Commercial Lipase Research Articles

Design of green DES-based aqueous two phase systems for lipolytic enzymes extraction

This study pioneers a novel green and cost-effective separation process using reline deep eutectic solvent (DES) for extracting Thermomyces lanuginosus lipase (TlL) from aqueous solutions. For the first time, it has been shown that various potassium organic salts combined with reline DES can induce phase separation in aqueous solutions of the biodegradable non-ionic surfactant Tergitol 15-S9, presenting a groundbreaking alternative to the banned surfactant Triton X-100. This work uniquely applies three different equations to describe the experimental solubility curves. The selection of reline DES is particularly innovative as it avoids significant deactivation effects with three commercial lipases: Candida antarctica lipases A and B, and TlL, with the latter undergoing a detailed study. This includes an unprecedented analysis of its thermodeactivation kinetics in reline DES aqueous solutions. Additionally, a novel investigation of immiscibility regions at different temperatures, involving comprehensive Tie-Line characterizations (slope and length), preceded the evaluation of lipase extraction. Remarkably, the study demonstrates that optimizing the feed composition to contain higher water concentrations can achieve exceptionally high extraction efficiencies (approximately 90%), suggesting a transformative potential for future applications in real aqueous cultures.

Ultrasound assisted enzymatic synthesis of OPO structured lipids by covalent organic framework immobilized lipase

In this study, the covalent organic framework immobilized Rhizomucor miehie lipase COF@RML as a novel biocatalyst was applied in the enzymatic synthesis of OPO structured lipids (1, 3-dioleoyl-2-palmitoylglycerol). The impact of reaction medium, substrate molar ratio, enzyme addition amount, reaction time and temperature on the enzymatic synthesis of OPO structured lipids were studied. Furthermore, the effects of ultrasonic power and ultrasonic time on the synthesis of OPO structural lipids were studied. The results showed that ultrasonication could increase the yeild of OPO structured lipids by improving substrate mass transfer and enzyme particle dispersion. The optimal process for the synthesis of OPO structured lipids was obtained. When the ultrasonic power was set at 90 W, ultrasonic time at 12 minutes, enzyme addition amount at 10 wt%, substrate molar ratio at 1:8, reaction temperature at 45 °C, and reaction time at 6 hours, the yield of OPO structured lipids reached a remarkable 51.27 %. Finally, the commercial lipase Lipozyme RM IM was compared with the COF@RML. The findings indicated that COF@RML immobilized enzyme had better application value in the synthesis of OPO structured lipids.

Sustainable Biocatalytic System for the Enzymatic Epoxidation of Waste Cooking Oil.

The present study is integrated in a global effort to capitalize waste cooking oil (WCO) into versatile compounds by introducing an oxirane ring into the unsaturated carbon chain of fatty acid residues (the epoxidation of double bound). Therefore, an enzymatic method was set up for the epoxidation of artificially adulterated WCO (SFw) and WCO under real conditions (SFr) derived from sunflower biomass. Commercial lipase (Novozyme, NZ) was used as a biocatalyst for generating the peracid requested by the epoxidation pathway. Optimum experimental conditions (e.g., 1.5 wt% NZ, 1:1:0.5 = H2O2/double bonds/peracid precursor (molar ratio) and 12 h reaction time) allowed for the conversion of 90% of the SFw substrate into products with an oxirane ring. Octanoic acid was selected as the best peracid precursor. The versatility of the developed system was tested for olive, milk thistle, hemp and linseed oils as both fresh and WCO samples. The characterization of the oil samples before and after the enzymatic epoxidation allowed for the evaluation of the system performance. SFw/SFr exhibited a better susceptibility to enzymatic epoxidation. In addition, the reusability of the biocatalytic system was investigated. Furthermore, different strategies, such as biocatalyst coating and the addition of organic solvents/buffers were applied, limiting enzyme leaching, for the better recovery of the biocatalyst activity.

Substrate specificity of commercial lipases activated by a hydration–aggregation pretreatment in anhydrous esterification reactions

Substrate specificity in non-aqueous esterification catalyzed by commercial lipases activated by hydration–aggregation pretreatment was investigated. Four microbial lipases from Rhizopus japonicus, Burkholderia cepacia, Rhizomucor miehei, and Candida antarctica (fraction B) were used to study the effect of the carbon chain length of saturated fatty acid substrates on the esterification activity with methanol in n-hexane. Hydration–aggregation pretreatment had an activation effect on all lipases used, and different chain length dependencies of esterification activity for lipases from different origins were demonstrated. The effects of various acidic substrates with different degrees of unsaturation, aromatic rings, and alcohol substrates with different carbon chain lengths on esterification activity were examined using R. japonicus lipase, which demonstrated the most remarkable activity enhancement after hydration–aggregation pretreatment. Furthermore, in the esterification of myristic acid with methanol catalyzed by the hydrated–aggregated R. japonicus lipase, maximum reaction rate (5.43 × 10−5 mmol/(mg-biocat min)) and Michaelis constants for each substrate (48.5 mM for myristic acid, 24.7 mM for methanol) were determined by kinetic analysis based on the two-substrate Michaelis-Menten model.

Lipase‐catalyzed transesterification of virgin and refined hemp seed oil with ferulic acid ethyl ester

AbstractThe transesterification of ethyl ferulate (EF) and unrefined, virgin, cold pressed hemp seed oil (HOV) and refined, bleached, deodorized cold pressed hemp seed oil (HOR) using a commercial lipase, Novozym 435 (Candida antarctica B lipase immobilized on an acrylic resin), was examined in 150‐mL, shaken, batch reactions at 60°C for 2 weeks. The reactions produced feruloylated hemp seed oils, FHOV and FHOR, respectively, and the reactions were monitored to determine the difference between virgin and refined hemp seed oil on the transesterifications. The FHOV and FHOR reactions both reached EF conversion equilibrium of 58% after ca. 168 h. Ultraviolet (UV) absorbing and antioxidant capacity of the FHOV and FHOR were determined. Both FHOV and FHOR (50 μM in ethanol) were excellent UVA II absorbers, λmax 322 nm, and exhibited absorption into the UVB. The DDPH* radical (200 μM) scavenging of the FHOV and FHOR (0.25–2.5 mM) were both shown to be rapid antioxidants (50% DDPH* radical scavenged in <5 min) at 1.0 and 2.5 mM suggesting that inherent components contained in the HOV did not adversely affect enzyme activity relative to transesterification using HOR. Overall, using less expensive, unrefined, virgin hemp seed oil versus more expensive, refined hemp seed oil did not appreciably affect the enzyme kinetics of the transesterification reactions nor the UV absorbing and antioxidant efficacy of the resultant feruloylated hemp seed oils, making FHOV a less expensively produced feruloylated hemp seed oil for cosmetic and personal care applications.

Effect of commercial lipase incorporation on technological properties of bread

Additives are used to improve bread quality; for this reason, lipase stands out, as it simulates the effect of emulsifiers. The present work aimed to verify the effect of commercial lipase addition on the physical and technological characteristics of the bread. The first stage used 33 factorial design namely three enzymes, five concentrations, and three fermentation times. The second stage used 23 factorial design, and stored over 28 days, with two formulations (control and with enzyme) and three storage temperatures. The PCA identified the variables with the most significance in PC1: specific volume, volume, and variables related to texture and expansion. In the second stage (shelf life), the PCA revealed that the bread samples with enzymes in the formulation differed from the control samples. Based on the circle of correlations, enzyme samples were distinguished by lower chewiness, gumminess, and hardness. Microscopic analysis showed that the starch molecules were more uniform in the first 14 days. However, the starch molecules lost their conformational structure afterwards. Therefore, the incorporation of lipase improved the bread’s technological parameters such as volume, texture, and structure.

Utilization of Fusarium Solani lipase for enrichment of polyunsaturated Omega-3 fatty acids.

Omega-3 fatty acids, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), offer numerous health benefits. Enriching these fatty acids in fish oil using cost-effective methods, like lipase application, has been studied extensively. This research aimed to investigate F. solani as a potential lipase producer and compare its efficacy in enhancing polyunsaturated omega-3 fatty acids with commercial lipases. Submerged fermentation with coconut oil yielded Lipase F2, showing remarkable activity (215.68 U/mL). Lipase F2 remained stable at pH 8.0 (activity: 93.84 U/mL) and active between 35 and 70°C, with optimal stability at 35°C. It exhibited resistance to various surfactants and ions, showing no cytotoxic activity in vitro, crucial for its application in the food and pharmaceutical industries. Lipase F2 efficiently enriched EPA and DHA in fish oil, reaching 22.1mol% DHA and 23.8mol% EPA. These results underscore the economic viability and efficacy of Lipase F2, a partially purified enzyme obtained using low-cost techniques, demonstrating remarkable stability and resistance to diverse conditions. Its performance was comparable to highly pure commercially available enzymes in omega-3 production. These findings highlight the potential of F. solani as a promising lipase source, offering opportunities for economically producing omega-3 and advancing biotechnological applications in the food and supplements industry.

A Whole-Process Visible Strategy for the Preparation of Rhizomucor miehei Lipase with Escherichia coli Secretion Expression System and the Immobilization.

Rhizomucor miehei (RM) lipase is a regioselective lipase widely used in food, pharmaceutical and biofuel industries. However, the high cost and low purity of the commercial RM lipase limit its industrial applications. Therefore, it is necessary to develop cost-effective strategies for large-scale preparation of this lipase. The present study explored the high-level expression of RM lipase using superfolder green fluorescent protein (sfGFP)-mediated Escherichia coli secretion system. The sfGFP(-15) mutant was fused to the C-terminus of RM lipase to mediate its secretion expression. The yield of the fusion protein reached approximately 5.1g/L with high-density fermentation in 5-L fermentors. Unlike conventional secretion expression methods, only a small portion of the target protein was secreted into the cell culture while majority of the fusion protein was still remained in the cytoplasm. However, in contrast to intracellular expression, the target protein in the cytoplasm could be transported efficiently to the supernatant through a simple washing step with equal volume of phosphate saline (PBS), without causing cell disruption. Hence, the approach facilitated the downstream purification step of the recombinant RM lipase. Moreover, contamination or decline of the engineered strain and degradation or deactivation of the target enzyme can be detected efficiently because they exhibited bright green fluorescence. Next, the target protein was immobilized with anion-exchange and macropore resins. Diethylaminoethyl sepharose(DEAE), a weak-basic anion-exchange resin, exhibited the highest bind capacity but inhibited the activity of RM lipase dramatically. On the contrary, RM lipase fixed with macropore resin D101 demonstrated the highest specific activity. Although immobilization with D101 didn't improve the activity of the enzyme, the thermostability of the immobilized enzyme elevated significantly. The immobilized RM lipase retained approximately 90% of its activity after 3-h incubation at 80°C. Therefore, D101 was chosen as the supporting material of the target protein. The present study established a highly efficient strategy for large-scale preparation of RM lipase. This innovative technique not only provides high-purity RM lipase at a low cost but also has great potential as a platform for the preparation of lipases in the future.

Use of hydroxyapatite as a support in the immobilization of Thermomyces lanuginosus lipase for application in the production of biodiesel using a by-product as lipid raw material.

The use of new materials in the field of biofuel production has been represented as a step in the development of remarkable catalysts. The use of lipases in the production of biodiesel is often seen as a cost-limiting step, as the operating expenses in recovering such catalysts can lead to unfeasible market expectations. In this study, hydroxyapatite (HAp) particles were evaluated as a support to immobilize commercial lipase, following application in ethyl ester synthesis. First, hydroxyapatite was synthesized through the co-precipitation method at constant pH and selected as a support to be used in enzyme immobilization. The characterization of the biocatalyst support materials produced was carried out using DRX, BET, FTIR, TGA, and SEM analysis. The lipase from Thermomyces lanuginosus was then immobilized in the matrices, and, subsequently, there was transesterification of the vegetable oil deodorization distillate (VODD). The biodiesel samples generated showed that they were within commercial standards, achieving ester conversion greater than 96.5%. Other properties such as density (0.87 g.cm-3) and viscosity (4.36 mm2.s-1) meet the specifications required by ASTM to be used as a biofuel. In the experiment planning technique, the results revealed an experimental trend and a defined behavior: a higher lipase loading in the immobilization and the use of temperatures in the range of 40-50 °C favor high conversions of ethyl esters. Thus, this confirms that the enzymatic chemical catalyst was able to form the main fatty acid esters even using a residual lipid raw material.

Sustainable one-pot solvent-free enzymatic synthesis of capric acid-rich structured lipids to enhance the nutritional value of grape seed oil

Grape seed oil (GSO) is a valuable resource for the pharmaceutical, cosmetic, and food industries due to its fatty acid composition, which provides anti-inflammatory, cardioprotective, antimicrobial, and anticancer properties. GSO is also recognized for its sensory attributes and serves as an essential component in our dietary intake, providing vital fatty acids for metabolic reactions. The enzymatic synthesis of structured lipids (SL) using GSO was investigated in this study. The SL was created by incorporating medium chain fatty acids (capric acid – C10) at the sn-1 and sn-3 positions and long chain fatty acids in the internal position of the triacylglycerols (TAG). GSO was selected based on its composition rich in unsaturated fatty acids, mainly linoleic acid. The acidolysis reactions were catalyzed by an immobilized commercial lipase Lipozyme TLIM®. The incorporation degree of C10 into GSO reached 36%. Health lipid indices were calculated to determine the C10 incorporation effect on the improvement of nutritional quality of oil. The SL showed lower oxidative stability index (OSI) values than the original oil and higher degree of fatty acid (DFA) values. The simpler, solvent-free method not only enhances the health benefits of oils but also gives valuable properties to GSO, making it an even more attractive option for food industries.

Investigation of Complexing Properties with Polyethyleneimine of Some Commercial Lipases

Lipases are enzymes used in various industrial process and are immobilized to increase their applicability as biocatalysts. Ionic polymers such as polyethyleneimine (PEI) make possible the co-precipitation of enzymes. In this study, complexation and aggregation with PEI of enzymes were investigated with commercial enzymes from Novozyme 51032 (Fusarium solani pisi), Palatase 20000 L (Rhizomucor miehei), Lipolase 100 L (Thermomyces lanuginosus), Lipozyme CAL B L (Candida antarctica B) and Amano (Pseudomonas fluorescens) using PEI as a linker and aggregation agent. The highest percentage of PEI-enzyme agregate was obtained for Novozyme 51032, Palatase 20000 L and Lipolase 100 L at the PEI/enzyme ratio of a 1/20-1/80 range. This study documented that Lipozyme CAL B L and (Amano) P. fluorescens enzyme preparations failed to occur precipitates resulting PEI-enzyme aggregates. The some commercial lipase preparations may contain various impurity components that prevent complexation or aggregation with PEI. Complexing with PEI of lipases is based on of basis electrostatic interaction of enzyme with PEI as a cationic polymer resulting in PEI-lipase aggregates.

Efficient enzymatic synthesis of vitamin E succinate using an organic solvent‐stable immobilized lipase

AbstractVitamin E succinate has gained substantial attention as a potential therapeutic agent for cancer treatment due to its biomedical activities. One of the prominent methods of synthesizing vitamin E succinate is through enzymatic processes, which, although advantageous, presents inherent challenges related to optimization, scalability, and particularly, the poor stability of lipases in organic solvents. Our study addresses these challenges by conducting a comprehensive comparative analysis between Lipase UM1 and three other immobilized commercial lipases, demonstrating Lipase UM1's enhanced resistance to organic solvents and its superior efficiency in vitamin E succinate production. Further optimization experiments with Lipase UM1 led to an unprecedented conversion of 99%. Additionally, we scaled the reaction to a proof‐of‐concept industrial level. The synthesized product was verified using Fourier transform infrared spectroscopy and nuclear magnetic resonance analysis, ensuring its quality and consistency. This study validates Lipase UM1 as an efficient catalyst for vitamin E succinate synthesis, offering a promising avenue for industrial production with potential applications in cancer therapy and beyond.

Evaluation of mesoporous silica particles as a support for lipase immobilization in biodiesel production: Enhanced ethyl ester synthesis from algal oil

The utilization of novel materials in the biofuel production domain has been regarded as a significant advancement in catalyst development. Incorporating lipases in biodiesel manufacturing is often viewed as a cost-restrictive phase since the expenses associated with recovering these catalysts can render market expectations unfeasible. Within this investigation, mesoporous silica (MPS) particles were assessed as a supporting medium for immobilizing commercial lipase, subsequent to their utilization in ethyl ester synthesis. Initially, the production of mesoporous silica was achieved through the co-precipitation technique at a consistent pH and was subsequently chosen as an immobilization support for the enzyme. The characterization of the biocatalyst support materials produced was performed using various analytical methods including X-ray diffraction (XRD), scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), and BET analysis. The Novozym 435 lipase was then immobilized within the matrices, followed by the transesterification process of algal oil derived from Nannochloropsis. The resulting biodiesel samples demonstrated compliance with commercial standards, exhibiting an ester conversion rate exceeding 94.8 %. Additionally, properties such as density (0.93 g cm−3) and viscosity (5.01 mm2 s−1) satisfied the specifications mandated by ASTM for biofuel application. Experimental planning techniques showed that increasing lipase loading and using temperatures between 38 and 48 °C resulted in higher conversion rates of ethyl esters. This confirms the effectiveness of the enzymatic catalyst in generating primary fatty acid esters, even with residual lipid feedstocks.

Lipase-Catalyzed Synthesis of Cetyl Palmitate in the Presence of n-Hexane and Ethyl Ether

Cetyl palmitate was produced by the esterification of palmitic acid with cetyl alcohol using n-hexane and ethyl ether as solvents and a commercial lipase as a catalyst. The effect of solvents to reagents mass ratio (0.5 : 1–3 : 1), percentage of n-hexane in the solvent mixture (0–100%), and reaction temperature (25–55°C) on the reaction rate were evaluated for a fixed amount of enzyme (1 wt% related to the total mass of substrates) in an equimolar mixture of palmitic acid and cetyl alcohol. Temperature and n-hexane percentage in the solvent had positive effects on the reaction rate. The total solvent-to-reagent mass ratio showed a negative effect on the reaction rate when a solvent mixture rich in ethyl ether was used. The higher the concentration of n-hexane, the lower the effect of the mass ratio of solvents to reagents on the reaction rate. Although the amount of ethyl ether in the solvent mixture had a negative effect on the reaction rate, it had a positive effect on the solubility of the system, that is, the more ethyl ether in the mixture, the lower the solvent to reagents mass ratio required to ensure a homogeneous mixture. A ping-pong bi-bi mechanism-based model was proposed to represent the system kinetics and was well fitted to the experimental data.

Green Enzymatic Synthesis of Geranyl Butyrate: Process Optimization and Mechanistic Insights.

Flavor esters are organic compounds widely used in the food industry to enhance the aroma and taste of products. However, most chemical processes for the production of these flavoring compounds use toxic organic solvents. Some organic solvents derived from petroleum can leave behind residual traces in food products, which may raise concerns about potential health risks and contamination. In this study, we employ Eversa Transform 2.0, a commercial lipase derived from the lipase from Thermomyces lanuginosus, to produce geranyl butyrate in aqueous media. The chemical process was optimized using the Taguchi method, and a conversion of 93% was obtained at the optimal reaction conditions of: 1:5 molar ratio (v/v), 15% biocatalyst load (w/w), at 50 °C, in 6 h. Classic (molecular dynamics) and quantum (density functional theory) simulations unveiled amino acid residues involved in the stabilization of the enzyme-substrate complex. Detailed QM/MM mechanistic studies identified the nucleophilic attack of the deacylation reaction as the rate-limiting step of the entire mechanism, which has a free energy barrier of 14.0 kcal/mol.

Lipase-catalyzed synthesis of laurate esters from puerarin and its β-D-fructofuranosyl-(2→6)-puerarin derivative

This study investigates the transesterification of isoflavones puerarin and β-D-fructofuranosyl-(2→6)-puerarin to enhance their lipophilicity for specific pharmacological applications. The process achieved high yields (80-100%) in the isoflavones conversion when commercial lipases from Candida antarctica (CALB) and Thermomyces lanuginosus (TL IM) were used. The acylation patterns between CALB and TL IM varied depending on the substrate; both enzymes yielded principally monoacylated products from puerarin and β-D-fructofuranosyl-(2→6)-puerarin. However, TL IM also produced di- and triacylated derivatives of β-D-fructofuranosyl-(2→6)-puerarin in substantial amounts. Structural characterization by NMR identified puerarin-6ʹʹ-O-laurate and β-D-fructofuranosyl-(2→6)-puerarin-6ʹʹʹ-O-laurate as the primary products in CALB and TL IM reactions. These acyl derivatives presented improved lipophilicity (Log P from −1.0 − 0.16 to 3.26 − 3.58) and antioxidant activity (67.2-97.3 to 102.5-131.4 µM TE/mmol). Therefore, these compounds have potential pharmacological applications.

Preparation of functional oils rich in phytosterol esters and diacylglycerols by enzymatic transesterification

Phytosterol esters (PEs) and diacylglycerols (DAGs) have various health benefits in humans. In this study, PEs and DAGs were synthesized by lipase-catalyzed transesterification between a natural oil and phytosterols. First, commercial lipases were screened for transesterification and were further verified using multiple-ligand molecular docking. AYS “Amano” (a lipase from Candida rugosa) was found to be the optimum lipase. Subsequently, the enzymatic transesterification conditions were optimized. The optimized conditions were determined to be a 1:2 M ratio of phytosterols to oil, 100 mmol/L phytosterols, and 9 % AYS “Amano”, and 50 °C for 24 h in 20 mL n-hexane. Under these conditions, over 70 % of phytosterols were converted to PEs. In this study, an efficient enzymatic process was developed to produce value-added functional oils rich in PEs and DAGs, with PEs content ≥ 31.6 %, DAGs content ≥ 11.2 %, acid value ≤ 0.91 mg KOH/g, and peroxide value ≤ 2.38 mmol/kg.

Lipases: market study and potential applications of immobilized derivatives

AbstractThe enzyme market is growing constantly, and lipases, in their free and immobilized forms, constitute an important part of this market. This study aimed to analyze and discuss current market scenarios using data from the literature, focusing on lipases and the feasibility of their use in the immobilized form. It also compares the cost of biodiesel production using two commercial lipases: Eversa Transform 2.0 (free lipase) and Novozyme 435 (immobilized lipase). The results show that the European and North American enzyme markets are the most prominent worldwide, with Denmark and the USA as the major exporters and importers, respectively. Lipases can be used in a wide variety of fields, and immobilization brings many advantages to this enzyme class. Finally, we show the use of immobilized enzymes and lipases in biorefinery and food industries and carry out a comparative cost analysis to produce biodiesel using a free and immobilized lipase. We concluded that the enzyme market, in which lipases play an important role, is extensive, and that immobilized lipases seem to be excellent biocatalysts with feasible costs for industrial applications.

Innovative approach for semi‐continuous production of puerarin palmitate via transesterification with novel immobilized lipase

BackgroundPuerarin, a flavonoid abundantly found in the roots of Pueraria lobata, exhibits antioxidant, anti-cancer, anti-inflammatory, and anti-aging properties. Despite its numerous benefits, the low bioavailability of puerarin hinders its widespread industrial application. To improve this, flavonoid acylation is required. Lipase is a biocatalyst primarily used for flavonoid acylation; immobilized lipase is preferred given the limited stability of free lipase. Although research on flavonoid acylation for structural modification is actively being conducted, there is a need to explore continuous production for enhancing the industrial application of flavonoid esters. Therefore, in this study, we aimed to produce puerarin palmitate innovatively by selecting an effective lipase, developing an immobilized lipase, characterizing the enzymatic reaction, and designing a semi-continuous reactor system.ResultsLipase from Thermomyces lanuginosus (TL) was found to be the most suitable for the synthesis of puerarin palmitate; it was successfully immobilized on glutaraldehyde-activated silica gel (GASG). TL GASG showed greater solvent, thermal, and operational stability than those of commercial immobilized lipases. Furthermore, TL GASG use resulted in a puerarin palmitate conversion rate of 97.17% within 3 h. Additionally, the reusability of TL GASG for this reaction was higher than that of commercially available immobilized lipases. Upon using a semi-continuous reactor, the cumulative conversion rate of puerarin palmitate remained above 85% after more than ten substrate injections.ConclusionsThe results support the feasibility of the continuous production of flavonoid esters, underscoring their diverse industrial applications. A biorefinery strategy is anticipated to be proposed to utilize P. lobata extract biomass for flavonoid ester production. Moreover, optimizing continuous reactors through statistical experimental design is expected to enhance flavonoid ester production efficiency; these aspects will be explored in future research.Graphical

Pretreatment of shrimp shells with an acidic deep eutectic solvent system for chitin extraction and its enhanced performance as a carrier for immobilized lipase

Existing methods for chitin extraction usually produce substantial waste, which poses ecological hazards. Natural deep eutectic solvent (NADES) offers a promising one-step pretreatment alternative, replacing the resource-intensive demineralization (DM) and deproteinization (DP) process. Hence, in this study, the influence of various acidic NADES, on achieving a simplified one-step DM and DP in the chitin extraction process was investigated. The study yielded chitin with 87.73 % purity, and microstructural analysis showed that NADES pretreatment minimally affected chitin quality without deacetylation. In addition, chitin extracted using choline chloride-oxalic acid as a carrier displayed excellent performance in the immobilization of Geobacillus thermocatenulatus lipase 2 (GTL2) because of obvious Ca2+ activation effect. This process contributed to enhancement of immobilized enzyme activity. The immobilized GTL2 showed excellent hydrolytic capabilities, with its highest activity reaching 547.80 ± 20.62 U/mg, significantly better than the five commercial lipases that exhibited <40 % of the enzyme activity. Furthermore, the hydrolytic capacity of immobilized GTL2 was notably high for 4-nitrophenyl butyrate, measuring 935.47 ± 51.60 U/mg. This study provided a constructive approach for the one-step pretreatment of shrimp shells with organic acid-based NADES to isolate and purify chitin and its potential application as an immobilized carrier to enhance enzyme activity.