Immobilization Of Candida Rugosa Lipase Research Articles

Enhanced catalytic performance of Candida rugosa lipase through immobilization on zirconium-2-methylimidazole: A novel biocatalyst approach

Immobilization of enzymes on suitable supports is a critical approach for enhancing enzyme stability, reusability, and overall catalytic efficiency. This study explores the immobilization of Candida rugosa lipase on zirconium-based 2-methylimidazole (ZrMI) nanoparticles, aiming to develop a stable and reusable biocatalyst. The ZrMI was produced via a solvothermal technique and analyzed using various characterization methods. Candida rugose lipase was immobilized using cross-linking agents, achieving an 87 % immobilization efficiency. The immobilized enzyme exhibited significantly enhanced thermal stability, broader pH tolerance, and increased catalytic efficiency compared to free C. rugose lipase. The ZrMI@lipase retained 69 % of its enzymatic activity following a 60-day storage period at 4 °C. Notably, it displayed significant reusability, maintaining 65 % of its original activity after undergoing 15 catalytic cycles. Examination of the kinetics revealed that the immobilized enzyme possessed a heightened substrate affinity (Km of 4.1 mM) and maximal reaction rate (Vmax of 85.7 μmol/ml/min) in comparison to the free enzyme (Km of 5.4 mM and Vmax of 69 μmol/ml/min), indicating enhanced catalytic efficiency. Validation through zeta potential and hydrodynamic size assessments verified the successful binding of the enzyme and the consistent colloidal characteristics. These results suggest that ZrMI is a promising support for C. rugose lipase immobilization, offering improved stability and reusability for various industrial applications. The study highlights the potential of ZrMI@lipase as an efficient and durable biocatalyst, contributing to advancements in enzyme immobilization technology and sustainable industrial processes.

Functionalized ZnFe2O4@Mesoporous silica nano-support for lipase enzyme immobilization: Enhanced biocatalysis and antibacterial activity for food industry applications

Enzymes, as highly efficient biocatalysts, play a crucial role in diverse biotechnological applications. However, enzyme (re)purification and recovery challenges pose substantial obstacles, rendering them costly for industrial utilization. Addressing this, our study developed mesoporous zinc ferrite nanoparticles coated with amine-functionalized mesoporous silica structure (ZnFe2O4@MS) through the solvothermal method. These nanoparticles are an effective nano-support for immobilizing Candida rugosa Lipase (CRL), ensuring easy separability and recoverability through a magnetic field and providing a significant surface area for high mass transference capacity. Immobilizing the lipase on the nano-support (ZnFe2O4@MS/CRL) through covalent bonds on the surface and within the pores led to a notable increase in enzyme activity from 324 U/mg for free enzyme to 689 U/mg for immobilized enzyme. This indicates unchanged active sites post-immobilization, accompanied by enhanced catalytic activity. The immobilized lipases demonstrated prolonged stability at elevated temperatures, maintaining over 59% of initial catalytic activity through five cycles. Furthermore, ZnFe2O4@MS/CRL exhibited substantial antibacterial activity against Staphylococcus aureus, more than that observed in Gram-negative bacteria. The immobilized lipase was also effective in synthesizing banana flavor (isoamyl acetate) in n-hexane, achieving 64% esterification at 45 °C after 4 h. Overall, the study underscores the industrial promise of this immobilized enzyme system, emphasizing its potential for sustainable and cost-effective biotechnological processes.

Co-immobilization of magnesium precursor and Candida rugosa lipase on alumina via covalent bonding for biodiesel production

Catalyst development is one of the most challenging aspects in biodiesel production due to the compatibility issue with the feedstocks and catalyst poisoning upon exposure to process conditions. In the present research, chemoenzymatic catalyst was prepared by impregnation of magnesium aluminate (MA) on alumina support, modifying the support using activating agent, and immobilization of Candida rugosa lipase (CRL) onto the support. The optimum immobilization condition was resulting in 85.07 % of activity recovery of immobilized lipase, equivalent to 30.53 % of immobilization efficiency. The catalysts were characterized through XRD, FTIR, FESEM-EDX, NAA and CO2-TPD confirming the mixture of AlO(OH), magnesium aluminate (MA), (MgNO3)2 and Mg2+ on the support. CRL/L-lysine/MA showed remarkable improvement in thermal stability (40 − 70℃), solvents tolerance (ethanol, methanol, isopropanol, tert-butanol), and storage stability at 30 ℃, as compared to free CRL. CRL/L-lysine/MA successfully produced biodiesel yield up to 87.10 % at 200.71 rpm of agitation speed, 13.58 % (w/w) of water content, 10 h reaction time, 15 % (w/w) of catalyst loading, at 50 ℃, using 1:12 of oil to methanol molar ratio, and retaining 46.60 % of biodiesel yield after six cycle. The CRL/L-lysine/MA demonstrated novel catalysts characteristics comprising chemical and biological active species on a single support for biodiesel production from waste cooking oil (WCO).

Scientific prospecting of lignocellulosic and inorganic supports used to immobilize Candida rugosa lipase in the biodiesel production process

Scientific prospecting of lignocellulosic and inorganic supports used to immobilize Candida rugosa lipase in the biodiesel production process

Thermo-Switchable Enzyme@Metal-Organic Framework for Selective Biocatalysis and Biosensing.

The stimulus-responsive regulation of enzyme catalytic activity and selectivity provides a new opportunity to extend the functionality and efficiency of immobilized enzymes. This work aims to design and synthesize a thermo-switchable enzyme@MOF for size-selective biocatalysis and biosensing through the immobilization of Candida rugosa lipase (CRL) within ZIF-8 functionalized with thermally responsive polymer, poly(N-isopropylacrylamide) (PNIPAM) (CRL@ZIF-8-PNIPAM). Unlike free CRL, which does not demonstrate substrate selectivity, we can reversibly tune the pore size of the ZIF-8-PNIPAM nanostructures (open pores or blocked pores) through temperature stimulus and subsequently modulate the substrate selectivity of CRL@ZIF-8-PNIPAM. CRL@ZIF-8-PNIPAM had the highest hydrolytic activity for small molecules (12 mM p-nitrophenol/mg protein/min, 4-nitrophenyl butyrate (p-NP Be)) and the lowest hydrolytic activity for large molecules (0.16 mM p-nitrophenol/mg protein/min, 4-nitrophenyl palmitate (p-NP P)). In addition, CRL@ZIF-8-PNIPAM demonstrated thermo-switchable behavior for large molecules (p-NP P). The p-NP P hydrolytic activity of CRL@ZIF-8-PNIPAM was significantly lower at 40 °C (blocked pores) than at 27 °C (open pores). However, the transition of blocked pores and open pores is a gradual process that resulted in a delay in the "thermo-switchable" catalytic behavior of CRL@ZIF-8-PNIPAM during thermal cycling. CRL@ZIF-8-PNIPAM was also successfully used for the fabrication of electrochemical biosensors for the selective biosensing of pesticides with different molecular sizes.

Investigation on solvent-free esterification of oleic acid by hemp tea waste-immobilized Candida rugosa lipase

This study aimed at Candida rugosa lipase immobilization on a low-cost and readily available support. Among agro-industrial crops, hemp tea waste was chosen as the carrier because it provides higher immobilization performance than hemp flower and leaf wastes. Support characterization by ATR-FTIR, SEM and elemental analysis and the optimization of the adsorption immobilization process were performed. The lipase adsorption immobilization was obtained by soaking the support with hexane under mild agitation for 2 h and a successively incubating the enzyme for 1 h at room temperature without removing the solvent. The esterification of oleic acid with n-decanol was tested in a solvent-free system by studying some parameters that influence the reaction, such as the substrates molar ratio, the lipase activity/oleic acid ratio, reaction temperature and the presence/absence of molecular sieves. The biocatalyst showed the ability to bring the esterification reaction to equilibrium under 60 min and good reusability (maintaining 60 % of its original activity after three successive esterification reactions) but low conversion (21 %) at the optimized conditions (40 °C, 1:2 substrates molar ratio, 0.56 lipase/oleic acid ratio, without sieves). Comparing the results with those obtained by free lipase form at the same activity (1 U) and experimental conditions, slightly higher conversion (%) appeared for the free lipase. All this highlighted that probably the source of lipase for its carbohydrate-binding pocket and lid structure affected the esterification of oleic acid but certainly, the immobilization didn’t induce any lipase conformational change also allowing the reuse of the catalytic material.

Functionalized Ionic Liquids-Modified Metal-Organic Framework Material Boosted the Enzymatic Performance of Lipase.

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.

Octadecyl and sulfonyl modification of diatomite synergistically improved the immobilization efficiency of lipase and its application in the synthesis of pine sterol esters.

Phytosterols usually have to be esterified to various phytosterol esters to avoid their disadvantages of unsatisfactory solubility and low bioavailability. The enzymatic synthesis of phytosterol esters in a solvent-free system has advantages in terms of environmental friendliness, sustainability, and selectivity. However, the limitation of the low stability and recyclability of the lipase in the solvent-free system, which often requires a relatively high temperature to induce the viscosity, also increased the industrial production cost. In this context, a low-cost material, namely diatomite, was employed as the support in the immobilization of Candida rugosa lipase (CRL) due to its multiple modification sites. The Fe3 O4 was also then introduced to this system for quick and simple separation via the magnetic field. Moreover, to further enhance the immobilization efficiency of diatomite, a modification strategy which involved the octadecyl and sulfonyl group for regulating the hydrophobicity and interaction between the support and lipase was successfully developed. The optimization of the ratio of the modifiers suggested that the -SO3 H/C18 (1:1.5) performed best with an enzyme loading and enzyme activity of 84.8mg·g-1 and 54 U·g-1 , respectively. Compared with free CRL, the thermal and storage stability of CRL@OSMD was significantly improved, which lays the foundation for the catalytic synthesis of phytosterol esters in solvent-free systems. Fortunately, a yield of 95.0% was achieved after optimizing the reaction conditions, and a yield of 70.0% can still be maintained after six cycles.

Adsorption study of isotherms, kinetics modelling, and thermodynamics of immobilized Candida rugosa lipase on metal oxide for esterification of waste cooking oil

The problem regarding improper disposal of waste cooking oil (WCO) can be solved via biotransformation of WCO to value added product using lipases as a biocatalyst. Thus, immobilization method was proposed for the recyclability of the biocatalyst. However, a comprehensive discussion on adsorption study is limited for lipase immobilization. In this study, Candida rugosa lipase (CRL) was immobilized on modified metal oxide (Mg2+/Al2O3) and the support was improved by addition of activating agent (L-lysine/Mg2+/Al2O3). Subsequently, the biocatalysts were used for esterification of WCO in the presence of methanol. Maximum adsorption capacity for the support was improved after addition of L-lysine resulting in 0.262 mg protein/g of support. The equilibrium adsorption data for both systems were fitted to the Redlich Peterson isotherm model while the kinetics study was most fitted with Elovich model, and the adsorption process were influenced by intraparticle and film diffusion mechanisms. Thermodynamic analysis explained that the adsorption process of CRL/L-lysine/Mg2+/Al2O3 was more spontaneous and stronger as compared to CRL/Mg2+/Al2O3. Furthermore, CRL/L-Lysine/Mg2+/Al2O3 exhibited its potential in esterification of WCO by converting 62.95% of free fatty acid (FFA), while CRL/Mg2+/Al2O3 converted lower FFA (53.62%). This study offers a fundamental understanding on designing an effective immobilization system by addition of activating agent to develop a good biocatalyst for biotransformation of WCO.

Green synthesis of iron-alginate nanoparticle for Taguchi-assisted immobilization of Candida rugosa lipase and its application in the synthesis of butyl butyrate ester

The synthesis of butyl butyrate, medium chain flavored esters by green chemistry is of immense importance to the food industry. In the present work, green synthesis of iron-alginate nanogels is done using a water-in-oil microemulsion method using food-grade oil for immobilization of Candida rugosa lipase (CrL). The morphological characterization of Fe-Alg nanogels was accomplished using dynamic light scattering (DLS) and scanning electron microscopy (SEM). Elongated tube-shaped Fe-Alg showed a hydrodynamic diameter of 100 ± 2 nm and a zeta potential of − 28 ± 4 mV. The nanogels exhibit an immobilization efficiency of 98%. Taguchi design-assisted immobilization of CrL onto Fe-Alg was effective in predicting the maximum response against immobilization parameters (0.7 µgm/ml of substrate, 20 min immobilization, 53⁰ C and pH 8.5). The esterification of butyl butyrate (∼90%) was completed in 6 h, which is confirmed by gas chromatography. The application of food-grade oil in the microemulsion method (water-in-oil) of nanogel synthesis is an attractive improvement for the development of support matrix in enzyme immobilization and ester synthesis.

Immobilization of Candida rugosa lipase on optimized polyamidoamine dendrimer functionalized magnetic multiwalled carbon nanotubes for green manufacture of butyl butyrate ester

Mineral acid catalysis in Fisher-Speier esterification is hazardous and have other disadvantages. The objective of the study was immobilization of Candida rugosa lipase onto iron oxide (Fe3O4) impregnated magnetic mutiwalled carbon naotubes (MMWCNTs) using amine functionalized polyamidoamine (PAMAM) dendrimers for manufacture of butyl butyrate ester. Dendrimers were synthesized from reaction of ethyl methacrylate with varying chain length of diamines 1, 4 diaminobutane (DAB) / 1, 3 diaminopropane (DAP) / ethylene diamine (ED). The effect of aliphatic alkyl chain length (CH2) of functionalizing agents ED, DAP and DAB, and dendrimer generations on amount of terminal NH2 group, surface charge, protein loading and immobilized enzyme activities were analysed. Magnetic MWCNTs support surface modified with third generation PAMAM using ethylene diamine showed highest protein load 220.1 mg g−1 and enzymatic activity 8,276.21 U g−1 support. The immobilized enzyme particles were characterized by HRTEM, FTIR, XRD, TGA, VSM and Zeta potential analysis. The immobilized enzyme showed improvement in the dispersion, activity, stability and reusability characteristics. Maximum esterification yield 92.67 % and reuse upto 11 cycles with 65.2 % yield obtained. The immobilized preparation showed 2.3 folds esterification than free enzyme. Developed nanobiocatalyst expects to possess potential for various other applications of lipase catalysed reactions.

Preparation and characterization of stable biopolymer-based nanogel for studying its application in immobilization of Candida rugosa lipase

Preparation and characterization of stable biopolymer-based nanogel for studying its application in immobilization of Candida rugosa lipase

The effect of immobilization parameters towards Candida rugosa lipase immobilization on magnesium oxide-aluminium oxide via adsorption

Candida rugosa lipase (CRL) can catalyze many chemical reactions and are widely used in industrial application. Nevertheless, its shortcomings include poor recyclability and a lack of stability. These limitations can be resolved using immobilization technique. The objective of this work was to evaluate the capability of magnesium oxide-aluminium oxide (MgO-Al2O3) as a support for CRL immobilization by physical adsorption. The effect of five immobilization parameters including agitation speed, temperature, CRL loading, immobilization time and weight of MgO-Al2O3 were investigated in this study. The optimal conditions were determined to be at 100 rpm, 30 °C, 1.5 g/L of CRL loading, 3-hour immobilization time and 2 g weight of MgO-Al2O3, resulted in 83.20 % of activity recovery correspond to 45.36 % of immobilization efficiency. Thus, CRL/MgO-Al2O3 was proven have a capability to be used as a biocatalyst for industrial applications.

Rapid mechanochemical production of biocomposites by encapsulating enzymes to zinc based-two metal organic frameworks (ZIF-8 and Zn-MOF-74) for enantioselective hydrolysis reaction of racemic Naproxen methyl ester

The mechanochemical encapsulation of enzymes during the synthesis of metal–organic frameworks (MOF) have been thought to be a natural, economical, new, and environmentally friendly step in synthesizing supported enzymatic catalysts. In this study, for the first time, a mechanochemical technique was employed to immobilize Candida rugosa lipase (CRL), and two zinc-based MOF types (Zn-MOF-74 and ZIF-8) were preferred. The enzyme kinetics and immobilization parameters (pH, temperature, thermal stability and reusability) of the prepared encapsulated enzymes (Zn-MOF-74@CRL and ZIF-8@CRL) were investigated. The prepared encapsulated enzymes were characterized by FTIR, SEM and XRD. It was observed that the catalytic activity of Zn-MOF-74 @CRL was six times higher than that of ZIF-8@CRL. After five reuses, the activities of Zn-MOF-74@CRL and ZIF-8@CRL were found to be 81% and 73% retained, respectively. After being subjected to 60 °C for 120 min, ZIF-8@CRL and Zn-MOF-74@CRL maintained their activities at 65% and 73%, respectively. The enantioselective hydrolysis reaction of racemic naproxen methyl ester was chosen as the model reaction. The results showed Zn-MOF-74@CRL possessed better enantioselectivity (E = 245) than ZIF-8@CRL (E = 136). This study demonstrated that it is possible to create biocatalysts with excellent catalytic properties for the pharmaceutical industry using MOFs produced by the mechanochemical method.

Optimization of microreactor-intensified transesterification reaction of sesame cake oil (sesame waste) for biodiesel production using magnetically immobilized lipase nano-biocatalyst

Enzyme-catalyzed transesterification to produce biodiesel is a time-consuming reaction. This study aimed to use a recoverable nanomagnetic immobilized enzyme and microreactor-intensified process to design a significantly rapid transesterification reaction. Immobilization of Candida Rugosa lipase (CRL) on GO-Fe3O4@TiO2 nanocomposite was optimized using response surface methodology-box–Behnken design (RSM-BBD). Results showed the highest immobilization efficiency of 92.73% at the optimum condition of CRL concentration of 2.59 mg/mL, time of 4.96 h, and temperature of 37.61 °C. Newly synthesized GO-Fe3O4@TiO2@CRL nano-biocatalyst (NBC) was characterized using FTIR, XRD, VSM, FE-SEM, EDX and elemental mapping. Immobilized CRL showed improved stability under different parameters, including pH, thermal, and methanol medium compared to free CRL. Freshly synthesized NBC was used in a microreactor to intensify the transesterification reaction of sesame cake oil to produce biodiesel. The RSM-BBD optimization of the transesterification reaction showed a maximum conversion rate of 97.03%, achieved at the NBC amount of 4.47 wt%, residence time of 13.29 min, and Me/oil molar ratio of 4.28. The generated biodiesel has all characteristics well within the ASTM D6751, demonstrating its good quality. The results of this study showed that using a microreactor could sufficiently intensify the transesterification reaction and reduce the reaction time using GO-Fe3O4@TiO2@CRL NBC.

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.

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.

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.

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.