Search Results (73)

Commercial fungal lipases from Rhizopus oryzae, Rhizopus niveus, Aspergillus niger, Rhizomucor miehei, and Candida rugosa were immobilized via physical adsorption onto Accurel MP 1000, a hydrophobic polypropylene support. The effects of enzyme concentration, pH, temperature, and glutaraldehyde post-treatment were systematically evaluated. Immobilization generally enhanced enzyme stability, which was further improved in several cases by glutaraldehyde crosslinking. The immobilized preparations retained over 50% of their initial activity for 3–6 cycles, and 7–10 cycles following glutaraldehyde treatment. While soluble enzymes lost nearly all activity within three months at 5 °C and 25 °C and retained only 5–20% at −20 °C, the immobilized forms preserved 50–100% of their activity under all storage conditions tested. Immobilized lipases also exhibited improved thermal stability at 60 °C by general increments between 1.3 and 1.8 times compared to soluble lipases. Increased tolerance to pH fluctuations was observed in most immobilized enzymes, particularly from R. oryzae, R. niveus, R. miehei, and C. rugosa. Organic solvent tolerance of the immobilized enzymes showed highest stability in hexane (66–100% residual activity after 4 h incubation). Glutaraldehyde treatment affected solvent stability of immobilized lipases in enzyme and solvent dependent manner. These findings demonstrate the improved stability and applicability of the produced biocatalysts in varying reaction environments. Full article

Enzyme entrapment in alginate hydrogel microbeads is an effective method of immobilization for industrial applications, but many fabrication methods for alginate microbeads involve oil, organic solvents, or high temperatures that reduce enzymatic activity. In this study, we employed an oil- and solvent-free gas-shearing technique to prepare alginate microbeads for the entrapment of Candida rugosa lipase (CRL), thereby minimizing thermal- and solvent-induced inactivation. To enhance immobilization efficiency and reusability, the effects of gas flow rate, alginate concentration, and cross-linking metal ions were systematically investigated. CRL entrapped in Ba- and Fe-alginate microbeads showed superior immobilization yield, activity retention, and activity recovery compared with CRL entrapped in conventional Ca-alginate microbeads. Notably, both Ba- and Fe-alginate microbeads exhibited significantly enhanced stability, with half-lives up to 127-fold greater than that of free CRL at 60 °C, and maintained substantially higher pH stability across the tested range. Ba-alginate microbeads provided greater pH stability and substrate affinity, whereas Fe-alginate microbeads demonstrated enhanced thermal stability and catalytic turnover. These findings highlight gas-shearing as a scalable and gentle fabrication method for producing high-performance alginate microbeads with tunable properties, making them suitable for enzyme entrapment in diverse biocatalytic applications. Full article

Deep eutectic solvents (DESs) have shown promise as a medium for extracting polar volatile fatty acids (VFAs) and in situ esterification of the extracted molecules using lipases. This solvent enhanced biocatalysis process can potentially streamline VFA separation from fermentation broth by integrating conversion and extraction steps. Two commercial lipases from Aspergillus oryzae (AoL) and Candida rugosa (CrL) were evaluated in reaction systems containing hydrophilic or hydrophobic DESs using a newly optimized lipase assay. The optimal pH for both lipases was around 5.0, with a slight reduction in activity at pH 8.0 and a significant inhibition at pH 2.0. The impact of DES concentration on lipase activity varied depending on the specific DES–lipase pairs. Most hydrophilic DESs show good compatibility with the tested lipases. Specifically for choline chloride/ethylene glycol (1:2) and choline chloride/levulinic acid (1:2), taking into account the influence of pH, CrL activity increased with DES concentration. However, the hydrophobic DES thymol/2,6-dimethoxyphenol (1:2) demonstrated enhanced inhibitory effects on both lipases. Docking simulation helped explain the ligand–protein interactions but showed limited capability in predicting the compatibility of specific DES–lipase pairs due to its constraints in simulating flexible protein structures and the complex interactions between DES components and water. Full article

Vitamin E succinate is a more mature vitamin E derivative, and its chemical stability and many effects have been improved compared with vitamin E, which can not only make up for the shortcomings of vitamin E application but also broaden the application field of vitamin E. At present, in developed countries such as Europe, America, and Japan, vitamin E succinate is widely used in health foods, and due to its good water solubility and stability, the vitamin E added to most nutritional supplements (tablets and hard capsules) is vitamin E succinate. At the same time, vitamin E succinate used in the food and pharmaceutical industries is mainly catalyzed by enzymatic catalysis. In this paper, Candida rugosa lipase (CRL) was studied. Chemical modification and immobilization were used to improve the enzymatic properties of CRL, and immobilized lipase with high stability and high activity was obtained. It was applied to the enzymatic synthesis of vitamin E succinate, and the reaction conditions were optimized to improve the yield and reduce the production cost. The review covered the research progress of the methods for enhancing the catalytic performance of immobilized enzymes and discussed its application in the synthesis of vitamin E succinate, providing new ideas and technical support for the catalytic performance enhancement of immobilized enzymes and its application in the synthesis of vitamin E succinate and promoting the production and application of vitamin E succinate. Full article

Phytosterol exhibits cholesterol-lowering, cardiovascular-protecting, anti-inflammatory, and anticancer efficacies but has low bioavailability due to its high melting point and poor solubility. Esterification with fatty acids enhances liposolubility, improving absorption and utilization fivefold. Industrial production of phytosterol esters mainly relies on chemical synthesis, which faces challenges in separation, purification, and quality assurance due to side reactions. Enzymatic synthesis, featuring mild conditions, environmental friendliness, and high selectivity, has gained attention from academia and industry. Candida rugosa lipase (CRL) is widely utilized due to its high efficiency in catalyzing the esterification of phytosterol. Natural lipases are highly sensitive to changes in temperature and pH and are difficult to reuse in continuous cycles; however, certain immobilization techniques can improve their catalytic activity and stability. Furthermore, the application of immobilized lipases in the synthesis of phytosterol esters can be combined with ultrasonic treatment to enhance the efficiency of enzymatic catalysis. This paper reviews the research progress on the catalytic esterification of phytosterol by immobilized lipases, highlighting the current challenges and future research directions, thereby fostering further advancements in the field of preparing phytosterol esters through immobilized lipase catalysis. Full article

The species Yarrowia lipolytica is an aerobic yeast that produces different lipase isoforms, including extracellular, intracellular, and membrane-bound ones. The immobilization of lipases, such as those from Y. lipolytica, increases enzyme stability and lowers operational costs, through its reuse. The characterization of those biocatalysts is highly important to orientate their technological applications. The present work aims to obtain different Y. lipolytica lipases, through fermentation and immobilization techniques, and to evaluate the ester synthesis and hydrolysis activity of these biocatalysts in comparison to a commercial lipase produced by Candida rugosa and test them for phytosterol ester production. High immobilization yield was achieved by microencapsulating Y. lipolytica lipase extract on magnetic nanoparticles (>99.7%). However, immobilization significantly reduced their activity (more than 90%). Lipases from Y. lipolytica showed greater 4-nitrophenyl laurate synthesis in relation to the lipase from C. rugosa. However, C. rugosa lipase was still the best biocatalyst for β-sitosterol oleate synthesis, with a conversion of more than 99%. Y. lipolytica lipases can be good catalysts for ester hydrolysis reactions, even for ester synthesis, but are not good catalysts specifically for phytosterol esters synthesis. Full article

This study describes the production of solketal esters from used soybean cooking oil (USCO) via enzymatic hydroesterification. This process consists of the complete hydrolysis of USCO into free fatty acids (FFAs) catalyzed by crude lipase extract from Candida rugosa (CRL). The resulting FFAs were recovered and utilized as the raw material for an esterification reaction with solketal, which was achieved via an open reaction. For this purpose, lipase Eversa^® Transform 2.0 (ET2.0) was immobilized via physical adsorption on treated epicarp particles from Acrocomia aculeata (macauba), a lignocellulosic residue. A protein loading of 25.2 ± 1.3 mg g⁻¹ with a support and immobilization yield of 64.8 ± 2.5% was achieved using an initial protein loading of 40 mg g⁻¹ of support. The influence of certain parameters on the esterification reaction was evaluated using a central composite rotatable design (CCRD). Under optimal conditions, a FFAs conversion of 72.5 ± 0.8% was obtained after 150 min of reaction at 46 °C using a biocatalyst concentration of 20% wt. and a FFAs–solketal molar ratio of 1:1.6. The biocatalyst retained 70% of its original activity after ten esterification batches. This paper shows the conversion of two agro-industrial waste into valuable materials (enzyme immobilization support and solketal esters). Full article

This study aimed to optimize the kinetic resolution of building blocks for the synthesis of β-blockers using Candida rugosa lipases, which could be potentially used to synthesize enantiomerically pure β-blockers further. Reaction mixtures were incubated in a thermostated shaker. Qualitative and quantitative analyses of the reaction mixtures were performed using chiral stationary phases and the UPLC-IT-TOF system. Of the 24 catalytic systems prepared, a system containing lipase from Candida rugosa MY, [EMIM][BF₄] and toluene as a two-phase reaction medium and isopropenyl acetate as an acetylating agent was optimal. This resulted in a product with high enantiomeric purity produced via biotransformation, whose enantioselectivity was E = 67.5. Using lipases from Candida rugosa enables the enantioselective biotransformation of the β-blockers building block. The biocatalyst used, the reaction environment, and the acetylating agent significantly influence the efficiency of performer kinetic resolutions. The studies made it possible to select an optimum system, a prerequisite for obtaining a product of high enantiomeric purity. As a result of the performed biotransformation, the (S)-enantiomer of the β-blocker derivative was obtained, which can be used to further synthesize enantiomerically pure β-blockers. Full article

In the present work, methanolic extracts from thyme and dittany plants were prepared and characterized in terms of their polyphenolic content through analytical and spectrophotometric techniques. Rosmarinic acid, thymol and carvacrol were found to be the main components of the extracts, which were further biologically assessed for their antioxidant, anti-tyrosinase, anti-lipase and antibacterial activity against Gram-negative and Gram-positive bacteria. As found, thyme extracts exhibited superior antioxidant activity (SC₅₀ at 33.9 μg mL⁻¹), while dittany extracts inhibited the microbial growth to a great extent against Bacillus subtilis strain (MIC at 0.5 mg mL⁻¹) and E. coli strain (MIC at 2 mg mL⁻¹). Furthermore, the thyme extract was proven to strongly inhibit the activity of lipase from Candida rugosa (IC₅₀ at 63.9 μg mL⁻¹), comparable to the standard inhibitor orlistat, while its inhibitory effect against mushroom tyrosinase was weak. On the other hand, the dittany extract presented an inhibitory effect against the tested lipase (IC₅₀ over 500 μg mL⁻¹) and an activation effect against tyrosinase (at concentrations > 500 μg mL⁻¹). Additionally, molecular docking studies of the main compounds of the extracts showed that rosmarinic acid plays a crucial role on the inhibitory activity of the extracts against lipase, while thymol has a stronger effect on inhibiting tyrosinase. Furthermore, both extracts were employed in the preparation of gelatin-deep eutectic solvent (DES) hydrogels that were further studied for their antioxidant and antibacterial activity. The results showed that the incorporation of the extracts offered antibacterial properties to the biopolymer-based hydrogels and enhanced the antioxidant activity of gelatin up to 85%. Full article

The development of immobilized enzymes with high activity and stability is critical. Metal–organic frameworks (MOFs) have attracted much academic and industrial interest in the field of enzyme immobilization due to their unique properties. In this study, the amino-functionalized ionic liquid (NIL)-modified metal–organic framework (UiO-66-NH₂) was prepared to immobilize Candida rugosa lipase (CRL), using dialdehyde starch (DAS) as the cross-linker. The results of the Fourier transform infrared (FT-IR) spectra, X-ray powder diffraction (XRD), and scanning electronic microscopy (SEM) confirmed that the NIL was successfully grafted to UiO-66-NH₂. The CRL immobilized on NIL-modified UiO-66-NH₂ (UiO-66-NH₂-NIL-DAS@CRL) exhibited satisfactory activity recovery (79.33%), stability, reusability, and excellent organic solvent tolerance. The research results indicated that ionic liquid-modified UiO-66-NH₂ had practical potential for application in enzyme immobilization. Full article

The use of lipase immobilized on an octyl-agarose support to obtain the optically pure enantiomers of chiral drugs in reactions carried out in organic solvents is a great challenge for chemical and pharmaceutical sciences. Therefore, it is extremely important to develop optimal procedures to achieve a high enantioselectivity of the biocatalysts in the organic medium. Our paper describes a new approach to biocatalysis performed in an organic solvent with the use of CALB-octyl-agarose support including the application of a polypropylene reactor, an appropriate buffer for immobilization (Tris base—pH 9, 100 mM), a drying step, and then the storage of immobilized lipases in a climatic chamber or a refrigerator. An immobilized lipase B from Candida antarctica (CALB) was used in the kinetic resolution of (R,S)-flurbiprofen by enantioselective esterification with methanol, reaching a high enantiomeric excess (ee_p = 89.6 ± 2.0%). As part of the immobilization optimization, the influence of different buffers was investigated. The effect of the reactor material and the reaction medium on the lipase activity was also studied. Moreover, the stability of the immobilized lipases: lipase from Candida rugosa (CRL) and CALB during storage in various temperature and humidity conditions (climatic chamber and refrigerator) was tested. The application of the immobilized CALB in a polypropylene reactor allowed for receiving over 9-fold higher conversion values compared to the results achieved when conducting the reaction in a glass reactor, as well as approximately 30-fold higher conversion values in comparison with free lipase. The good stability of the CALB-octyl-agarose support was demonstrated. After 7 days of storage in a climatic chamber or refrigerator (with protection from humidity) approximately 60% higher conversion values were obtained compared to the results observed for the immobilized form that had not been stored. The new approach involving the application of the CALB-octyl-agarose support for reactions performed in organic solvents indicates a significant role of the polymer reactor material being used in achieving high catalytic activity. Full article

Biomimetic ligands are synthetic compounds that mimic the structure and binding properties of natural biological ligands. The first uses of textile dyes as pseudo-affinity ligands paved the way for the rational design and de novo synthesis of low-cost, non-toxic and highly stable triazine-scaffolded affinity ligands. A novel method to assess and enhance protein stability, employing triazine-based biomimetic ligands and using cutinase from Fusarium solani pisi as a protein model, has been previously reported. This innovative approach combined the concepts of molecular modeling and solid-phase combinatorial chemistry to design, synthesize and screen biomimetic compounds able to bind cutinase through complementary affinity-like interactions while maintaining its biological functionality. The screening of a 36-member biased combinatorial library enabled the identification of promising lead ligands. The immobilization/adsorption of cutinase onto a particular lead (ligand 3′/11) led to a noteworthy enhancement in thermal stability within the temperature range of 60–80 °C. In the present study, similar triazine-based compounds, sourced from the same combinatorial library and mimicking dipeptides of diverse amino acids, were selected and studied to determine their effectiveness in binding and/or improving the thermal stability of several lipases, enzymes which are closely related in function to cutinases. Three ligands with different compositions were screened for their potential thermostabilizing effect on different lipolytic enzymes at 60 °C. An entirely distinct enzyme, invertase from Saccharomyces cerevisiae, was also assessed for binding to the same ligands and functioned as a ‘control’ for the experiments with lipases. The high binding yield of ligand 3′/11 [4-({4-chloro-6-[(2-methylbutyl)amino]-1,3,5-triazin-2-yl}amino)benzoic acid] to cutinase was confirmed, and the same ligand was tested for its ability to bind lipases from Aspergillus oryzae (AOL), Candida rugosa (CRL), Chromobacterium viscosum (CVL), Rhizomucor miehei (RML) and Rhizopus niveus (RNL). The enzymes CRL, CVL, RNL and invertase showed significant adsorption yields to ligand 3′/11—32, 29, 36 and 94%, respectively, and the thermal stability at 60 °C of free and adsorbed enzymes was studied. CVL and RNL were also stabilized by adsorption to ligand 3′/11. In the case of CRL and invertase, which bound but were not stabilized by ligand (3′/11), other ligands from the original combinatorial library were tested. Between the two alternative ligands, one was effective at stabilizing C. rugosa lipase, while none stabilized invertase. Full article

The present work proposes the optimization of enzymatic synthesis of alkyl stearates using stearic acid, alkyl alcohols (C₁-OH, C₂-OH, C₄-OH, C₈-OH and C₁₆-OH) and Candida rugosa lipase by a L₉ (3⁴) Taguchi-type design of experiments. Four variables were evaluated (reaction time, temperature, kU of lipase and alcohol:stearic acid molar ratio), ensuring that all variables were critical. In optimal conditions, five stearates were obtained with conversions > 90%. The obtained products were characterized by nuclear magnetic resonance (NMR). Additionally, the defoaming capacity of the five stearates was evaluated, obtaining better performance for the compound synthesized from C₈-OH alcohol. Full article

This study starts from the need to remove a mix of proteins, oils and natural resin, called beverone in the Italian literature, from the back of canvas paintings. The aim of this study is to develop and evaluate the effectiveness of two different agarose/enzyme gels containing, respectively, a trypsin derived from porcine pancreas and a lipase from Candida rugosa, both in an aqueous solution of deoxycholic acid-triethanolamine soap. Enzymes were selected because of their action on peptide and ester bonds, effectiveness at maintaining a weak alkaline pH and low cost. Several series of model samples, resulting from a combination of rabbit skin glue, linseed oil and colophony, were prepared to test the enzyme gels with two different values for each of the following variables: agarose concentration, application modes and time of application. Measurements of weight loss after the gel application and Fourier transform infrared analysis were conducted to underline the hydrolysis occurring due to the enzyme gels and their effectiveness. Results confirmed what has been found in the literature and improved our knowledge about the action of agarose enzyme gels on complex substrates (hydrophilic/hydrophobic). The gels applied fluidly, with a longer contact time and a lower agarose concentration, are more effective. Furthermore, trypsin gels provided better results on substrates with oil and glue, while lipase gels turned out to be more effective on substrates made of a mix of oil, glue and colophony. Full article

A significant bottleneck for the industrial application of lipases stems from their poor stability in the presence of commercial triglycerides. This is mainly due to the inactivating effect of the products of triglyceride oxidation (PTO), which are usually produced when oils and fats, being imported from far countries, are stored for long periods. In this study, the immobilization of a lipase from Candida rugosa on chitosan hydrogels has been carried out following two alternative approaches based on the enzyme adsorption and entrapment to increase the lipase stability under the operating conditions that are typical of oleochemical transformations. The effect of model compounds representing different classes of PTO on a lipase has been studied to optimize the enzyme immobilization method. Particular attention has been devoted to the characterization of the inactivating effect of PTO in nonaqueous media, which are adopted for most industrial applications of lipases. Full article