Rugosa Lipase Research Articles

Interfacial Hyperactivation of Candida rugosa Lipase onto Ca2Fe2O5 Nanoparticles: pH and Ionic Strength Fine-Tuning to Modulate Protein-Support Interactions.

The current study provides a comprehensive look of the adsorption process of Candida rugosa lipase (CRL) on Ca2Fe2O5 iron oxide nanoparticles (NPs). Protein-support interactions were identified across a broad range of pH and ionic strengths (mM) through a response surface methodology, surface charge determination, and spectroscopic and in silico analyses. The maximum quantity of immobilized protein was achieved at an ionic strength of 50 mM and pH 4. However, this condition did not allow for the greatest hydrolytic activity to be obtained. Indeed, it was recorded at acidic pH, but at 150 mM, where evaluation of the recovered activity revealed hyperactivation of the enzyme. These findings were supported by adsorption isotherms performed under different conditions. Based on zeta potential measurements, electrostatic interactions contributed differently to protein-support binding under the conditions tested, showing a strong correlation with experimentally determined immobilization parameters. Raman spectra revealed an increase in hydrophobicity around tryptophan residues, whereas the enzyme immobilization significantly reduced the phenylalanine signal in CRL. This suggests that this residue was involved in the interaction with Ca2Fe2O2 and molecular docking analysis confirmed these findings. Fluorescence spectroscopy showed distinct behaviors in the CRL emission patterns with the addition of Ca2Fe2O5 at pH 4 and 7. The calculated thermodynamic parameters indicated that the contact would be mediated by hydrophobic interactions at both pHs, as well as by ionic ones at pH 4. In this approach, this work adds to our understanding of the design of biocatalysts immobilized in iron oxide NPs.

Study on the Synthesis of Pine Sterol Esters in Solvent-free Systems Catalyzed by Candida rugosa Lipase Immobilized on Hydrophobic Macroporous Resin.

Pine sterol ester is a kind of novel food source nutrient with great advantages in lowering blood cholesterol levels, inhibiting tumors, preventing prostate enlargement, and regulating immunity. Macroporous resins with large specific surface area, stable structures, and various functional groups (epoxy, amino, and octadecyl groups) have been selected for immobilization of Candida rugosa lipase (CRL) to improve its stability and efficiency in the synthesis of pine sterol esters. A solvent-free strategy using oleic acid (substrate) as an esterification reaction medium is an important alternative for avoiding the use of organic solvents. The immobilization conditions of CRL immobilized on several kinds of commercial macroporous resins were optimized. Fortunately, by adsorption (hydrophobic interaction), a high immobilization efficiency of CRL was obtained using macroporous resins with hydrophobic octadecyl groups with an immobilization efficiency of 86.5%, enzyme loading of 138.5 mg/g and enzyme activity of 34.7 U/g. The results showed that a 95.1% yield could be obtained with a molar ratio of oleic acid to pine sterol of 5:1, an enzyme amount of 6.0 U/g (relative to pine sterol mass) at 50°C for 48 hours. The hydrophobic macroporous resin (ECR8806M) with a large specific surface area and abundant functional groups was used to achieve efficient immobilization of CRL. CRL@ECR8806M is an efficient catalyst for the synthesis of phytosterol esters and has the potential for further large-scale applications. Therefore, this simple, green, and low-cost strategy for lipase immobilization provides new possibilities for the high-efficiency production of pine sterol esters and other food source nutrients. This article is protected by copyright. All rights reserved.

Optimizing the First Step of the Biocatalytic Process for Green Leaf Volatiles Production: Lipase-Catalyzed Hydrolysis of Three Vegetable Oils.

Green leaf volatiles (GLVs), including short chain volatile aldehydes, are widely used in the flavor and food industries because of their fresh aroma. To meet the growing demand for natural GLVs with high added value, the use of biocatalytic processes appears as a relevant application. In such processes, vegetable oils are bioconverted into GLVs. First, the triacylglycerols of the oils are hydrolyzed by a lipase. Then, the free polyunsaturated fatty acids are converted by a lipoxygenase. Finally, volatile C6 or C9 aldehydes and 9- or 12-oxoacids are produced with a hydroperoxide lyase. Optimization of each biocatalytic step must be achieved to consider a scale-up. In this study, three oils (sunflower, hempseed, and linseed oils) and three lipases (Candida rugosa, Pseudomonas fluorescens, and Rhizomucor miehei lipases) have been tested to optimize the first step of the process. The experimental design and response surface methodology (RSM) were used to determine the optimal hydrolysis conditions for each oil. Five factors were considered, i.e., pH, temperature, reaction duration, enzyme load, and oil/aqueous ratio of the reaction mixture. Candida rugosa lipase was selected as the most efficient enzyme to achieve conversion of 96 ± 1.7%, 97.2 ± 3.8%, and 91.8 ± 3.2%, respectively, for sunflower, hempseed, and linseed oils under the defined optimized reaction conditions.

Lipase‐mediated plant oil hydrolysis—Toward a quantitative glycerol recovery for the synthesis of pure allyl alcohol and acrylonitrile

AbstractPlant based triacylglycerides are a sustainable feedstock for the chemical, bioenergy, food and cosmetic sector. While fatty acids conversion has been intensively studied, processes for glycerol valorization have been scarce. In contrast to chemical hydrolysis of plant triacylglycerides enzymatic hydrolysis methods provide a cleaner glycerol stream. This study focuses on the selection of a commercial lipase capable of quantitative hydrolysis of rapeseed‐ and high oleic sunflower oils. Enzymatic process using only water as the reaction medium allows simplified recovery of pure glycerol. Among the six commercial lipase preparations tested, Candida rugosa lipase was identified as the most effective biocatalyst. Catalytic behavior in buffer and pure water was equivalent. Glycerol generated using a washed lipase was recovered by just three purification steps. The FTIR spectrum of the recovered glycerol was equivalent to pure glycerol standard. Over the entire recovery process, 82%–88% of the theoretical glycerol yield could be obtained. Purified glycerol was further didehydroxylated to allyl alcohol by a formic acid mediated open distillation process. The enriched allyl alcohol had a quality, which allows to use it for the synthesis of bio‐based acrylonitrile.

Accelerated acidolysis for triglyceride modification using commercial lipases activated by a hydration–aggregation pretreatment

The modification of triglycerides (TGs) via acidolysis with fatty acids (FAs) was investigated using three commercial lipases activated via hydration–aggregation pretreatment. Lipase powder was dispersed in n-fatty alcohols and hydrated with a small volume of buffer solution under mechanical agitation. Subsequently, paste-like lipase aggregates were recovered from the mixture and lyophilized. Rhizopus japonicus lipase (RJL) exhibited acidolysis activity after hydration–aggregation pretreatment in free powder form without immobilization on solid supports, whereas non-activated lipase showed no detectable activity: the incorporation ratio of stearic acid in tripalmitin during acidolysis reached 33% within 24 h using activated RJL, whereas the acidolysis products were not detected using non-activated RJL. Morphological analysis demonstrated that the hydration of lipase powder and formation of a large oil–water interface area are essential for obtaining highly activated lipases. Decrease in chain length and increase in the degree of TG substrate unsaturation tended to increase the acidolysis activity of activated RJL, whereas the effect of chain length and degree of FA substrate unsaturation showed different trends. Chromobacterium viscosum lipase and RJL were activated via hydration–aggregation pretreatment, whereas Candida rugosa lipase was not.

Surfactant-based metal-organic frameworks (MOFs) in the preparation of an active biocatalysis.

Metal-organic frameworks (MOFs) are used as ideal support materials thanks to their unique properties and have become the focus of interest in enzyme immobilization studies, especially in recent years. In order to increase the catalytic activity and stability of Candida rugosa lipase (CRL), a new fluorescence-based MOF (UiO-66-Nap) derived from UiO-66 was synthesized. The structures of the materials were confirmed by spectroscopic techniques such as FTIR, 1H NMR, SEM, and PXRD. CRL was immobilized on UiO-66-NH2 and UiO-66-Nap by adsorption technique and immobilization and stability parameters of UiO-66-Nap@CRL were examined. Immobilized lipases UiO-66-Nap@CRL exhibited higher catalytic activity (204U/g) than UiO-66-NH2 @CRL (168U/g), which indicates that the immobilized lipase (UiO-66-Nap@CRL) carries sulfonate groups, this is due to strong ionic interactions between the surfactant's polar groups and certain charged locations on the protein surface. The Free CRL lost its catalytic activity completely at 60°C after 100min, while UiO-66-NH2 @CRL and UiO-66-Nap@CRL retained 45 % and 56 % of their catalytic activity at the end of 120min, respectively. After 5 cycles, the activity of UiO-66-Nap@CRL remained 50 %, while the activity of UiO-66-NH2 @CRL was about 40 %. This difference is due to the surfactant groups (Nap) in UiO-66-Nap@CRL. These results show that the newly synthesized fluorescence-based MOF derivative (UiO-66-Nap) can be an ideal support material for enzyme immobilization and can be used successfully to protect and increase the activities of enzymes.

Rapid prediction method of ZIF-8 immobilized Candida rugosa lipase activity by near-infrared spectroscopy

Candida rugosa lipase (CRL, EC3.1.1.3) is one of the main enzymes synthesizing esters, and ZIF-8 was chosen as an immobilization carrier for lipase. Enzyme activity testing often requires expensive reagents as substrates, and the experiment processes are time-consuming and inconvenient. As a result, a novel approach based on near-infrared spectroscopy (NIRs) was developed for predicting CRL/ZIF-8 enzyme activity. The absorbance of the immobilized enzyme catalytic system was evaluated using UV–Vis spectroscopy to investigate the amount of CRL/ZIF-8 enzyme activity. The powdered samples' near-infrared spectra were obtained. The sample’s enzyme activity data were linked with each sample’s original NIR spectra to establish the NIR model. A partial least squares (PLS) model of immobilized enzyme activity was developed by coupling spectral preprocessing with a variable screening technique. The experiments were completed within 48 h to eliminate inaccuracies between the reduction in enzyme activity with increasing laying-aside time throughout the test and the NIRs modeling. The root-mean-square error of cross-validation (RMSECV), the correlation coefficient of validation set (R) value, and the ratio of prediction to deviation (RPD) value were employed as assessment model indicators. The near-infrared spectrum model was developed by merging the best 2nd derivative spectral preprocessing with the Competitive Adaptive Reweighted Sampling (CARS) variable screening method. This model's root-mean-square error of cross-validation (RMSECV) was 0.368 U/g, the correlation coefficient of calibration set (R_cv) value was 0.943, the root-mean-square error of prediction (RMSEP) set was 0.414 U/g, the correlation coefficient of validation set (R) value was 0.952, and the ratio of prediction to deviation (RPD) was 3.0. The model demonstrates that the fitting relationship between the predicted and the reference enzyme activity value of the NIRs is satisfactory. The findings revealed a strong relationship between NIRs and CRL/ZIF-8 enzyme activity. As a result, the established model could be implemented to quantify the enzyme activity of CRL/ZIF-8 quickly by including more variations of natural samples. The prediction method is simple, rapid, and adaptable to be the theoretical and practical basis for further studying other interdisciplinary research work in enzymology and spectroscopy.

Sensitive and rapid electrochemical detection of nitroaromatic pesticides by the ordered macro-microporous ZIF-8 immobilized lipase-based sensor

The high toxicity and bioaccumulation effects of nitroaromatic pesticides and their serious environmental risks have led to a sharp increase in public concern about pesticide residues. Present work developed a direct, rapid, sensitive and reliable Candida rugosa lipase (CRL)@ the ordered macro-microporous ZIF-8 (MAC-ZIF-8) nanofibers biosensor (CRL@MAC-ZIF-8 nanofibers) for methyl parathion and chlornitrofen residues detection in lake water. The biosensors fabricated by the CRL@MAC-ZIF-8 nanofibers demonstrated wide linear range for both methyl parathion (0.5–114 µM) and chlornitrofen (1–40 µM) detection. The biosensors also demonstrated good reusability for methyl parathion detection with retainment of more than 97% of its initial response after scanning continuously for 100 times. During 1 month storage stability test at 4 °C, the fabricated biosensor demonstrated excellent stability for methyl parathion detection with retainment of 86%-100% of its initial response. Meanwhile, the biosensors also demonstrated good recovery rates when the biosensors were used to detect methyl parathion and chlornitrofen residues in lake water sample.

Ion Exchange Resin and Entrapped Candida rugosa Lipase for Biodiesel Synthesis in the Recirculating Packed-Bed Reactor: A Performance Comparison of Heterogeneous Catalysts

Ion exchange resins and immobilized lipase as heterogeneous catalysts are used to synthesize biodiesel for alternative fossil fuels. The use of ion exchange resins in the solid and activated phase can ease the separation process. Furthermore, resins can be reactivated and used repeatedly, reducing the need for catalysts. On the other hand, an immobilized enzyme is biodegradable and can catalyze the transesterification process to produce biodiesel with a lower alcohol-to-oil ratio, minimizing side reactions and impurities. Therefore, the catalysts used in this study are ion exchange resins, such as Lewatit MP-64, Amberlite IRA410Cl, and Diaion PK208LH, as well as immobilized Candida rugosa lipase. By using vegetable oil as a feedstock and methanol for the transesterification, biodiesel production was carried out in a packed bed reactor. The present study aims to investigate the optimum process parameters, including the concentration of resin and enzyme, resin activation time, resin types, flowrate, and stability of resin and enzyme on the biodiesel yield. The results showed that the optimum conditions for biodiesel production with ion exchange resin were 4 g of resin, activated for 3 h, and synthesized for 3 h; Lewatit obtained a biodiesel yield of 94.06%, Amberlite obtained 90.00%, and Diaion obtained 73.88%. Additionally, the stability test of the reactivated Lewatit resin showed that it still has the capability of producing biodiesel with a yield of more than 80% after three regeneration cycles. In contrast, Candida rugosa lipase as was immobilized by entrapment in sodium alginate before being used in the biodiesel production for 12 h. The results showed that lower flowrate in enzymatic biodiesel synthesis produced a higher amount of biodiesel, of up to 71.1%. Nonetheless, immobilized lipases can be used up to three times without a significant loss in biodiesel yield.

Improvement of the enantioselectivity of lipase catalyzed reactions with deoxycholic acid‐based molecular tweezers

AbstractBACKGROUNDLipase as a hydrolase has important applications in the synthesis of chiral drugs. Based on the special interfacial activity of lipase, bile salt as a biosurfactant can effectively improve the catalytic activity of lipase.RESULTSIn this work, the strong selective recognition of the molecular tweezers and guest molecules helps improve the guest molecule concentration near the lipase catalytic center. However, this strong recognition will also make it difficult for the guest molecules to break away from the molecular tweezers. Therefore, there is an optimal value for this recognition that can effectively improve the chiral resolution effect of lipase.CONCLUSIONSThe two hydroxyl groups on deoxycholic acid were modified to prepare deoxycholic acid‐based molecular tweezers with two ‘arms’ and one ‘crack.’ The recognition performance between the molecular tweezers and amino acid methyl ester was determined by UV spectrophotometry. According to the binding constant, Ka, and free energy, −ΔG0, a complex formed between the deoxycholic acid‐based molecular tweezers (host) and amino acid methyl ester (guest) due to chiral recognition. Molecular tweezers were subsequently mixed with porcine pancreatic lipase (PPL) and candida rugosa lipase (CRL), respectively, to catalyze the transesterification of racemic tryptophan methyl ester. © 2023 Society of Chemical Industry.

Vacuum-assisted adsorption of Candida rugosa lipase with nanotubes for monooleoglycerol synthesis

A surplus of crude glycerol has been produced as a by-product due to the escalating production of biofuels. Utilization of crude glycerol for monoolein production which offers a different approach using the abandoned crude glycerol from the biodiesel sector is the main aim of this study. Immobilized lipase was used in this investigation to catalyse the direct esterification of oleic acid and glycerol, which resulted in monoolein. The immobilized Candida rugosa lipase onto the halloysite nanotubes (HNTs) has shown remarkably promising attainment of lipase activity and loading capacity. The immobilised lipase was optimised for crucial parameters (HNT to lipase ratio, pH, temperature, and pressure) via response surface methodology (RSM). The recommended optimum conditions obtained are pH 6, 38.6 °C, and 0.33 atm (81.92% lipase activity). To analyse influences of reaction period, temperature, mixing rate, and crude glycerol to oleic acid molar ratio on the esterification reaction was carried out using the optimized conditions of immobilization process. The optimum conditions for physically immobilised lipase were to yield 88.97% monoolein produced at 42 °C in a substrate ratio of 1:4 (oleic acid: glycerol) stirred at 200 rotations per minute (rpm) with 16 h’ incubation time. Based on the research's findings, it was confirmed that HNT has good application potential for enzyme immobilization carriers.

The effect of ethanol solvent on thermostability and esterification activity of immobilized Candida rugosa lipase on celite

Lipase is an enzyme commonly applied as a catalyst in the food industry, the synthesis of bioplastics, and the production of biodiesel. Candida rugosa lipase (CRL) is one of the widely used lipases due to its high selectivity and activity. However, high operating temperatures can denature enzymes, affecting enzyme activity due to structural changes. An enzyme can be immobilized on a supporting matrix to improve stability. Ethanol as an immobilization solvent may also increase the thermostability and esterification activity. This study examines the effect of ethanol solvent on the thermostability of immobilized Candida rugosa lipase. Immobilization was carried out on the Celite-545 matrix in buffer/ethanol solution. Different operating conditions such as enzyme concentrations, immobilization temperatures, immobilization time, and immobilization pH were observed. The immobilized enzyme activity was determined by esterification reaction using butyric acid and 1-butanol as substrates and isooctane as reaction medium. The results showed that immobilization increased the esterification activity by 76% at 37°C. Thermostability of immobilized enzyme was also enhanced by 29-51%, depending on the esterification temperature.

Stability and Esterification Activity of Candida rugosa Lipase Immobilized on Celite with Acetone Solvent

Candida rugosa lipase (CRL) is an enzyme that is widely used in biopharmaceutical and biopesticide industries due to its high catalytic ability and reusability. However, conditions from industrial processes that are often outside CRL stability range may cause denaturation or destabilization of the enzyme, causing it to be not reusable, hence lowering its economic value. To increase its catalytic activity, stability, and reusability, CRL enzyme can be immobilized in a matrix. In this study, the CRL was physically adsorbed onto Celite-545. The immobilization process was done using buffer solution with the addition of acetone. The influence of initial enzyme concentration and immobilization condition (time, temperature, and pH) were optimized using OFAT method. The data were taken in terms of esterification activity, thermal stability, and protein content. The result showed good thermal stability of the immobilized biocatalysts and an increase in esterification activity at optimum conditions that supported the efficiency of Celite-545 as lipase immobilization supports.

Enzymatic hydrolysis for the removal of 3- monochloropropanediol esters in edible oils using Candida rugosa lipase in the presence of deep eutectic solvents and nanocellulose

3-monochloropropanediol ester (3-MCPDE) is identified as a food-borne contaminant in edible oils and are classified as a possible carcinogen. This study reports an efficient enzymatic technique for the removal of 3-MCPDE from extra virgin olive oil (EVO) using Candida rugosa lipase (CRL) as the biocatalyst in the presence of choline chloride and fructose-based natural deep eutectic solvent (NADES) and nanocellulose (NC) extracted from almond shells. The validity of the method was confirmed by gas chromatography mass spectrometry (GC-MS) showing adequate precision with relative standard deviation values ≤ 2.37%. The quantification and detection limit are within the permissible levels of 3-MCPD in edible oils. Under optimized conditions of 30 min at 90 °C with 60 µL of phenylboronic acid (PBA), the enzymatic hydrolysis resulted in the removal of 79.8% of spiked 3-MCPD in EVO. No adverse effects of the EVO were detected from this technique with respect to the oil quality testing. The application of DESs and NCs as the support material for the CRL biocatalyst for the removal of 3-MCPDE has yet to be explored. This could have a significant impact on the edible oil industry for producing oils of higher quality free from 3-MCPDE.

Wax esters from waste fish oil catalysed by immobilized Candida rugosa lipase

Herein, the evaluation of waste fish (WF) oils as feedstock for the production of wax esters through transesterification reactions was reported. In particular, the synthesis of an emollient ester in the presence of oleyl alcohol and using an immobilized lipase catalyst was proposed. Lipase from Candida rugosa (CRL) was immobilized (0.5 enzyme/support wt. ratio) on a magnetic amino-functionalized hypercross-linked resin (M-HCLR-NH2) by ion exchange, interfacial activation, and covalent anchoring, mediated by 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide (EDC). The experimental results demonstrated the successful immobilization of lipase on the as-produced M-HCLR-NH2 with a high immobilization yield of 90%. The immobilized CRL showed very high activity and an activity recovery of ∼ 94%. The immobilized enzyme on the magnetic resin results in more stability and less sensitivity to temperature change than the free counterpart. In addition, during emollient ester synthesis in a solvent system, the immobilized enzyme exhibited very high activity, stability, and excellent reusability, with a yield of 94% after 12 h at 45 °C at an oil:alcohol molar ratio of 1:4 and an immobilized enzyme concentration of 15 wt%./wt of oil. Esters synthesized showed excellent physicochemical properties.

Conjugation of Candida rugosa lipase with hydrophobic polymer improves esterification activity of vitamin E in nonaqueous solvent

We described a novel polymer-lipase conjugate for high-efficient esterification of vitamin E using vitamin E and succinic anhydride as the substrates in nonaqueous media. In this work, the monomer, N-isopropylacrylamide (NIPAM), was grafted onto Candida rugosa lipase (CRL) to synthesize poly(NIPAM) (pNIPAM)-CRL conjugate by atom transfer radical polymerization via the initiator coupled on the surface of CRL. The result showed that the catalytic efficiencies of pNIPAM-CRL conjugates (19.5–30.3 L·s−1·mmol−1) were at least 7 times higher than that of free CRL (2.36 L·s−1·mmol−1) in DMSO. It was attributed to a significant increase in Kcat of the conjugates in nonaqueous media. The synthesis catalyzed by pNIPAM-CRL conjugates was influenced by the length and density of the grafted polymer, water content, solvent polarity and molar ratio of the substrates. In the optimal synthesis, the reaction time was shortened at least 7 times, and yields of vitamin E succinate by pNIPAM-g-CRL and free CRL were obtained to be 75.4% and 6.6% at 55 °C after the reaction for 1.5 h. The result argued that conjugation with pNIPAM induced conformational change of the lid on CRL based on hydrophobic interaction, thus providing a higher possibility of catalysis-favorable conformation on CRL in nonaqueous media. Moreover, pNIPAM conjugation improved the thermal stability of CRL greatly, and the stability improved further with an increase of chain length of pNIPAM. At the optimal reaction conditions (55 °C and 1.5 h), pNIPAM-g-CRL also exhibited good reusability in the enzymatic synthesis of vitamin E succinate and kept ∼70% of its catalytic activity after ten consecutive cycles. The research demonstrated that pNIPAM-g-CRL was a more competitive biocatalyst in the enzymatic synthesis of vitamin E succinate and exhibited good application potential under harsh industrial conditions.

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.

Solid‐Phase Lipase‐CuNPs Biohybrids as Catalysts for One‐Pot Parallel Synthesis of 2,3,4‐Triacetyl‐D‐Gluconic Acid

AbstractSolid‐phase lipase/metal nanobiohybrids, generated by growth of copper nanoparticles on enzyme matrixes immobilized on graphene, were used as heterogeneous catalysts with dual‐activity for the regioselective production of 2,3,4‐triacetyl‐D‐gluconic acid from α‐peracetylated‐glucose in a one‐pot parallel process combining a lipase‐mediated regioselective hydrolytic monodeprotection with a metal‐catalyzed oxidation in aqueous media. A novel synthetic strategy, based on the in situ fabrication of Cu nanoparticles induced by lipase molecules specifically immobilized on a multi‐layer graphene material by interfacial adsorption fixing them in the active open conformation, has been described. Thermomyces lanuginosus lipase was firstly used to prepare the functionalized multi‐layer graphene from graphite as a biographene preparation (Biographene, BIOG), support used to successfully immobilize Candida rugosa lipase (CRL). This immobilized form BIOG‐CRL was further used to create successful active bifunctional enzyme‐metal nanoarchitectures. Two different Cu‐lipase hybrids were synthesised, where Cu species and nanoparticles size were different depending on the methodology. Regioselectivity and stability of the hybrids were evaluated successfully in the production of monosaccharide building blocks, besides the robustness of the hybrids in recyclability experiments. These findings highlight the potential of these solid‐phase nanoarchitectures as useful tools in the synthesis of complex glycoderivatives for use in food, medicine, and cosmetics.

Concentrating omega-3 fatty acids in Nannochloropsis oceanica oil by using enzyme immobilized nano-silica systems

Burning agricultural waste causes local air pollution and global warming. Agricultural waste, such as rice husks, contains silica reserves that can be processed into nano silica and used as a substrate for the immobilization of enzymes. The present study attempts to develop efficient silica-based nano-biocatalysts for concentrating omega-3 fatty acids in algal oil by hydrolyzing specific short-chain fatty acids. In this regard, tetraethyl orthosilicate (TEOS) and rice husk ash (RHA) were used as precursors to obtain nano-silica supports. Candida rugosa lipase (CRL) was then physically adsorbed and chemically cross-linked onto the created silica nano supports. The immobilized nano-systems created using the physical adsorption approach showed the maximum binding efficiency and enzymatic activity of CRL. The protein content of the CRL physically adsorbed in TEOS was 925 mg/g, while the protein loading in the RHA systems was 938 mg/g. However, the enzymatic activity of the selected systems for hydrolyzing para nitro-phenol palmitate (100 mM PBS buffer at pH-7.2, for 10 min at 37 °C) was 56 units/mg for the RHA system and 707 units/mg for the TEOS system. These two nano-enzyme systems were utilized further to hydrolyze the Nannochloropsis oceanica oil, and a physically adsorbed CRL TEOS system was found to have a 2.5-fold enrichment in eicosapentaenoic acid (EPA) which is used in food and pharmaceutical applications.

Chicken tallow, a low-cost feedstock for the two-step lipase-catalysed synthesis of biolubricant

Biolubricants are oleochemical esters that are non-toxic to the environment, and they are preferred because of their high-performance activities in various automobile and industrial fields. In this study, chicken tallow has been explored as an alternative to a non-renewable source, which acts as a low-cost feedstock for synthesizing biolubricants in a two-step hydro esterification process catalysed by Candida rugosa lipase (CRL). The enzymatic hydrolysis of chicken tallow is optimized at different time intervals at 40 °C, and the maximum release of 160.2 µmol min−1 free fatty acids (FFAs) is observed at 100 min. The hydrolysed FFAs are characterized by the gas chromatography-mass spectrometry (GC-MS) analysis that has depicted the fatty acid methyl esters (FAMEs) composition of the tallow oil. The recovered FFA is then enzymatically esterified with trimethylolpropane (TMP) to produce TMP esters. Optimization studies are performed to explore the effect of factors such as time, temperature, amount of the enzyme, and TMP: FFA molar ratio for the esterification process. Thus, the optimal conditions for achieving a maximal hydroxyl group conversion of 60% during TMP ester production include incubation time (120 min), temperature (35 °C), lipase concentration (3% w/w), and TMP: FFA molar ratio (1:4). Fourier transform infrared spectroscopy (FT-IR), and nuclear magnetic resonance (NMR) studies confirm the synthesized TMP esters. Moreover, physicochemical properties such as viscosity, cloud point (CP), and calorific value (CV) are found to be 36 mm2/s, 7159.14 cal/g, and 20 °C, respectively.