Stability Of Candida Rugosa Lipase Research Articles

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.

Highly effective Candida rugosa lipase immobilization on renewable carriers: Integrated drying and immobilization process to improve enzyme performance

The overall stability of Candida rugosa lipase (CRL) was improved via immobilization onto low-cost eco-friendly carriers (agroindustrial wastes) followed by using spouted bed dryer or spray dryer. Lipase covalently immobilized on glutaraldehyde-activated rice husk proved the greatest performance, retaining 94.1% of the initial activity, followed by sugarcane bagasse (90.3%) and green coconut fiber (89.3%). On the other hand, the enzyme activity retention of CRL-lipase bioencapsulated in magnetic chitosan treated with glutaraldehyde or sodium tripolyphosphate ranged from 73.6% to 84.6%. The dried product moisture content, and water activities ranged from 4.1% and 6.5% and from 0.2 to 0.35, respectively. After storage for six months at 5 °C, the immobilized enzyme systems retained at least 70.0% of its initial activity (vs 18.3% retained activity for the free lipase). After ten reuse cycles, enzyme immobilized onto lignocellulosic carriers retained on average 72.7% of its initial activity. The thermostability of all immobilized derivatives was significantly improved and lipase immobilized onto rice husk showed a stabilization factor of 20.44 at 80 °C. The kinetic data showed that the CRL-lipase suffered alterations in catalytic behavior after the immobilization/drying, mainly a slight decrease in affinity to the substrate (↑KM). Among all the assessed immobilized lipase for aroma synthesis the rice husk immobilized lipase displayed the best result towards the production of isoamyl caprylate (62.40 g.L−1). In conclusion, this report reinforces the urgent need for the economic development of eco-sustainable immobilized biocatalysts to boost the use of enzymatic technology on industrial scale, and the feasibility of the association of immobilization and drying processes to improve the enzyme-quality features.

Synergistic role of carbon quantum dots in the activity and stability of Candida rugosa lipase encapsulated within metal–organic frameworks (ZIF-8)

Carbon quantum dots (CQDs) are particularly attractive in biosensing, bioimaging and drug delivery applications due to their luminescent properties, water dispersibility and favorable biocompatibility. In the study, by combining CQDs derived from edible mushroom Agaricus bisporus and zeolitic imidazolate frameworks (ZIFs) crystals, a new support material was prepared for encapsulation of Candida rugosa lipase. The study aimed to shed light on the activity, thermal and storage stability, and reusability of physically adsorbed or in-situ encapsulated enzyme. The enzyme loading efficiency of the in-situ encapsulation method was 3.11-fold higher (371 mg g−1). Also, catalytic activities were 119.8 U/g (CQDs@ZIF- 8@CRLenc) and 34.2 U/g (CQDs@ZIF-8@CRLads). The CQDs@ZIF-8@CRLenc with the biocatalytic activity was 3.5-folds higher than that of the CQDs@ZIF-8@CRLads. In the in-situ encapsulation, the CQDs and the enzyme were physically entrapped within three-dimensional structures of ZIF-8 that were constructed from tetrahedral zinc ions bridged by imidazolate. The structural, morphological, and spectrophotometric analyses were performed using TEM, SEM-EDX, FT-IR, XRD, UV–vis, fluorescence, and confocal microscopy. The effects of pH (4.0–9.0) and temperature (30–60 °C) on the enzymatic activity were studied and optimized. The coexistence of the CQDs and ZIF-8 crystals remarkably enhanced the activity, reusability, thermal resistance, and storage stability of the enzyme encapsulated within ZIF-8 crystals (the relative enzyme activity; after 6 re-uses: 73%, 2-hour-incubation at 60 °C: 70%, 5-week-storage at 4 °C: 70%). The CQDs from A. bisporus entrapped within ZIF-8 can synergistically enhance the catalytic performance and thermal/storage stability of immobilized C. rugosa lipase.

Fabrication and characterization of epoxylated zwitterionic copolymer-grafted silica nanoparticle as a new support for lipase immobilization

Abstract Our previous work proved that the thermal stability of Candida rugosa lipase (CRL) immobilized on zwitterionic polymer (poly(carboxybetaine methacrylate)) grafted silica nanoparticle (SNP) was much higher than that on poly(glycidyl methecrylate) (pGMA) grafted SNP, while the latter showed significantly increased activity. Inspired by the research, we have herein proposed to synthesize copolymers of zwitterionic sulfobetaine methacrylate (SBMA) and GMA for CRL immobilization. The copolymers were grafted onto SNP surface at three GMA/SBMA (G/S) molar ratios (G100/S0, G50/S50, G10/S90), followed by the covalent coupling of CRL to the surface copolymers. The immobilized CRLs on the corresponding supports were denoted as p(G100-S0)-CRL, p(G50-S50)-CRL and p(G10-S90)-CRL. The enzyme loading increased with the increase of GMA content in the copolymer, while the activity varied with the grafted copolymer composition. Kinetic study proved the improvement of enzyme-substrate affinity after immobilization. In comparison to p(G100-S0)-CRL, p(G50-S50)-CRL and p(G10-S90)-CRL presented remarkably enhanced thermal stability and pH tolerance, and p(G10-S90)-CRL showed the highest stability. These results suggest that the copolymer design is promising for development as a versatile platform for enzyme immobilization.

Effect of deep eutectic solvent mixtures on lipase activity and stability

DESs (deep eutectic solvents) have many potential applications as cosolvents or anhydrous reaction media for biocatalytic reactions, owing to their non-volatility, non-flammability, non-toxicity, biocompatibility, biodegradability, and low cost. In this work, choline chloride ([Ch]Cl)-based DESs and DES mixtures containing two hydrogen bond donors were used as cosolvents to enhance the activity and stability of Candida rugosa lipase in aqueous reactions. The activity of lipase in an aqueous solution of [Ch]Cl:urea:glycerol was 155% higher than that in buffer. The half-life time of lipase at 40°C in an aqueous solution of [Ch]Cl:glycerol was enhanced by 9.2 times. The lipase showed the highest acid stability and base stability in the aqueous solutions of [Ch]Cl:glycerol:thiourea and [Ch]Cl:ethylene glycol:formamide, respectively. In general, glycerol-containing DES mixtures were very useful in enhancing the activity and stability of lipase, while formamide-containing DES mixtures could not efficiently enhance the activity and stability of lipase. To understand the effect of DES mixtures on the activity and stability of lipase in aqueous solution, four solvatochromic parameters of DES mixtures were determined. When the solvatochromic parameters of DES mixtures were correlated with the stability of lipase in aqueous solutions of DES mixtures, it was found that thermal stability and storage stability of lipase were associated with the hydrogen bond acidity of DES mixtures. Acid stability and base stability of lipase were correlated with polarity based on Reichardt’s dye and the dipolarity/polarizability of DES mixtures, respectively.

Candida rugosa Lipase Immobilized onto Acid-Functionalized Multi-walled Carbon Nanotubes for Sustainable Production of Methyl Oleate.

The chemical production of methyl oleate using chemically synthesized fatty acid alcohols and other toxic chemicals may lead to significant environmental hazards to mankind. Being a highly valuable fatty acid replacement raw material in oleochemical industry, the mass production of methyl oleate via environmentally favorable processes is of concern. In this context, an alternative technique utilizing Candida rugosa lipase (CRL) physically adsorbed on multi-walled carbon nanotubes (MWCNTs) has been suggested. In this study, the acid-functionalized MWCNTs prepared using a mixture of HNO3 and H2SO4 (1:3 v/v) was used as support for immobilizing CRL onto MWCNTs (CRL-MWCNTs) as biocatalysts. Enzymatic esterification was performed and the efficiency of CRL-MWCNTs was evaluated against the free CRL under varying conditions, viz. temperature, molar ratio of acid/alcohol, solvent log P, and enzyme loading. The CRL-MWCNTs resulted in 30-110 % improvement in the production of methyl oleate over the free CRL. The CRL-MWCNTs attained its highest yield (84.17 %) at 50 °C, molar ratio of acid/alcohol of 1:3, 3 mg/mL of enzyme loading, and iso-octane (log P 4.5) as solvent. Consequently, physical adsorption of CRL onto acid-functionalized MWCNTs has improved the activity and stability of CRL and hence provides an environmentally friendly means for the production of methyl oleate.

Effect of the physicochemical properties of binary ionic liquids on lipase activity and stability

In the present study, the lipase-catalyzed hydrolysis of p-nitrophenyl butyrate is used as a model reaction to determine the activity and stability of Candida rugosa lipase in binary ionic liquids (ILs). The binary ILs consist of hydrophobic 1-butyl-3-methylimidazolium hexafluorophosphate ([Bmim]PF6) and a small amount of hydrophilic 1-butyl-3-methylimidazolium nitrate ([Bmim]NO3) or 1-butyl-3-methylimidazolium trifluoromethanesulfonate ([Bmim]CF3SO3) or 1-butyl-3-methylimidazolium tetrafluoroborate ([Bmim]BF4). The activity and the stability of lipase are first correlated with the physicochemical properties of the binary ILs. In the three binary IL systems, both the hydrophilicity and the polarity of the systems increase with the increase of the content of hydrophilic ILs (HILs). At a fixed concentration of HIL, they vary in a descending order of [Bmim]PF6/[Bmim]NO3>[Bmim]PF6/[Bmim]CF3SO3>[Bmim]PF6/[Bmim]BF4. This order is in contrast with the order of the lipase conformation stability, i.e., the higher the polarity of ILs, the more unstable the lipase conformation. However, both the activity and the stability of lipase depend on the type and the content of the HIL in binary ILs, showing a complex dependency. Analysis shows that the catalytic performance of lipase in the binary ILs is affected not only by the direct influence of the ILs on lipase conformation, but also through their indirect influence on the physicochemical properties of water. The present study helps to explore binary IL mixtures suitable for lipase-based biocatalysis.

A facile enzymatic synthesis of geranyl propionate by physically adsorbed Candida rugosa lipase onto multi-walled carbon nanotubes

In view of several disadvantages as well as adverse effects associated with the use of chemical processes for producing esters, alternative techniques such as the utilization of enzymes on multi-walled carbon nanotubes (MWCNTs), have been suggested. In this study, the oxidative MWCNTs prepared using a mixture of HNO3 and H2SO4 (1:3 v/v) were used as a supportive material for the immobilization of Candida rugosa lipase (CRL) through physical adsorption process. The resulting CRL-MWCNTs biocatalysts were utilized for synthesizing geranyl propionate, an important ester for flavoring agent as well as in fragrances. Enzymatic esterification of geraniol with propionic acid was carried out using heptane as a solvent and the efficiency of CRL-MWCNTs as a biocatalyst was compared with the free CRL, considering the incubation time, temperature, molar ratio of acid:alcohol, presence of desiccant as well as its reusability. It was found that the CRL-MWCNTs resulted in a 2-fold improvement in the percentage of conversion of geranyl propionate when compared with the free CRL, demonstrating the highest yield of geranyl propionate at 6h at 55°C, molar ratio acid: alcohol of 1:5 and with the presence of 1.0g desiccant. It was evident that the CRL-MWCNTs biocatalyst could be reused for up to 6 times before a 50% reduction in catalytic efficiency was observed. Hence, it appears that the facile physical adsorption of CRL onto F-MWCNTs has improved the activity and stability of CRL as well as served as an alternative method for the synthesis of geranyl propionate.

Effects of Additives on Lipase Immobilization in Microemulsion-Based Organogels

An inexpensive, facile, and environmentally benign method was developed to improve the activity and stability of Candida rugosa lipase (triacylglycerol acylhydrolase) immobilized on microemulsion-based organogels (CRL MBGs) via the addition of additives during immobilization. The additives used were polyethylene glycol (PEG) or polysaccharides. This study is the first report on the effect of additives in CRL MBGs. Among the tested additives, PEG produced the most improvement in the immobilized CRL, enhancing its stability in organic solvents (specifically polar solvents). The results of circular dichroism and fluorescence spectra experiments indicated that exposure of the acidic CRL to electronegative additives in the buffer, such as polyethylenimine and the electropositive surfactant cetyltrimethylammonium bromide, may change the lipase secondary structure, ultimately causing enzyme inactivation. However, sodium bis(2-ethylhexyl)sulfosuccinate and PEG 2000 had minimal effects on the secondary structure of CRL. The CRL MBGs containing PEG 2000 demonstrated remarkable retention of their catalytic activity during the recycling test. No significant changes in enzymatic activity were observed, even after nine runs, and 90% of the original yield was maintained after 15 cycles.

Structure–activity relationships of Candida rugosa lipase immobilized on polylactic acid nanoparticles

Interfacing proteins with nanostructured materials offers the possibility to obtain novel bioconjugates for many applications. We report herein the ability of nanostructured poly-DL-lactic acid (PDLLA) based carriers to enhance enzymatic activity and stability. PDLLA was processed using an innovative patented methodology that permitted to obtain spherical nanoparticles with an average diameter of 220 nm that were used as carrier for the physical adsorption of Candida rugosalipase (CRL). Enzymatic activity and stability of CRL before and after conjugation to the nanopolymeric support were evaluated in different conditions (pH, T, organic solvents) and the conformational changes of CRL produced by its interaction with the nanopolymeric carrier were investigated by using Fourier Transform Infrared (FTIR) spectroscopy. A comparative study between X-ray diffraction data in the literature and experimental FTIR results gave deeper insight into the conformational features of the immobilized protein.

Esterification activity and operational stability of Candida rugosa lipase immobilized in polyurethane foams in the production of ethyl butyrate

Ethyl butyrate is a fruity flavor ester widely used in food and pharmaceutical products. The synthesis of ethyl butyrate in n -hexane, catalyzed by Candida rugosa lipase immobilized in two hydrophilic polyurethane foams (“HYPOL FHP 2002” and “HYPOL FHP 5000”) was performed. In this study, the effects of (i) the immobilization supports, (ii) the initial substrate concentrations and (iii) the water content of the system, on the activity and operational stability of C. rugosa lipase in both foams, during the esterification in continuous packed-bed reactor (PBR) and in repeated batches, were investigated. When low substrate concentrations were used, no deactivation was observed for both biocatalysts, along the continuous 30-d PBR operation. Conversely, under high substrate concentrations, a fast deactivation of the biocatalysts was observed. In consecutive batches, the deactivation was faster for the lipase in the less hydrophilic foam (“FHP 5000”) with a half-life of 53 h against 170.3 h for the other counterpart. Water molecules in the microenvironment did not present a deactivation effect on the biocatalysts. The low operational stability can be ascribed to the inhibitory effect of ethanol, which tends to accumulate inside the foams.

Increased activity and stability of Candida rugosa lipase in reverse micelles formed by chemically modified AOT in isooctane

AbstractSodium bis (2‐ethylhexyl polyoxyethylene) sulfosuccinates, which can be structurally viewed as chemically modified AOT (MAOT), were prepared and successfully used to form reverse micelles in isooctane. The activity and stability of Candida rugosa lipase (glycerol‐ester hydrolase, EC 3.1.1.3) appreciably increased in the MAOT/isooctane reverse micellar system compared with the AOT/isooctane system taking the hydrolysis of olive oil as a model reaction. The best polymerization number of oxyethylene group was found to be 2.0, which corresponded to a maximum enzyme activity of twice that in the AOT/isooctane system. The half‐life of lipase in the MAOT micellar system was twice that in the AOT micellar system. The optimal operational conditions remained unchanged. The Michaelis kinetics showed that the maximum reaction rate obviously increased in the MAOT micellar system compared with that in the AOT micellar system, while the change in the Michaelis constant was insignificant.© 2001 Society of Chemical Industry

Activity and batch operational stability of Candida rugosa lipase immobilized in different hydrophilic polyurethane foams during hydrolysis in a biphasic medium

The aim of this study was to investigate eventual relationships between some physico-chemical properties (e.g. porosity, aquaphilicity, partition coefficient for oleic acid and drying curves) of relatively hydrophilic polyurethane foams and the activity and batch operational stabiliy of Candida rugosa lipase immobilized within these foams. Two biocompatible polyurethane pre-polymers (“HYPOL FHP 2002TM” and “Hypol FHP X4300TM” from Hampshire Chemical GmbH, Germany) were tested as immobilization supports. The model reaction was the hydrolysis of crude olive residue oil in a biphasic aqueous/n-hexane medium.

Activity and stability of lipase in Aerosol-OT/isooctane reverse micelles

The stability of Candida rugosa lipase, which catalyzes the hydrolysis reaction of olive oil in AOT/isooctane reverse micelles, decreased with the increase of ω0 (defined as the molar ratio of water to surfactant) and Aerosol-OT concentration. The addition of a non-ionic cosurfactant, tetraethylene glycol dodecyl ether (C12E4), preserved enzymatic activity. The residual activity of the lipase was 53% after 24 h, while the enzyme completely lost its activity within 6 h in the absence of C12E4 addition. The stabilizing effect of C12E4 resulted in the increase of conversion. The enhancement of the activity and stability of lipase in reverse micelles by the addition of C12E4 may contribute to increase the rigidity of the micellar matrix stabilizing the enzyme structure.

Esterification activity and stability of Candida rugosa lipase in AOT microemulsion-based organogels

AOT microemulsion-based organogels (MBGs) were investigated as a reaction medium for the esterification of lauric acid with butanol catalyzed by Candida rugosa lipase. Gelatin was used as the gelling component of the MBGs. In a reactor fitted with a basket-type impeller, the initial reaction rate remained constant when the agitation rate was 0.83 s −1 or above. The reaction rate of immobilized lipase was enhanced with a high gelatin concentration. Under a fixed W G ( = [H 2O]/[AOT]) value of 100, the maximal reaction rate was obtained at an AOT concentration of 150 mM. The diffusion coefficient changed as a function of the gelatin and AOT concentrations, and its profile corresponded to that of the reaction rate. Immobilized lipase was also reused in a batch reaction system. In this case, the lipase activity was maintained successfully for 19 batch runs, that is, for 912 h. Water that accumulated in the MBGs was removed using a concentrated amphiphile solution.

Glycerolysis of olive oil: batch operational stability of Candida rugosa lipase immobilized in hydrophilic polyurethane foams

The operational stability of the Candida rugosa lipase immobilized in a hydrophilic polyurethane foam was evaluated in consecutive batches for the glycerolysis of olive oil in n-hexane, aimed at the production of monoglycerides. Glycerol controlled the glycerolysis in the system under study, since it is both a substrate and a powerful water binder that reduces the water activity of the reaction medium and of the microenvironment. Two sets of experiments were carried out under different glycerol/triglyceride ratios. After 345 hours of consecutive 23 hours batches no lipase inactivation was observed.

Activity decay and conformational change of lipase in presence of organic solvents. A fluorescence study of Candida rugosa lipase

Different factors that modify the stability of Candida rugosa lipase in presence of organic solvents have been investigated. Distinct aqueous and, mono- or biphasic organic media have been studied comparatively. Stabilities of crude and purified isospecies from Candida rugosa lipase in a polar organic medium have been compared, and the effect of contaminants and depressors of water activity discussed. In addition, factors typically tested in studies of enzyme activity have been considered: water content, solvent properties, protein aggregation, enzyme concentration, etc. The crude lipase suspended in acetonitrile is more stable than in n-heptane with the same water content, but the opposite is true when the protein is solubilized in these organic media-water systems. Similar changes of enzyme conformation during the inactivation process of crude and pure lipase, in acetonitrile-water media and in water have been detected by fluorescence spectroscopy. Inactivation curves in presence of acetonitrile are biphasic. The first activity decay was found to be favored by acetonitrile, but the fluorescence study reflects that conformational changes causing the rest of the inactivation are decelerated in presence of this co-solvent.

Glycerolysis of olive oil: batch operational stability of Candida rugosa lipase immobilized in hydrophilic polyurethane foams

Glycerolysis of olive oil: batch operational stability of Candida rugosa lipase immobilized in hydrophilic polyurethane foams