 °C and shifted its optimal pH from 7.0 to 8.0, along with enhanced thermostability. The immobilized MAS1-H108W lipase demonstrated superior efficiency in DAG synthesis compared to non-regiospecific immobilized MAS1 lipase and commercial lipases (Novozym 435 and Lipozyme RM IM). Under the optimized reaction conditions (reaction temperature 60 °C, olive oil/glycerol molar ratio 1:2, adding amount of immobilized MAS1-H108W lipase 1.0 wt.%), a maximum DAG content of 49.3% was achieved within 4 h. The immobilized lipase also exhibited excellent operational stability, retaining 81.9% of its initial production capacity after 10 reuse cycles. Furthermore, in the glycerolysis of various vegetable oils (corn oil, rapeseed oil, peanut oil, sunflower oil, and soybean oil), the DAG content catalyzed by immobilized MAS1-H108W lipase consistently exceeded 48%. This work provides a highly efficient and economical immobilized biocatalyst for DAG production, and highlights the significant potential of regioselective lipases in promoting efficient DAG synthesis via glycerolysis. Full article

Tetrasubstituted furans and their derivatives represent a versatile class of important heterocyclic frameworks widely distributed in natural products. These scaffolds also demonstrate significant potential in pharmaceutical chemistry, materials science, and organic synthesis methodologies. In this study, we successfully established a synergistic catalytic system utilizing benzoylacetonitriles, aldehydes, and benzoyl chlorides as substrates, facilitated by tributylphosphine and immobilized lipase (Novozym 435), to achieve efficient synthesis of cyano-containing tetrasubstituted furans. Under optimized conditions, we obtained a series of target products exhibiting exceptional substrate tolerance with good to excellent isolated yields ranging from 80% to 94%. Additionally, we proposed a reasonable reaction mechanism and verified it through controlled experiments. This methodology not only expands the synthetic utility of lipase in non-natural transformations but also establishes a paradigm of green chemistry for the construction of tetrasubstituted furans. Full article

The concept of combi-lipases is herein explored in the full hydrolysis of babassu oil. The commercially immobilized lipases from Candida antarctica (form B) (Novozym^® 435), Rhizomucor miehei (Lipozyme^® RM-IM), and Thermomyces lanuginosus (Lipozyme^® TL-IM) were evaluated as single and combined biocatalysts by a mixture design with triangular surface. As a result, after evaluating the response desirability profiling for all biocatalysts, the best biocatalyst in the reaction was the combi-lipases composed of 75% of Lipozyme^® RM-IM, 17% of Novozym^® 435, and 8% of Lipozyme^® TL-IM, reaching full hydrolysis (>99%) after 4 h of reaction. Subsequently, such combi-lipases were employed as biocatalysts in the optimization of the reaction in a shorter reaction time (3 h). After optimization by the Taguchi method, full hydrolysis (>99%) was reached under optimized reaction conditions (9 wt.% of biocatalyst content, 1:2 (oil/water), 40 °C, and 180 rpm). Under such conditions, the combi-lipases maintained 70% of their initial activity after 10 reaction cycles. The antimicrobial activity against some of the most common environmental bacteria of the obtained free fatty acids (FFAs) was also evaluated. The FFAs inhibited more than 90% of the growth of S. aureus, E. coli, and P. aeruginosus when using 10 mg FFAs/mL. Full article

Triacylglycerols containing medium-chain fatty acids at the sn-1,3 positions and a long-chain fatty acid at the sn-2 position (MLM-TAG) are of nutritional interest. However, they are scarce in common food sources and are usually synthesized by chemical or enzymatic methods. In this work, the enzymatic synthesis of MLM-TAG was attempted using sn-2 monoacylglycerols (sn-2 MAG) from the ethanolysis of an arachidonic acid-rich fraction from ARASCO and fatty acid ethyl esters from the ethanolysis of coconut oil as substrates. The highest yield of sn-2 MAG (23.3 mol%) was obtained after 1 h of ethanolysis with Novozym 435 lipase at 25 °C, and the best profile of the ethanolysis products of coconut oil was obtained after 24 h of reaction catalyzed by the lipase from Thermomyces lanuginosus. Regarding the enzymatic synthesis of structured TAG, the lipase from Rhizopus oryzae gave better results than those from Thermomyces lanuginosus and Rhizomucor miehei, with the sn-2 position mainly esterified with arachidonic acid (34.8%) and the sn-1,3 positions mainly esterified with capric and lauric acids (35.1%). This work focuses on a simple process for the enzymatic production of structured TAG without prior purification of the sn-2 MAG. Full article

Tyrosol and hydroxytyrosol are powerful phenolic antioxidants occurring in olive oil and in by-products from olive processing. Due to their high polarity, esterification or other lipophilization is necessary to make them compatible with lipid matrices. Hydroxytyrosol methyl carbonate is a more effective antioxidant than dibutylhydroxytoluene or α-tocopherol and together with tyrosol methyl carbonate exerts interesting pharmacological properties. The purpose of this work was the enzymatic preparation of alkyl carbonates of tyrosol and hydroxytyrosol. A set of 17 hydrolases was tested in the catalysis of tyrosol methoxycarbonylation in neat dimethyl carbonate to find an economically feasible alternative to the recently reported synthesis of methyl carbonates catalyzed by Novozym 435. Novozym 435 was, however, found to be the best performing catalyst, while Novozym 735, pig pancreatic lipase, lipase F-AK and Lipex 100T exhibited limited reactivity. No enzyme accepted 1,2-propylene carbonate as the acylation donor. Under optimized reaction conditions, Novozym 435 was used in the batch preparation of tyrosol methyl carbonate and hydroxytyrosol methyl carbonate in quantitative yields. The enzymatic methoxycarbonylation of tyrosol and hydroxytyrosol can also be used as a method for their selective protection in enzymatic syntheses of phenylethanoid glycosides catalyzed with enzymes comprising high levels of acetyl esterase side activity. Full article

Marine microalgae Schizochytrium sp. have a high content of docosahexaenoic acid (DHA), an omega-3 fatty acid that is attracting interest since it prevents certain neurodegenerative diseases. The obtention of a bioactive and purified DHA fatty acid ester using a whole-integrated process in which renewable sources and alternative methodologies are employed is the aim of this study. For this reason, lyophilized Schizochytrium biomass was used as an alternative to fish oil, and advanced extraction techniques as well as enzymatic modification were studied. Microalgal oil extraction was optimized via a surface-response method using pressurized liquid extraction (PLE) obtaining high oil yields (29.06 ± 0.12%) with a high concentration of DHA (51.15 ± 0.72%). Then, the enzymatic modification of Schizochytrium oil was developed by ethanolysis using immobilized Candida antarctica B lipase (Novozym^® 435) at two reaction temperatures and different enzymatic loads. The best condition (40 °C and 200 mg of lipase) produced the highest yield of fatty acid ethyl ester (FAEE) (100%) after 8 h of a reaction attaining a cost-effective and alternative process. Finally, an enriched and purified fraction containing DHA-FAEE was obtained using open-column chromatography with a remarkably high concentration of 93.2 ± 1.3% DHA. The purified and bioactive molecules obtained in this study can be used as nutraceutical and active pharmaceutical intermediates of marine origin. Full article

Puerarin is a flavonoid known as a natural antioxidant found in the root of Pueraria robata. Its antioxidant, anticancer, and anti-inflammatory effects have attracted attention as a potential functional ingredient in various bioindustries. However, puerarin has limited bioavailability owing to its low lipid solubility and stability. Acylation is proposed as a synthesis method to overcome this limitation. In this study, lipase-catalyzed acylation of puerarin and various acyl donors was performed, and the enzymatic synthetic condition was optimized. Under the condition (20 g/L of Novozym 435, palmitic anhydride, 1:15, 40 °C, tetrahydrofuran (THF)), the synthesis of puerarin ester achieved a significantly high conversion (98.97%) within a short time (3 h). The molecule of the synthesized puerarin palmitate was identified by various analyses such as liquid chromatography–mass spectrometry (LC–MS), Fourier-transform infrared spectroscopy (FT-IR), and carbon-13 nuclear magnetic resonance (¹³C NMR). The lipid solubility and the radical scavenging activity were also evaluated. Puerarin palmitate showed a slight decrease in antioxidant activity, but lipid solubility was significantly improved, improving bioavailability. The high conversion achieved for puerarin esters in this study will provide the foundation for industrial applications. Full article

Ionic additives affect the structure, activity and stability of lipases, which allow for solving common application challenges, such as preventing the formation of protein aggregates or strengthening enzyme–support binding, preventing their desorption in organic media. This work aimed to design a biocatalyst, based on lipase improved by the addition of ionic additives, applicable in the production of ethyl esters of fatty acids (EE). Industrial enzymes from Thermomyces lanuginosus (TLL), Rhizomucor miehei (RML), Candida antárctica B (CALB) and Lecitase^®, immobilized in commercial supports like Lewatit^®, Purolite^® and Q-Sepharose^®, were tested. The best combination was achieved by immobilizing lipase TLL onto Q-Sepharose^® as it surpassed, in terms of %EE (70.1%), the commercial biocatalyst Novozyme^® 435 (52.7%) and was similar to that of Lipozyme TL IM (71.3%). Hence, the impact of ionic additives like polymers and surfactants on both free and immobilized TLL on Q-Sepharose^® was assessed. It was observed that, when immobilized, in the presence of sodium dodecyl sulfate (SDS), the TLL derivative exhibited a significantly higher activity, with a 93-fold increase (1.02 IU), compared to the free enzyme under identical conditions (0.011 IU). In fatty acids ethyl esters synthesis, Q-SDS-TLL novel derivatives achieved results similar to commercial biocatalysts using up to ~82 times less enzyme (1 mg/g). This creates an opportunity to develop biocatalysts with reduced enzyme consumption, a factor often associated with higher production costs. Such advancements would ease their integration into the biodiesel industry, fostering a greener production approach compared to conventional methods. Full article

In lipase-catalyzed kinetic resolutions (KRs), the choice of immobilization support and acylating agents (AAs) is crucial. Lipase B from Candida antarctica immobilized onto magnetic nanoparticles (CaLB-MNPs) has been successfully used for diverse KRs of racemic compounds, but there is a lack of studies of the utilization of this potent biocatalyst in the KR of chiral amines, important pharmaceutical building blocks. Therefore, in this work, several racemic amines (heptane-2-amine, 1-methoxypropan-2-amine, 1-phenylethan-1-amine, and 4-phenylbutan-2-amine, (±)-1a–d, respectively) were studied in batch and continuous-flow mode utilizing different AAs, such as diisopropyl malonate 2A, isopropyl 2-cyanoacetate 2B, and isopropyl 2-ethoxyacetate 2C. The reactions performed with CaLB-MNPs were compared with Novozym 435 (N435) and the results in the literature. CaLB-MNPs were less active than N435, leading to lower conversion, but demonstrated a higher enantiomer selectivity, proving to be a good alternative to the commercial form. Compound 2C resulted in the best balance between conversion and enantiomer selectivity among the acylating agents. CaLB-MNPs proved to be efficient in the KR of chiral amines, having comparable or superior properties to other CaLB forms utilizing porous matrices for immobilization. An additional advantage of using CaLB-MNPs is that the purification and reuse processes are facilitated via magnetic retention/separation. In the continuous-flow mode, the usability and operational stability of CaLB-MNPs were reaffirmed, corroborating with previous studies, and the results overall improve our understanding of this potent biocatalyst and the convenient U-shape reactor used. Full article

Branched-chain esters (BCEs) have found a large number of applications in cosmetics. Among them, neopentyl glycol dilaurate (NPGDL) stands out as an emollient, emulsifier, and skin-conditioning agent. This work presents the synthesis of NPGDL in a solvent-free medium using the two most common immobilized lipases: Novozym^® 40086 (Rml) and Novozym^® 435 (CalB). Results proved that the former biocatalyst has lower activity and certain temperature deactivation, although conversions ≥ 90% were obtained at 60 °C and 7.5% of catalyst. On the other hand, optimal reaction conditions for Novozym^® 435 are 3.75% w/w of the immobilized derivative at 80 °C. Under optimal conditions, the process productivities were 0.105 and 0.169 kg NPGDL/L h, respectively. In order to select the best conditions for NPGDL production, studies on the reuse of the derivative and cost estimation have been performed. Economic study shows that biocatalytic processes can be competitive when lipases are reused for five cycles, yielding biocatalyst productivities of 56 and 122 kg NPGDL/kg biocatalyst using Novozym^® 40086 and Novozym^® 435, respectively. The final choice will be based on both economic and sustainability criteria. Green metric values using both biocatalysts are similar but the product obtained using Novozym^® 40086 is 20% cheaper, making this alternative the best option. Full article

The enzymatic acetylation in the organic solvents of a number of the important bioactive cardiac glycosides was investigated. With the bufanolide proscillaridin A and the cardenolide lanatoside C, acylation, as expected, occurred at the secondary 4′-OH of the rhamnopyranosyl unit of the former (by the action of Novozym 435 lipase) and the primary 6′′′′-OH of the terminal glucopyranosyl unit of the latter (best results obtained by the action of the lipase PS). Only lipase PS was found to be able to acylate the cardenolides digitoxin and digoxin at the 4‴-OH of their terminal digitoxose unit. The corresponding monoacetyl derivatives, both of which are commercialized drugs, could be isolated with good yields. The investigation of the Novozym 435-catalyzed acetylation of free D-digitoxose provided a possible explanation for the inability of this lipase to acylate digitoxin and digoxin. Full article