Encapsulated Lipase Research Articles

Improving the Encapsulation Efficiency of Lipase in Molecular Cages and Its Application.

Here, lipase encapsulation is constructed by locking enzyme molecules in nanomolecular cages on the surface of SH-PEI@PVAC magnetic microspheres. To improve the encapsulation efficiency in enzyme loading, the thiol group is efficiently modified on the grafted polyethyleneimine (PEI) using 3-mercaptopropionic acid. N2 adsorption-desorption isotherms reveal the existence of mesoporous molecular cages on the microsphere surface. The robust immobilizing strength of carriers to lipase demonstrates the successful encapsulation of enzymes in nanomolecular cages. The encapsulated lipase shows high enzyme loading (52.9 mg/g) and high activity (51.4 U/mg). Different sizes of molecular cages are established, and the cage size showed important effects on lipase encapsulation. It shows that enzyme loading is low at a small size of molecular cages, which is attributed to that the nanomolecular cage is too small to house lipase. The investigation in lipase conformation suggests that the encapsulated lipase retains its active conformation. Compared with the adsorbed lipase, the encapsulated lipase shows higher thermal stability (4.9 times) and higher resistance to denaturants (5.0 times). Encouragingly, the encapsulated lipase shows high activity and reusability in lipase-catalyzed synthesis of propyl laurate, suggesting the potential application value of encapsulated lipase.

Encapsulation of Lipase Enzyme in Silica Gel Matrix from Rice Husk Ash and Transesterification Reaction Activity Test on Palm Oil

Encapsulation is one of the immobilization methods to increase the stability of the enzyme or other biomolecules by binding or trapping the molecules in a certain matrix. Silica gel was mostly chosen as a matrix to encapsulate the enzyme due to its high thermal stability. In this research, silica gel was used to encapsulate of lipase enzyme. Silica gel was synthesized from sodium silicate by the sol-gel technique. Silica from rice husk ash was extracted using 2 M NaOH to produce a sodium silicate solution. The silica gel was synthesized using the acidification of sodium silicate solution. The various mass and volume ratios of the enzyme and sodium silicate solution were 1:1; 1:2, and 2:1 (w/v). The amount of encapsulated enzyme was analyzed with UV-Vis spectrophotometry at a wavelength of 540 nm after complexing with biuret. The encapsulated enzyme activity test was carried out for the transesterification reaction of palm oil under various conditions. Fatty Acid Methyl Esther (FAME) analysis and surface analysis of the encapsulated enzyme was performed using GC-MS and SEM-EDX, respectively. The results showed that the lipase enzyme could be well encapsulated in silica gel after aging for eight days, at a lipase and sodium silicate ratio of 1:2 (w/v) with a percentage of 93.72%, and there were still 85.76% encapsulated lipase even after being washed seven times. The transesterification reaction resulted in the highest conversion of oil to FAME (33.87%) at a mole ratio of 1:3 oil: methanol and a lipase mass of 0.5 g.

Molecular cages encapsulating lipase and the effect of cage hydrophobicity and cage size

To firmly immobilize enzyme on carrier surface without covalent attachment as well as improve enzyme stability, an effective method of enzyme immobilization was developed by encapsulating enzyme into nano-molecular cages on the surface of magnetic microspheres. Compared with physical adsorption, molecular cage encapsulation could fix lipase much more firmly and make lipase more active. Then, this study focused on investigating the effects of cage hydrophobicity and cage size on lipase encapsulation respectively by adjusting the log P value (from −0.85 to 1.28) and chain length (from 2.93 to 5.41 nm) of crosslinkers. It showed that lipase activity was dependent on the hydrophobicity of cage. Moreover, the size of cage had important effect on lipase loading amount, and a big cage was conducive to house lipase molecule. The structural analysis indicated that molecular cages could retain the active conformation of lipase and increase its structural rigidity. Molecular dynamics simulation revealed the interaction between the lid domain of lipase and molecular cage, leading to the higher activity of encapsulated lipase (63.6 U/mg) than that of free lipase (50.6 U/mg). Besides, the encapsulated lipase showed more than twice higher thermal stability and almost 8 times higher tolerance to denaturant than the adsorbed lipase.

Encapsulation of lipases by nucleotide/metal ion coordination polymers: enzymatic properties and their applications in glycerolysis and esterification studies.

In the present study, lipases of TLL (lipase from Thermomyces lanuginosus), AOL (lipase from Aspergillus oryzae), RML (lipase from Rhizomucor miehei), BCL (lipase from Burkholderia cepacia), CALA (Candida antarctica lipase A) and LU (Lecitase® Ultra) were encapsulated into nucleotide-hybrid metal coordination polymers (CPs). Enzyme concentration was optimized for encapsulation and the enzymatic properties of the obtained lipases were investigated. In addition, their performance in glycerolysis and esterification was evaluated, and glycerolysis conditions (water content, temperature and time) were optimized. Hydrolysis activity over 10 000 U g-1 and activity recovery over 90% were observed from AOL@GMP/Tb, TLL@GMP/Tb and RML@GMP/Tb. GMP/Tb encapsulation (of AOL, TLL, RML and LU) improved their thermostability when incubated in air. The encapsulated lipases exhibited moderate [triacylglycerols (TAG) conversion 30-50%] and considerable glycerolysis activity (TAG conversion over 60%). TAG conversions from 69.37% to 82.35% and diacylglycerols (DAG) contents from 58.62% to 64.88% were obtained from CALA@GMP/metal samples (except for CALA@GMP/Cu). Interestingly, none of the encapsulated lipases initiated the esterification reaction. AOL@GMP/Tb, TLL@GMP/Tb, RML@GMP/Tb and CALA@GMP/Tb showed good reusability in glycerolysis, with 88.80%, 94.67%, 89.85% and 78.16% of their initial glycerolysis activity, respectively, remaining after five cycles of reuse. The relationships between temperature and TAG conversion were LnV0 =6.5364-3.7943/T and LnV0 =13.8820-6.4684/T for AOL@GMP/Tb and CALA@GMP/Tb, respectively; in addition, their activation energies were 31.55 and 53.78 kJ mol-1 , respectively. Most of the present encapsulated lipases exhibited moderate and considerable glycerolysis activity. In addition, AOL@GMP/Tb, TLL@GMP/Tb, RML@GMP/Tb and CALA@GMP/Tb exhibited good reusability in glycerolysis reactions and potential in practical applications. © 2022 Society of Chemical Industry.

Ethyl cellulose-chitosan complex particles stabilized W/O Pickering emulsion as a recyclable bio-catalytic microreactor

Water-in-oil (W/O) emulsions have numerous applications in many industries like foods, pharmaceuticals, in particular biphasic bio-catalysis. However, most of them are stabilized by surfactants or synthetic particles, which lead to limited uses in foods. Here, a W/O type Pickering emulsion was successfully prepared firstly by ordered ethyl cellulose-chitosan complex particles (ECCPs) and used for a recyclable biocatalytic micro-reactor. The performances of emulsions with different ethyl cellulose-chitosan ratios, water phase fraction and particle concentration were discussed. The type of emulsions could be shifted from W/O to W/O/W and O/W as water fraction increased. Interestingly, a recyclable bio-catalytic microreactor platform was constructed using such novel W/O emulsions, and the activity of lipase was significantly increased by encapsulating it into Pickering emulsions, and was 6.3-times higher than that of free lipase in biphasic system. In particular, the encapsulated lipase remained stable and could be recycled over 15 times without the loss of activity.

Reaction-diffusion model to describe biodiesel production using lipase encapsulated in ZIF-8

Lipase has successfully been encapsulated into hexagonal zeolitic imidazolate frameworks (ZIF-8) for use in biodiesel production. It was shown that the lipase encapsulation did not have a significant effect on the morphology, surface properties and crystallinity. The effects of methanol ratio, temperature, oil concentration and water content, on the biodiesel production yield and rate of reaction, were tested. The highest yield was obtained at a methanol ratio and temperature of 6:1 and 40 °C, respectively, and a drop was observed by increasing both. It was also shown that the yield decreased with the increase in water content. The activity and stability of the immobilized lipase in ZIF-8 by encapsulation was compared to that immobilized by surface adsorption. Although the adsorbed lipase on ZIF-8 showed higher activity, the stability of the encapsulated one was higher. At methanol ratio of 12:1, the encapsulated lipase in ZIF-8 maintained 83% residual activity after 5 cycles, compared to only 34% attained by the adsorbed lipase at the same conditions. The experimental results were used to determine the kinetics parameters of modified Ping Pong Bi Bi model, and the accuracy of the prediction were compared to those obtained by the Michaelis Menten model. To gain a better insight into how the reaction occurs inside the ZIF-8 crystal with encapsulated lipase, a diffusion–reaction model was developed and numerically solved. The results clearly show that the substrate did not diffuse deeply into the crystal, which further confirmed the mass transfer limitation that resulted in the lower activity of the encapsulated lipase as compared to the adsorbed one. The developed numerical model can be applied to any diffusion–reaction systems.

Collapsible charge induced aggregation behaviour of cerium oxide nanoparticles for treating pancreatic enzyme deficiency

We report weakly bound collapsible aggregates of CeO2 nanoparticles (NPs) for successful encapsulation and release of lipase enzyme to treat lipase deficiency via oral delivery. The collapsible aggregates of CeO2 NPs formed during the encapsulation process not only improved the encapsulation efficiency, but also protected the encapsulated lipase from the harsh gastrointestinal conditions. The released lipase maintained its activity in native form and the remaining activity was estimated to be 50% indicating their ability in retaining the activity of encapsulated biomolecules. Thus, the CeO2 NPs demonstrated here have high potential for use as oral delivery system for the encapsulation and release of lipase to treat exocrine pancreatic insufficiency.

Encapsulation of lipase using magnetic fluorescent calix[4]arene derivatives; improvement of enzyme activity and stability.

In this study, iron magnetic nanoparticles capped with fluorescent calixarene derivatives (Fe3O4@Calix-2 and Fe3O4@Calix-3) were prepared in one-step using coprecipitation of Fe(II) and Fe(III) in basic solution. Different techniques were used to characterize the synthesized magnetic nanoparticles, such as Fourier Transform Infrared spectroscopy (FTIR), powder X-ray diffraction (XRD), vibrating sample magnetometer (VSM) and confocal microscopy. Candida rugosa was encapsulated on synthesized nanoparticles following sol-gel method. It has been observed that under the optimum conditions, the activity of encapsulated lipase (Fe3O4@Calix-2E) was 119 U/g of support which is 4.1 times more that of the encapsulated lipase without calix[4]arene derivative (Fe3O4@E). Comparative study show that the encapsulated lipase on nanoparticles has higher thermal and operational stability than encapsulated lipase without calix[4]arene derivative. Among these encapsulated lipase nanoparticles, Fe3O4@Calix-2 was capable of effectively catalyze hydrolysis of racemic Flurbiprofen methyl ester with high conversion of 49% and substrate enantiomeric excess (ees) of 85% at optimum pH and temperature. The efficiency of these nanoparticles was assessed by their reusability, for that after five consecutive operational uses these encapsulated lipase nanoparticles retained their conversion ratios up to 38% and 30% respectively, in the hydrolysis of (R,S)-Flurbiprofen methyl ester. The results showed that encapsulated lipases nanoparticle with calixarene moieties lead to increased activity, stability, reusability and enhanced stereoselectivity in kinetic resolution.

Candida rugosa lipase encapsulated with magnetic sporopollenin: design and enantioselective hydrolysis of racemic arylpropanoic acid esters

The aim of this study was to prepare the encapsulation of Candida rugosa lipase (CRL) with magnetic sporopollenin. The sporopollenin was covalent immobilized onto magnetic nanoparticles (Fe3O4), grafted amino (APTES), or epoxy groups (EPPTMS). CRL was sol-gel encapsulated in the presence of magnetic sporopollenin/Fe3O4 nanoparticles. The influence of activation agents ([3-(2,3-epoxypropoxy) propyl] trimethoxysilane (EPPTMS), (3-Aminopropyl)triethoxysilane (APTES) and pH and thermal stabilities of the biocatalyst were assessed. Experimental data showed the improved catalytic activity at different pH and temperature values. At 60 °C, free lipase lost its initial activity within 80 min of time, although the encapsulated lipases retained their initial activities of about 65% by APTES and 60% by EPPTMS after 120 min of heat treatment at 60 °C. The catalytic properties of the encapsulated lipases were utilized to hydrolysis of racemic aromatic carboxylic acid methyl esters (Naproxen and 2-phenoxypropionic acid). The results show that the sporopollenin-based encapsulated lipase (Fe-A-Spo-E) has greater enantioselectivity and conversion in comparison with the encapsulated lipase without supports (lipase-enc).

Submicron Inverse Pickering Emulsions for Highly Efficient and Recyclable Enzymatic Catalysis.

Herein, we report the utilization of a submicron Pickering emulsion (SPE) for the encapsulation of enzymes (e.g., lipase from Candida sp.) in water droplets that were solely stabilized by hydrophobic solid or mesoporous silica nanoparticles in toluene for use in biphasic reactions. The catalytic performance of encapsulated lipase was evaluated in the esterification of 1-hexanol and hexanoic acid under stirring-free conditions, which was favorable for maintaining enzymatic activity. Remarkably, the SPE significantly increased the specific activity of encapsulated lipase, owing to the exceptionally high water/oil interfacial area and short diffusion distance of the reagents in the SPE. With mesoporous silica nanoparticles, the activity of lipase was approximately 25.5- and 2.8-times higher than that of free lipase and encapsulated lipase in the micron Pickering emulsion, respectively. The higher water/toluene interfacial area was attributed to the smaller submicron-scale water droplets and the increase in the mass transfer of enzymes or substrates was further improved by using mesoporous Pickering stabilizers. In addition, the encapsulated lipase in SPE also demonstrated excellent stability and could be recycled up to 15 times without significant loss of activity.

<b>Effect of parameters on butyl butyrate synthesis using novel <i>Aspergillus niger</i> lipase as biocatalyst

A novel “green” Aspergillus niger lipase, obtained from the fermentation of pumpkin seeds, was used in a free form and encapsulated in sol-gel matri x in butyl butyrate (pineapple flavor) synthesis. Esterification reactions were performed with varying substrate molar ratio (butanol: butyric acid) ranging between 1:1 and 5:1; temperature between 30 and 60°C and biocatalyst mass between 0 and 1g, respectively, according to experimental design 23 with 6 axial and 3 central points. Maximum butyl butyrate production was obtained when substrate molar ratio (butanol:butyric acid) 3:1, temperature at 60°C and 0.5 g free or encapsulated lipase as biocatalyst, were used. Temperature was the most significant parameter for production with the two biocatalysts, indicating that higher rates mean greater compound synthesis. Response surface plots showed that higher butyl butyrate production may be obtained with higher temperature and molar ratio rates (butanol:butyric acid) and with lower rates of biocatalyst mass in reactions catalyzed by free or encapsulated lipase. Aspergillus niger lipase obtained from agro-industrial waste could be employed as biocatalyst in esterification reactions in the production of natural aroma as butyl butyrate.

Encapsulation of enzymes in metal ion-surfactant nanocomposites for catalysis in highly polar solvents.

We described a method to encapsulate enzymes in metal ion-surfactant nanocomposites. The nanobiocatalyst displayed highly retained activity (∼100%) and much improved stability in protein-denaturing organic solvents. As a demonstration, the encapsulated lipase exhibited much higher activity and stability in the synthesis of vitamin E succinate in dimethyl sulfoxide.

Utilization of Catalytic Properties of the Encapsulated Lipase with Calix[4]arene-Adorned Sporopollenin

ABSTRACTThe dihydrazide calix[4]arene was immobilized onto sporopollenin in order to encapsulate Candida rugosa lipase (CRL) via sol-gel entrapment. The kinetic resolution of the new encapsulated lipase was investigated for enantioselective hydrolysis of racemic naproxen methyl ester and 2-phenoxypropionic acid methyl ester. The results demonstrated that the activity and enantioselectivity of the lipase were improved when the lipase was encapsulated in the presence of calix[4]arene-immobilized sporopollenin. The encapsulated lipase showed an excellent rate of enantioselectivity against the (R/S)-naproxen methyl and (R/S)-2-phenoxypropionic acid methyl esters, with E = 350 and 295, respectively, compared to the free enzyme.

Encapsulation of protease from Aspergillus oryzae and lipase from Thermomyces lanuginoseus using alginate and different copolymer types

Although the application of enzymes in food as a food processing aid and enzyme supplement is of interest and widely used, the enzymes can be easily deactivated or lose their activity due to many causes such as pH and moisture as well as through the introduction of incompatible ingredients during food processing and storage. These problems can be solved by the encapsulation technique, especially in a gel matrix. The influences were studied of the alginate concentration, types of copolymer and their concentrations on the bead size, encapsulation yield (EY), encapsulation efficiency (EE), leakage and the retention of enzyme activity during storage period of encapsulated protease from Aspergillus oryzae and lipase from Thermomyces lanuginosus beads. A solution of purified protease or lipase was encapsulated in calcium alginate-chitosan beads (CACB), calcium alginate-xanthan gum beads (CAXB) and calcium alginate-maltodextrin beads (CAMB) using the extrusion method. Increasing the alginate and copolymer concentrations in the solution increased the bead size, EY, EE and the retention of enzyme activity during the storage period and reduced leakage of both the encapsulated protease and lipase. In addition, different types of copolymer significantly (p ≤ 0.05) affected these properties of both encapsulated enzymes. Furthermore, protease encapsulated using 2.0% alginate and 0.2% chitosan provided the highest EY (81.7%) and EE (77.2%) with a bead size of 1.85 mm and 8.1% leakage. The retention of encapsulated protease activity and the shelf-life of encapsulated enzyme which was expressed as half-life, the time required for the enzyme activity to decrease by half (thalf life) were 75.8% and 27.2 wk, respectively after storage at 4 °C for 10 wk. For lipase, encapsulation using 2.0% alginate and 0.4% xanthan gum provided the highest EY (42.5%) and EE (43.9%) and the bead size and leakage were 1.81 mm and 6.2%, respectively. The retention of encapsulated lipase activity and the thalf life were 77.9% and 27.8 wk, respectively after storage at 4 °C for 10 wk. CACB was a suitable complex polymer for encapsulating protease while CAXB was suitable for lipase. EY and EE values of CACB-protease were higher than those of CAXB-lipase. Therefore, the encapsulation method, gelling conditions and interactions between carriers and lipase should be further studied.

Improvement of catalytic activity of Candida rugosa lipase in the presence of calix[4]arene bearing iminodicarboxylic/phosphonic acid complexes modified iron oxide nanoparticles

In the present study, iron oxide magnetite nanoparticles, prepared through a co-precipitation method, were coated with phosphonic acid or iminodicarboxylic acid derivatives of calix[4]arene to modulate their surfaces with different acidic groups. Candida rugosa lipase was then directly immobilized onto the modified nanoparticles through sol–gel encapsulation. The catalytic activities and enantioselectivities of the two encapsulated lipases in the hydrolysis reaction of (R/S)-naproxen methyl ester and (R/S)-2-phenoxypropionic acid methyl ester were assessed. The results showed that the activity and enantioselectivity of the lipase were improved when the lipase was encapsulated in the presence of calixarene-based additives; the encapsulated lipase with the phosphonic acid derivative of calix[4]arene had an excellent rate of enantioselectivity against the (R/S)-naproxen methyl and (R/S)-2-phenoxypropionic acid methyl esters, with E=350 and 246, respectively, compared to the free enzyme. The encapsulated lipases (Fe-Calix-N(COOH)) and (Fe-Calix–P) showed good loading ability and little loss of enzyme activity, and the stability of the catalyst was very good; they only lost 6–11% of the enzyme’s activity after five batches.

Enhancing Effect of Calix[4]arene Amide Derivatives on Lipase Performance in Enantioselective Hydrolysis of Racemic Arylpropionic Acid Methyl Esters

Calix[4]arene amide derivatives were employed as new additives within the sol-gel encapsulation of lipase from Candida rugosa (CRL) to improve its catalytic properties. Evaluation of catalytic activity of the encapsulated lipases was acheived by enantioselective hydrolysis of both racemates, Naproxen methyl ester and 2-phenoxypropionic acid methyl ester, in aqueous buffer solution/isooctane reaction system. Results show that enantioselectivity was improved by using calix[4]arene amide derivatives-based encapsulated lipases. The reaction of naproxen methyl ester resulted in 47.6% conversion (x) in 24 h with 88.9% enantiomeric excess of substrate (ees), analogous to an enantioselectivity (E) value of 297 (E = 137 for the encapsulated free enzyme). The conversion of 2-phenoxypropionic acid methyl ester, obtained was 48.4% with E value of 327, enantiomeric excess of substrate (ees) of 92% for the reaction time of 1 h (E = 211 for the encapsulated free enzyme).

An alternative method for production of microalgal biodiesel using novel Bacillus lipase.

In this study, enzymatic interesterification is carried out using encapsulated lipase as biocatalyst with methyl acetate as acyl acceptor in a solvent-free system. Lipase, isolated from a marine bacterial isolate, Bacillus sp.S23 (KF220659.1) was immobilized in sodium alginate beads. This investigation elaborated on the effects of various parameters, namely enzyme loading, temperature, water, molar ratio, reaction time and agitation for interesterification. The study resulted in the following optimal conditions: 1.5 g immobilized lipase, 1:12 molar ratio of oil to methyl acetate, 35 °C, 8 % water, 60 h reaction time, 250 rpm of agitation. With the standardized condition, the maximum conversion efficiency was 95.68 %. The immobilized beads, even after ten cycles of repeated usage showed high stability in the presence of methyl acetate and no loss of lipase activity. The microalgal biodiesel composition was analyzed using gas chromatography. The current study was efficient in using immobilized lipase for the interesterification process, since the method was cost-effective and eco-friendly, no solvent was involved and the enzyme was encapsulated in a natural polymer.

Improvement of catalytic activity of lipase in the presence of calix[4]arene valeric acid or hydrazine derivative

Sol-gel encapsulation is a simple but powerful method to enhance the enantioselectivity of lipase-catalyzed transformations in an isooctane/aqueous buffer solution. Candida rugosa lipase was encapsulated according to a sol-gel procedure in the presence and absence of calix[4]arene hydrazine or carboxylic acid derivatives with Fe3O4 magnetic nanoparticles as an additive. The activity of the encapsulated lipases was evaluated for the enantioselective hydrolysis of racemic Naproxen methyl ester and the hydrolysis of p-Nitrophenylpalmitate. The results indicate that the encapsulated lipase without calix[4]arene derivative has lower conversion and enantioselectivity compared to the encapsulated lipase with calix[4]arene derivative. It was found that the calix[4]arene hydrazine and carboxylic acid-based encapsulated lipases have excellent activity and enantioselectivity (E >300) compared to encapsulated lipase without the calix[4]arene derivatives.

Enantioselective hydrolysis of (R,S)-Naproxen methyl ester using Candida rugosa lipase with calix[4]arene derivatives

Candida rugosa lipase has been immobilized on a variety of calix[4]arene derivatives using the sol–gel encapsulation technique, and the catalytic activities of the resulting encapsulated lipases towards the hydrolysis of p-nitrophenyl palmitate and the hydrolytic kinetic resolution of racemic Naproxen methyl ester were evaluated using standard techniques. The results revealed that the calix[4]arene-based immobilized encapsulated lipases 5-CRL and 6-CRL gave higher levels of enantioselectivity and conversion than the free encapsulated lipase.

Resolution of 1,1,1-trifluoro-2-octanol by Pseudomonas sp. lipase encapsulated in aggregated silica nanoparticles

Lipase from pseudomonas sp. (PSL) was encapsulated in stacked silica nanoparticles and used for resolution of 1,1,1-trifluoro-2-octanol. The effects of reaction conditions, such as solvent, temperature, water activity and type of acyl donor were investigated. Compared with the free lipase, the encapsulated lipase exhibits better performance (enzyme activity, 20.32 μmol g−1 min−1; E value, 33.83) under optimum conditions. Most importantly, almost no enzyme leakage phenomenon takes place during continuous batch operation, which suggests that this new encapsulation method has great potential for application.