Activity Of Candida Rugosa Lipase Research Articles

Enhanced triolein and ethyl ferulate interesterification performance by CRL-AuNPs

This study established a Candida rugosa lipase (CRL) system to catalyze triolein and ethyl ferulate interesterification. The products were identified, and the binding mode between the substrates and CRL was predicted through molecular docking. Three methods for preparing CRL-AuNPs were proposed and characterized. It was found that the addition of 40 mL of 15 nm gold nanoparticles increased the CRL activity from 3.05 U/mg to 4.75 U/mg, but the hybridization efficiency was only 32.7 %. By using 4 mL of 0.1 mg/mL chloroauric acid, the hybridization efficiency was improved to 50.7 %, but the enzyme activity was sharply decreased. However, when the molar ratio of Mb to HAuCl4 was 0.2, the hybridization efficiency increased to 71.8 %, and the CRL activity was also enhanced to 5.98 U/mg. Under optimal conditions, the enzyme activity of CRL-AuNPs③ was maintained at 95 % after 6 repetitions and 85.6 % after 30 days at room temperature.

Titration vs. GC-MS Analysis to Determine the Factors Affecting the Esterification Activity of Candida rugosa Lipase Immobilized onto Celite

The activity of lipases to catalyze the synthesis of esters in a non-aqueous environment can be assessed by performing a simple esterification study. In such tests, titration of the remaining acid has been one of the most used methods to determine the reaction progress due to its simplicity. Nonetheless, the execution of the titration is not always as simple as it sounds. In this study, Candida rugosa lipase is immobilized onto celite matrix, and its esterification activity was evaluated by catalyzing the reaction between butanol and butyric acid. Among the factors affecting the esterification activity of the immobilized enzymes are celite:CRL ratio, buffer pH during immobilization, and post-immobilization drying time. The titration results were analyzed using factorial design, ANOVA test, and Pareto chart. Here, the ambiguity of the titration results is showcased. On the other hand, analyzing the esterification results using GC-MS allowed optimization of the immobilization method to be performed. Finally, factors affecting the activity of the immobilized enzyme can be better assessed when the esterification results were analyzed using GC-MS.

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

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

β-Sitosterol Oleate Synthesis by Candida rugosa Lipase in a Solvent-Free Mini Reactor System: Free and Immobilized on Chitosan-Alginate Beads

Candida rugosa lipase (CRL) was immobilized by the ionic gelling technique using alginate and chitosan as encapsulating agents. An immobilization yield of 99% and an immobilization efficiency of 51% were obtained. Maximum hydrolytic activity for free and immobilized CRL was detected at 40 °C and for synthesis activity at 35 °C. The optimum pH for immobilized and free CRL hydrolysis activity was 8.0. The Vmax obtained for the hydrolysis reaction was higher for free CRL (4121.4 μmol/min/g) compared to immobilized CRL (2359.13 μmol/min/g). A Vmax of 2.24 μmol/min/g was detected for the synthetic activity of free CRL. The Km obtained for the hydrolysis reaction was higher (660.02 μmol/L) for immobilized CRL than for free CRL (403.06 μmol/L). For the synthetic activity, a Km of 234.44 μmol/L was calculated. The conversion of β-sitosterol oleate ranged from 80.85 to 96.84% for free CRL, higher than the maximum found for immobilized CRL (32%). The scale-up (scale factor: 50) with the free CRL was successfully performed, achieving a high conversion value (92%) in a 500 mL bioreactor. This conversion value was within the range predicted by the mathematical model obtained using mini reactors. These mini reactors are good models to test several conditions of enzyme reactions that are intended for large scales.

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

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

Hollow Mesoporous Carbon-Based Enzyme Nanoreactor for the Confined and Interfacial Biocatalytic Synthesis of Phytosterol Esters.

Rationally designing carriers to obtain efficient and stable immobilized enzymes for the production of food raw materials is always a challenge. In this work, hollow cube carbon (HMC) as a carrier of Candida rugosa lipase (CRL) was prepared to construct a Pickering interfacial biocatalysis system, which was applied to biphasic biocatalysis. For comparison, the nonporous carbon (HC) and porous MoS2 (HMoS2) were also designed. On these grounds, p-NPP and linolenic acid were selected as the representative substrates for hydrolysis and esterification reactions. Under the optimal conditions, the protein loading amount, specific activity, and expressed activity of CRL immobilized on HMC (HMC@CRL) were 167.2 mg g-1, 5.41 U mg-1, and 32.34 U/mg protein, respectively. In the "oil-water" biphase, the relative hydrolytic activity of HMC@CRL was higher than that of HC@CRL, HMoS2@CRL, and CRL by 50, 68, and 80%, respectively, as well as itself in one phase. Compared to other reports (1.13%), HMC@CRL demonstrated a satisfactory hydrolysis rate (3.02%) and was the fastest among all other biocatalysts in the biphase. Moreover, compared with the free CRL in one-phase system, the Pickering interfacial biphasic biocatalyst, HMC@CRL, exhibited a higher esterification rate (85%, 2.7-fold enhancement). Therefore, the HMC@CRL nanoreactors had more optimal performance in the field of biomanufacturing and food industry.

Synthesis of pyridinium-based ionic liquids and their application to improve Candida rugosa lipase stability in a methanol-water solvent system.

This paper studied the effect of pyridinium-based ionic liquids as cosolvents in a methanol-water solvent system on the hydrolytic activity of Candida rugosa lipase. These ionic liquids were successfully synthesized using imidazolium-based ionic liquid synthesizing methods with a certain adjustment. The hydrolytic activity of Candida rugosa lipase was analyzed using 4-nitrophenol acetate (pNPA) and 4-nitrophenol palmitate (pNPP) as substrates. The addition of ionic liquids had no significant effect on the hydrolytic activity of lipase in a water solvent, and it had a greater effect in methanol. The addition of [C6Py] Br ionic liquid as a methanol cosolvent (methanol: ionic liquid, 10:5) could increase the hydrolytic activity of lipase. The use of ionic liquid as a cosolvent could increase the hydrolytic activity of lipase by about 15.61% while using pNPP as a substrate in the methanol system. A molecular dynamics study for the interaction between lipase and ionic liquids supported the experimental results. The ionic liquid using bromide as an anion provided more stability on lipase conformation. It tends to form the short-range interaction between the lipase and bromide anion.

Effect of triblock copolymers on the lipase catalytic behavior at the interface of conventional O/W emulsions

The ‘interfacial quality’ is the key influencing factor for lipase activities at interfaces, and thus, interfacial engineering has become an effective strategy to modulate lipase catalysis. In the current research, the triblock copolymers poly (ethylene oxide)-poly (propylene oxide)-poly (ethylene oxide) (PEO-PPO-PEO) were adopted to form conventional oil-in-water (O/W) emulsion systems. The results showed the different effects of PEO-PPO-PEO molecules on the spatial barriers, emulsion particle size, and ultimately interfacial adsorption properties of lipases. Since the molecular weight of Candida antarctica lipase B (CALB) is approximately half that of Candida rugosa lipase (CRL), it can more easily pass through the triblock copolymer adsorption layer, resulting in a higher adsorption ratio. The results confirmed that the catalytic efficiency of CALB at the emulsion interfaces was much lower than that of CRL due to its lack of a typical ‘lid’ structure. Both the effective amount of lipase molecules at the interfaces (controlled by the interfacial adsorption ratio) and the effective catalytic interface area (influenced by the specific surface area controlled by the average particle size of the O/W emulsion) impacted the catalytic activities of CRL. Na+ ions were found to have a certain inhibitory effect on the catalytic activity of CRL.

Probing the interaction of superparamagnetic iron oxide nanoparticles with lipase and their interacting consequences at the molecular level.

Although superparamagnetic iron oxide nanoparticles (SPIONs) are used as carriers for candida rugosa lipase (CRL) in biomedical fields, their interactions and the influences on CRL are still unknown. Consequently, SPIONs were synthesized, characterized, and incubated with CRL to explore their molecular interactions and interacting consequences in this study. The toxic effects of SPIONs on CRL and their molecular interactions were explored through transmission electron microscope, isothermal titration calorimetry, zeta potential measurements, multi-spectroscopic techniques, and biological enzyme activity tests. Results revealed the adsorption of SPIONs to CRL and the reduction of CRL aggregation. The unfolding and loosening of CRL structure as well as the change of secondary structure with the decrease of α-helix were found under SPIONs exposure. Moreover, higher SPIONs concentrations contributed to larger conformational changes and less aggregation of CRL. Meanwhile, it showed that hydrophobic forces were the dominant driving forces in the binding process, with the participation of electrostatic forces. CRL binds to SPIONs with the stoichiometry of 20.7 and the binding constant of 9.9×106M-1. No obvious changes were found in CRL activity due to no interference to Ser-209, Glu-341, and His-449 residues. This study examined the biological compatibility of SPIONs at the molecular level and provided important information about the structure and function of CRL upon binding to SPIONs. Our work might contribute to comprehend the molecular toxicity of SPIONs and the risks of engineered nanoparticles to human health.

Taguchi orthogonal design assisted immobilization of Candida rugosa lipase onto nanocellulose-silica reinforced polyethersulfone membrane: physicochemical characterization and operational stability

A greener processing route to replace the current environmentally-unfriendly esterification technique to produce biofuels such as pentyl valerate (PeVa) was explored. This study statistically optimized the covalent immobilization of Candida rugosa lipase (CRL) onto biomass-based nanocellulose-silica (NC-SiO2) reinforced polyethersulfone (PES) membrane to synthesize PeVa. Raman spectroscopy, field emission scanning electron microscopy, high-resolution transmission electron microscopy, and atomic force microscopy of NC-SiO2-PES/CRL proved that CRL was successfully conjugated to the membrane. The optimized Taguchi Design-assisted immobilization of CRL onto NC-SiO2-PES membrane (5% glutaraldehyde, 4 h of immobilization, 20 mg/mL CRL concentration, 40 °C and pH 5) gave 90% yield of PeVa in 3 h. The thermal stability of NC-SiO2-PES/CRL was ~ 30% greater over the free CRL, with reusability for up to 14 successive esterification cycles. In a nutshell, the greener NC-SiO2-PES membrane effectively hyperactivated and stabilized the CRL for the esterification production of PeVa. This research provides a promising approach for expanding the use of sustainably sourced NC and SiO2 nanoparticles, as fillers in a PES for improving CRL activity and durability for an extended catalytic process.

Novel, natural allosteric inhibitors and enhancers of Candida rugosa lipase activity

Candida rugosa lipase (CRL) is an enzyme commonly used in medicinal and biotechnological applications. Allosteric modulators of CRL could aid in modifying lipase-related diseases as well as improving biotechnological processes.Thus, a combinatorial approach of computational in-silico and high-throughput in-vitro screening was used to identify allosteric modulators of CRL. The screening of natural product libraries resulted in 132 compounds of which 53 were tested in-vitro. Subsequently, four inhibitors and three enhancers were identified of which rutin and cynaroside represented the strongest inhibitors of CRL activity (IC50: 227 ± 26 µM and 446 ± 15 µM, respectively) and NP-008496 the strongest enhancer (EC50: 425 ± 18 µM). All three compounds were predicted to bind the same allosteric site suggesting a common mechanism.Therefore, the present study demonstrated a reliable work-flow, identified an allosteric site of CRL and determined inhibitors and enhancers with numerous potential medical and biotechnological applications.

Candida rugosa lipase alters the gastrointestinal environment in wild-type mice

Diet and commercially available supplements can significantly impact the gut microbial composition; however, the effects of supplements often lack scientific data demonstrating the effects on healthy and diseased individuals. Hence, it was investigated, whether a frequently used supplement in humans, Candida rugosa lipase (CRL), gets delivered active beyond the stomach in the intestinal tract of C57BL/6 J mice and its impact on the gut microbial community and environment.We showed for the first time the movement of CRL in an active state through the mouse digestive tract by determination of intestinal CRL activity and free fatty acids concentrations. The short- and long-term administration of CRL resulted in significant alterations of the gut microbiome, favoring the growth of, for instance, Verrucomicrobia but also other species associated with normal body mass index (BMI) or butyrate expression, both considered beneficial. In addition, we showed that these changes persisted after supplementation and that gut barrier integrity was unaffected by the treatment.In conclusion, CRL can be delivered in an active state beyond the stomach and supplementation altered the murine gut microbiome favoring beneficial bacterial species, which may be of relevance in humans in healthy but also potentially in disease states.

Effect of cyclic and acyclic surfactants on the activity of Candida rugosa lipase.

The active site of Candida rugosa lipase (CRL) is mainly hydrophilic on its external face and hydrophobic on the internal side, and calix[n]arene-based surfactants form complexes with protein residues or with strong hydrogen bonds to open up the lid. Therefore, the activity of lipase persists for a long time. In this work, a series of cyclic and acyclic anionic surfactants (sodium dodecyl sulfate (SDS), p-sulfonatocalix[4]arene, and p-sulfonatocalix[8]arene) and zwitterionic surfactants (L-proline and L-proline derivative of calix[4]arene) were used to examine the relationship between the surfactants' molecular structures and their effects on the hydrolytic activity of CRL. We explored the effects of different surfactant concentrations, ring effects, and mixing times on CRL activity and several kinetic parameters. The results demonstrated that cyclic compounds were more effective than linear structures for increasing CRL activity and the highest enzyme activity was obtained by the addition of the calix[4]-L-proline derivative. This zwitterionic compound (calix[4]-L-proline derivative) maintains the active center of enzyme and conformation by enabling electrostatic interactions and hydrogen bonding with both the acidic and basic amino acid groups in the structure of the enzyme. The results indicated that, compared with the other surfactants, activating CRL with calix[4]-L-proline resulted in hyperactivation at all concentrations (a relative increase of 230%).

Enhanced biological activity of Candida rugosa lipase via immobilization on magnetic nanoparticles

Magnetic nanoparticles Fe3O4 were synthesized by the co-precipitation method. Magnetic nanoparticles were activated and modified with (3-aminopropyl) triethoxysilane (APTES) and glutaraldehyde (GA), respectively. Then, these nanoparticles were characterized by transmission electron microscope and Fourier transform infrared. Candida rugosa lipase (CRL) was successfully immobilized on GAMNPs by the chemical method via forming a covalent bond between a functional group on an enzyme protein molecule and a reactive group on the surface of solid support by chemical interaction. CRL-Fe3O4 displayed an elevate recovery rate of upward to 1348%, which was a 13-fold improvement in its free one. Compared to a free enzyme, the pH and thermal properties of the immobilized lipase were also increased. This study clearly indicates that GAMNPs could be deemed good support for the immobilization of enzymes

Effect of Glutaraldehyde Concentration on Catalytic Efficacy of <i>Candida rugosa</i> Lipase Immobilized onto Silica from Oil Palm Leaves

Till date, studies that investigated the effect of glutaraldehyde concentration on catalytic efficacy of biocatalyst developed with silica-derived from oil palm leaves (OPL) as support, are unknown. The study presents the preparation of a support consisting of silica extracted from OPL coated over magnetite (G/A/SiO2-M) for the immobilization of Candida rugosa lipase (CRL). Herein, the effect of glutaraldehyde concentration on the catalytic efficacy of immobilized CRL was assessed by the enzymatic production of butyl butyrate as a model. Fourier transform infrared (FTIR) spectra and immobilization parameters indicated that covalent bound CRL on functionalized OPL-derived silica-magnetite composite activated with 4% (v/v) glutaraldehyde solution (100 mM, pH 7.0) (CRL/G/A/SiO2-M) and pretreated in toluene, resulted in a protein loading and an immobilization yield of 68.3 mg/g and 74.3%, respectively. The resultant CRL/G/A/SiO2-M biocatalyst which specific activity was 61.9 U/g catalyzed the esterification production of 76.5% butyl butyrate in just 3 h, as confirmed by analyses of the purified ester using FTIR and 1H NMR spectroscopy. Hence, the finding envisages the promising use of G/A/SiO2-M support fabricated from discarded OPL as a carrier for immobilization and activation of CRL, in conjunction to being a good alternative source of renewable silica.

Two phase enzymatic membrane reactor for the production of biodiesel from crude Eruca sativa oil

In the present study, a novel two-phase enzymatic membrane bioreactor (TP-EMR) was developed for the enzymatic biodiesel production from crude Eruca sativa oil. Commercial poly acrylonitrile (PAN) ultrafiltration membrane was examined with a molecular weight cut offs (MWCO) of 30 and 100 kilo Dalton (kDa). Using enzymatic membrane bioreactor with hydrophilic PAN membrane increased the yield of biodiesel production. In this regards, PAN membrane with a MWCO of 100 kDa gave the better results. The inactivation and inhibition effects of methanol and produced glycerol were reduced with the use of membrane bioreactor. It was found that by methanol feeding via aqueous phase, methanol has been penetrated across membrane pores to the organic phase. Afterwards, methanol has reacted with oil at the interface of the membrane at the presence of Candida rugosa lipase (CRL) as a biocatalyst. Produced glycerol, which surrounded the CRL, could be removed from organic phase and conveyed to the aqueous phase to reduce the inhibitory effect of glycerol on the activity of CRL. The yield of about 100% from Eruca sativa oil was achieved in TP-EMR with PAN 100 and organic phase flow rate of 40 ml/min along with 40% initial water content in organic phase.

Effect of Surfactant Structure on the Superactivity of Candida rugosa Lipase.

In this work, we present the effects of ionic and zwitterionic surfactants on the hydrolytic activity of Candida rugosa lipase (CRL), one of the most important and widely used microbial lipases. A series of amine N-oxide surfactants was studied to explore the relationship between their molecular structures and their effect on catalytic properties of CRL. These zwitterionic amphiphiles are known for their ability to form aggregates that can increase their size, thanks to a sphere-rod transition, without any additive. Enzyme activity seemed to be improved by morphological changes of micelles from spherical to rod-like, and the structure of the monomers played a crucial role in this transition. In fact, all the amine oxides investigated provoked superactivation, but the CRL activity increased by lengthening the alkyl chain of N-oxide surfactants, whereas it decreased in the presence of bulky head groups. Superactivity was mainly because of an increase in kcat (0.57 s-1 in buffer, 0.80-1.99 s-1 in surfactant solutions) and, in some cases, a decrease in KM (2 × 10-3 M in buffer, 1.08-4.28 × 10-3 M in surfactant solutions). Micelles seemed to play a dual role: superactivity occurred at surfactant concentrations higher than their critical micelle concentration, but, on the other hand, micelles subtracted the substrate from the bulk, making it unavailable for the catalysis.

The binding interaction between cadmium-based, aqueous-phase quantum dots with Candida rugosa lipase.

As a promising biolabeling biomaterials, quantum dots (QDs) present a great potential. However, the toxicity of QDs to organisms has attracted wide attention. In our research, we introduced an in vitro method to study the molecular mechanisms for the structure and activity alterations of Candida rugosa lipase (CRL) with the binding of 3-mercaptopropionic acid-capped CdTe QDs. Multiple spectroscopic methods, isothermal titration calorimetry, and enzyme activity measurements were used in this paper. QDs statically quenched the intrinsic fluorescence of CRL with the quenching constant decreases from 2.46×1013 to 1.64×1013 Lmol-1 second-1 (298 to 310K). It binds to CRL through hydrophobic force with 1 binding site, unfolding and loosening the skeleton and changed its secondary structure. Rather than aggregating on the surface, it enters the pocket of the CRL to interact with Ser-209 (2.43Å) and the residues surrounding Ser-209, making the catalytic triad more exposed. Furthermore, the activity of CRL was inhibited by approximately 15%. This work demonstrates that 3-mercaptopropionic acid-capped CdTe QDs may cause negative effects to CRL and obtains a molecular mechanism on QD-induced toxicity to proteins in vitro.

Characterization, optimization and stability studies on Candida rugosa lipase supported on nanocellulose reinforced chitosan prepared from oil palm biomass

The contribution of chitosan/nanocellulose (CS-NC) to the enzymatic activity of Candida rugosa lipase covalently bound on the surface of CS-NC (CRL/CS-NC) was investigated. Cellulosic material from oil palm frond leaves (OPFL) were bleached, alkaline treated and acid hydrolyzed to obtain the purified NC and used as nano-fillers in CS. XRD, Raman spectroscopy and optical fluorescence microscopic analyses revealed existence of strong hydrogen bonds between CS and the NC nanofillers. The CRLs were successfully conjugated to the surface of the CS-NC supports via imine bonds that occurred through a Schiff's based mechanism. Process parameters for the immobilization of CRL were assessed for factors temperature, concentration of glutaraldehyde and pH, to afford the highest enzyme activity to achieve maximum conversion of butyl butyrate within 3h of incubation. Conversion as high as 88% was reached under an optimized condition of 25°C, 0.3% glutaraldehyde concentration and buffer at pH7. Thermal stability of CRL/CS-NCs was 1.5-fold greater than that of free CRL, with biocatalysts reusability for up to 8 successive esterification cycles. This research provides a promising approach for expanding the use of NC from OPFL for enhancing enzyme activity in favour of an alternative eco-friendly means to synthesize butyl butyrate.

Exploring the binding interaction between copper ions and Candida rugosa lipase.

The wide range of applications of copper have caused widespread concern about its toxicity. However, few studies have reported the mechanism of the binding interaction between copper ions and digestive enzymes, which play an important role in physiological health and industrial production. In this study, the effects of copper ions on the conformation and activity of Candida rugosa lipase (CRL) were evaluated by isothermal titration calorimetry (ITC), multiple spectral techniques, molecular simulation and enzyme activity assays. The results showed that copper ions can be combined with lipase, the binding affinity constant (K) was (2.91 ± 0.619) × 10-3 M-1, the binding process was a spontaneous process, and the main force was the hydrophobic force. Rather than increasing the hydrophobicity of the amino acid microenvironment of CRL, the spectral methods demonstrated that copper can also make the protein peptide bond structure compact, changing its secondary structure. In addition, molecular simulation results showed that copper ions opened the "lid" of the CRL and entered the active center, which consequently changed the conformation of the CRL molecule. Structural changes may cause changes in enzyme activity. The increased activity of CRL with the addition of copper ions was verified by enzyme activity assay. In summary, copper showed an effect on CRL at the molecular level, which means its toxicity should receive more attention.