Immobilization Of Candida Antarctica Lipase Research Articles

L-cysteine-coated magnetite nanoparticles as a platform for enzymes immobilization: Amplifying biocatalytic activity of Candida antarctica Lipase A

This study presents the synthesis of magnetite nanoparticles coated with L-cysteine (Fe3O4@LC) and their subsequent utilization as a support matrix for the immobilization of Candida antarctica Lipase A (CALA). The immobilization process involved physical interactions and covalent bonding mediated by glutaraldehyde. Comprehensive characterization was conducted using techniques including X-ray diffraction (XRD), transmission electron microscopy (TEM), scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), and vibrating sample magnetometry (VSM), providing compelling evidence of a successful synthesis. Under optimized conditions, employing 1 mg of protein per gram of support, pH 7 in a 25 mM phosphate buffer, and a 5 h reaction at 25 °C, immobilization on glutaraldehyde-activated support achieved an impressive yield of 85.0 % ± 2.6, accompanied by a specific activity of 212.5 ± 1.3 U/g, outperforming the physical adsorption approach. Remarkably, the immobilized enzyme exhibited higher activity than the free enzyme at alkaline and acidic pH levels. Furthermore, thermal and pH inactivation studies revealed that the biocatalyst's half-life exceeded that of free CALA by more than 8 times at pH 10. These results underscore the potential of the Fe3O4@LC-GLU-CALA system as a robust biocatalytic matrix with promising applications in biodiesel production, ester synthesis, and pharmaceutical manufacturing.

Isocyanate-mediated immobilization of Candida antarctica lipase B onto resin for efficient 1,3-diacylglycerol bio-synthesis

In this work, isocyanate-based strategy was developed for efficient immobilization of Candida antarctica lipase B (CALB). Of 41 commercial resins, EP109 was chosen and hydrophobized with hydroxyl-terminated polydimethylsiloxane (HTPDMS) for tert-butyl isocyanate (tBIC)-based CALB immobilization due to high loading amount and specific activity. The tBIC-based strategy exhibited shorter immobilization time, higher loading and activity than adsorption strategy by hydrophobic interaction. When compared with free CALB, EP109-HTPDMS-tBIC@CALB owned improved stability (against acid, alkali, and heat) and storability. Subsequently, EP109-HTPDMS-tBIC@CALB was employed in the bio-synthesis of 1,3-diacylglycerol in a solvent-free system with a yield of 76.31%, which was higher than commercial Novozym 435 (53.93%). EP109-HTPDMS-tBIC@CALB exhibited 76.34% of residual activity after 20 repeat uses.

Improved lipase performance by covalent immobilization of Candida antarctica lipase B on amino acid modified microcrystalline cellulose as green renewable support

Development of immobilized lipase with excellent catalytic performance and low cost is the major challenge for large-scale industrial applications. In this study, green renewable microcrystalline cellulose (MCC) that was hydrophobically modified with D-alanine (Ala) or L-lysine (Lys) was used for immobilizing Candida antarctica lipase B (CALB). The improved catalytic properties were investigated by experimental and computational methods. CALB immobilized on MCC-Ala with higher hydrophobicity showed better catalytic activity than CALB@MCC-Lys because the increased flexibility of the lid region of CALB@MCC-Ala favored the formation of open conformation. Additionally, the low root mean square deviation and the high β-sheet and α-helix contents of CALB@MCC-Ala indicated that the structure became more stable, leading to a significantly enhanced stability (54.80% and 90.90% relative activity at 70 °C and pH 9.0, respectively) and good reusability (48.92% activity after 5 cycles). This study provides a promising avenue to develop immobilized lipase with high catalytic properties for industry applications.

Immobilization of Candida antarctica lipase B on ILs modified CNTs with different chain lengths: Regulation of substrate tunnel “Leucine gating”

Ionic liquids (ILs) have been widely used as chemical modifiers to modify the carriers and thus improve the efficiency, activity and stability of the enzymes. However, as thousands of ILs have been found up to date, it's a huge work for screening and designing suitable ILs for immobilization of enzymes. Moreover, the mechanism of improving enzymes catalytic performance is still remain ambiguous. Thus, this study investigated the impact of ILs with different chain lengths on the enzymatic properties of Candida antarctica lipase B (CALB). Molecular dynamics simulations were employed to examine the interaction between ILs modified CNTs and CALB, as well as their effects on CALB's structure. The results revealed that ILs with different chain lengths significantly influenced the absorption orientation of CALB. Tunnel analysis identified a key role for Leu278 in regulating the open or closed state of Tunnel 2 during CALB's catalytic cycle. The weak interaction analysis demonstrated that ILs with suitable chain lengths provided spatial freedom and formed strong interactions with CNTs and ILs (vdW and hbond). This led to a conformational flip of Leu278, stabilizing the open state of Tunnel 2 and improving the activity and stability of immobilized CALB. This study provides novel insights into the design of new green modifiers to modulate carrier performance and obtain immobilized enzymes with better performance, and establishes a theoretical basis for the design and selection of modifiers for ILs in future work.

Biodiesel Production Using Palm Oil with a MOF-Lipase B Biocatalyst from Candida Antarctica: A Kinetic and Thermodynamic Study

This research presents the results of the immobilization of Candida Antarctica Lipase B (CALB) on MOF-199 and ZIF-8 and its use in the production of biodiesel through the transesterification reaction using African Palm Oil (APO). The results show that the highest adsorption capacity, the 26.9 mg·g−1 Lipase, was achieved using ZIF-8 at 45 °C and an initial protein concentration of 1.20 mg·mL−1. The results obtained for the adsorption equilibrium studies allow us to infer that CALB was physically adsorbed on ZIF-8 while chemically adsorbed with MOF-199. It was determined that the adsorption between Lipase and the MOFs under study better fit the Sips isotherm model. The results of the kinetic studies show that adsorption kinetics follow the Elovich model for the two synthesized biocatalysts. This research shows that under the experimental conditions in which the studies were carried out, the adsorption processes are a function of the intraparticle and film diffusion models. According to the results, the prepared biocatalysts showed a high efficiency in the transesterification reaction to produce biodiesel, with methanol as a co-solvent medium. In this work, the catalytic studies for the imidazolate, ZIF-8, presented more catalytic activity when used with CALB. This system presented 95% biodiesel conversion, while the biocatalyst formed by MOF-199 and CALB generated a catalytic conversion percentage of 90%. Although both percentages are high, it should be noted that CALB-MOF-199 presented better reusability, which is due to chemical interactions.

Comparison of covalent and in situ immobilization of Candida antarctica lipase A on a flexible nanoporous material.

In this study, Candida antarctica lipase A, which has a unique applicability for the conversion of highly branched and bulky substrates, was subjected to immobilization on the flexible nanoporous MIL-53(Fe) by two approaches: covalent coupling and in situ immobilization method. The pre-synthesized support under ultrasound irradiation was incubated with N,N-dicyclohexylcarbodiimide to mediate the covalent attachment between the carboxylic groups on the support surface and amino groups of enzyme molecules. The in situ immobilization in which the enzyme molecules directly were embedded into the metal-organic framework was performed under mild operating conditions in a facile one-step manner. Both immobilized derivatives of the enzyme were characterized by scanning electron microscopy, X-ray diffraction, thermogravimetric analysis, FT-IR spectra, and energy-dispersive X-ray spectroscopy. In the in situ immobilization method, the enzyme molecules were efficiently encapsulated within the support with a high loading capacity (220 ± 5mg/g support). On the other hand, the covalent attachment resulted in immobilizing much lower concentrations of the enzyme (20 ± 2.2mg/g support). Although both immobilized derivatives of lipase showed broader pH and temperature tolerance relative to the soluble enzyme, the biocatalyst, which was prepared through in situ method, was more stable at elevated temperatures than the covalently immobilized lipase. Furthermore, in situ immobilized derivatives of Candida antarctica lipase A could be efficiently reused for at least eight cycles (> 70% of retained activity). In contrast, its covalently immobilized counterpart showed a drastic decrease in activity after five cycles (less than 10% of retained activity at the end of 6 rounds).

Sortase-mediated immobilization of Candida antarctica lipase B (CalB) on graphene oxide; comparison with chemical approach

In this study, Candida antarctica lipase B (CalB) was covalently immobilized on the surface of graphene oxide (GO) nanoparticles by sortase-mediated immobilization as well as a chemical attachment approach. Sortase is a transpeptidase that provides one-step purification and targeted immobilization of CalB from one specific site, presenting oriented attachment of the enzyme to a solid support. Chemical immobilization, on the other hand, is considered as a random immobilization, in which the protein can bind to the support from different regions of the protein surface. In this approach, amine-functionalized GO was further modified with glutaraldehyde to facilitate the covalent binding of CalB via its amine residues. The applied methods produced 60% and 100% immobilization yields and presented 0.106 U/mg and 0.085 U/mg of specific activities for the oriented and random immobilization, respectively. The stabilized enzyme with the sortase-mediated approach retained approximately 80% of its initial activity at 50°C.

Sortase-Mediated Immobilization of Candida Antarctica Lipase B (Calb) on Graphene Oxide; Comparison with Chemical Approach

Sortase-Mediated Immobilization of Candida Antarctica Lipase B (Calb) on Graphene Oxide; Comparison with Chemical Approach

Sortase-Mediated Immobilization of Candida Antarctica Lipase B (Calb) on Graphene Oxide; Comparison with Chemical Approach

Sortase-Mediated Immobilization of Candida Antarctica Lipase B (Calb) on Graphene Oxide; Comparison with Chemical Approach

Enhancement of the catalytic efficiency of Candida antarctica lipase A in enantioselective hydrolysis through immobilization onto a hydrophobic MOF support

Immobilization of Candida antarctica lipase A (CAL-A) onto MOF (metal-organic framework) supports were investigated to enhance the efficiency of CAL-A in catalysis of an enantioselective hydrolysis reaction. CAL-A@ZIF-8 prepared through physical adsorption of CAL-A onto ZIF-8 was characterized and further employed as the catalyzer in kinetic resolution of carprofen (CP) enantiomers through enantioselective hydrolysis of (R,S)-CP methyl ester. Results show that CAL-A is immobilized on ZIF-8 with a loading amount of 227 mg/g. Efficiency of CAL-A is significantly improved after immobilization, which is evaluated by the yield (YS) and enantiomeric excess (eep) of (S)-CP in the hydrolysis products, and the enhancement is ascribed for the hydrophobic nature of ZIF-8. The YS of 90.62 % and eep of 97.42 % are achieved with CAL-A@ZIF-8, while only 59.79 % of YS and 92.61 % of eep are obtained with free CAL-A under the identical conditions. Moreover, CAL-A@ZIF-8 possesses superior thermal stability and pH-tolerance than free CAL-A. Immobilization of the lipase onto a hydrophobic MOF is therefore proposed for enhancement of a reaction of lipase-catalyzed ester hydrolysis.

Immobilization of Candida antarctica Lipase on Nanomaterials and Investigation of the Enzyme Activity and Enantioselectivity.

This study defines the lipase immobilization protocol and enzymatic kinetic resolution of 1-phenyl ethanol with the use of immobilized lipases (LI) as a biocatalyst. Commercially available lipase Candida antarctica B (Cal-B) was immobilized onto graphene oxide (GO), iron oxide (Fe3O4) nanoparticles, and graphene oxide/iron oxide (GO/Fe3O4) nanocomposites. Characterization of pure and enzyme-loaded supports was carried out by scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectroscopy. The influences of pH, temperature, immobilization time, crosslinker concentration, glutaraldehyde (GLA), epichlorohydrin (EPH), and surfactant concentrations (Tween 80 and Triton X-100) on the catalytic activity were evaluated for these three immobilized biocatalysts. The highest immobilized enzyme activities were 15.03U/mg, 14.72U/mg, and 13.56U/mg for GO-GLA-CalB, Fe3O4-GLA-CalB, and GO/Fe3O4-GLA-CalB, respectively. Moreover, enantioselectivity and reusability of these immobilized lipases were compared for the kinetic resolution of 1-phenyl ethanol, using toluene as organic solvent and vinyl acetate as acyl donor. The highest values of enantiomeric excess (ees = 99%), enantioselectivity (E = 507.74), and conversion (c = 50.73%) were obtained by using lipase immobilized onto graphene oxide (GO-GLA-CalB). It was obtained that this enzymatic process may be repeated five times without important loss of enantioselectivity.

Chemoenzymatic Epoxidation of Limonene Using a Novel Surface-Functionalized Silica Catalyst Derived from Agricultural Waste.

Limonene is one of the most important terpenes having wide applications in food and fragrance industries. The epoxide of limonene, limonene oxide, finds important applications as a versatile synthetic intermediate in the chemical industry. Therefore, attempts have been made to synthesize limonene oxide using eco-friendly processes because of stringent regulations on its production. In this regard, we have attempted to synthesize it using a cost-effective and eco-friendly process. Chemoenzymatic epoxidation of limonene to limonene oxide was carried out using in situ generation of peroxy octanoic acid from octanoic acid and H2O2. In this study, agricultural-waste rice husk ash (RHA)-derived silica was surface-functionalized using (3-aminopropyl) triethoxysilane (APTS), which was cross-linked using glutaraldehyde for immobilization of Candida antarctica lipase B. Furthermore, the immobilized enzyme was entrapped in calcium alginate beads to avoid enzyme leaching. Thus, limonene oxide was prepared using this catalyst under conventional and microwave heating. The microwave irradiation intensifies the process, reducing the reaction time under the same conditions. Maximum conversion of limonene to limonene oxide of 75.35 ± 0.98% was obtained in 2 h at 50 °C using a microwave power of 50 W. In the absence of microwave irradiation, the conventional heating gave 44.6 ± 1.14% conversion in 12 h. The reaction mechanism was studied using the Lineweaver–Burk plot, which follows a ternary complex mechanism with inhibition due to peroxyoctanoic acid (in other words H2O2). The prepared catalyst shows high reusability and operational stability up to four cycles.

Epoxy functionalized polymer grafted magnetic nanoparticles by facile surface initiated polymerization for immobilization studies of Candida Antarctica lipase B

Immobilization of Candida Antarctica lipase B (CalB) was done on the epoxy functionalized polymer (EFP) grafted magnetic nanoparticles (MNPs) via covalent attachment with the active epoxy groups. The EFP brushes were grafted on iron-oxide based MNPs by a facile surface-initiated atom transfer radical polymerization (ATRP) using activators generated by electron transfer (AGET) of glycidyl methacrylate (GMA). Each step of the surface modification, polymer grafting, and enzyme immobilization process on the polymer grafted MNPs was studied using Fourier transform infrared spectroscopy (FTIR). A thermogravimetric analysis (TGA) calculated the amount of engineered organic components, a transmission electron microscopy (TEM) visualized the core-shell formation of the MNPs, and a vibrating sample magnetometer (VSM) validated their magnetic properties at various modification stages. The lipase immobilization efficiency was described as a function of immobilization time, as well as, enzyme amount. The activity was characterized within a range of pH, temperature, kinetic parameters, resusability and storage stability, for both the free and immoblized Cal-B enzyme. The results of this study suggested that poly(GMA) grafted MNPs can be successfully used for the immobilization of Cal-B with improved efficiencies compared to those obtained with free soluble lipase. The reported enzyme immobilization method appears to be reproducible and scalable for industrial production.

The Immobilization of Candida antarctica lipase B by ZIF-8 encapsulation and macroporous resin adsorption: preparation and characterizations.

A novel enzyme immobilization method employing metal-organic framework (MOF) encapsulation and macroporous resin adsorption was developed in this study. Candida antarctica lipase B (CALB) was firstly encapsulated onto metal-organic frame structures (Zeolitic imidazole framework-8, ZIF-8) and further bonded to macroporous resin by physical adsorption. Under optimized immobilization conditions, the activity of the prepared immobilized lipase (CALB-ZIF-8@D101) determined via the methyl esterification of oleic acid was 38.4U/mg. Compared with free lipase, the immobilized lipase exhibited improved thermal and operational stability and organic solvent tolerance. These results demonstrate that the immobilization method of ZIF-8 encapsulation and macroporous resin adsorption enhanced enzyme properties at a superior level.

Formation lipase cross-linked enzyme aggregates on octyl-modified mesocellular foams with oxidized sodium alginate

Supported cross-linked enzyme aggregates were prepared by immobilization of Candida antarctica lipase B onto hydrophobic surface of octyl-modified mesocellular foams (MCFs-C8). Oxidized sodium alginate was used as a substitute for traditional glutaraldehyde. Supported cross-linked enzyme aggregates using oxidized sodium alginate (SA-CLEAs@MCFs-C8) exhibited significantly improved thermal stability and organic solvents tolerance compared to the free lipase, lipase adsorbed onto MCFs-C8 and supported cross-linked enzyme aggregates using glutaraldehyde (G-CLEAs@MCFs-C8). Then immobilized lipases were employed for biodiesel production by transesterification of soybean oil with methanol. In the optimization condition, SA-CLEAs@MCFs-C8 were quite stable and still showed high fatty acid methyl esters (FAME) yield after 5 repeated cycles (from 89% to 78%), whereas MCFs-C8-CALB retained 67% FAME yield (about 72% for G-CLEAs@MCFs-C8).

The synthesis of sulfonated polyethersulfone (SPES) and the preparation of its membranes as matrix in the immobilization of Candida antarctica lipase B (Cal-B)

The immobilization of lipases is often used in biotechnology to improve the performance, reusability, and stability of the enzyme. Candida antarctica lipase B (Cal-B) has been used as a catalyst in trans-esterification reaction. To improve the utilization of Cal-B, therefore the immobilization of Cal-B onto a polymer matrix became crucial. In this study, polyethersulfone (PES) and sulfonated polyethersulfone (SPES) membranes were used as matrices of Cal-B immobilization. SPES was previously synthesized by modifying PES using chlorosulfonic acid (ClSO3H) to improve the immobilization of Cal-B. SPES-PSf (polysulfone) blended membranes were also successfully prepared by blending SPES, PSf, and PEG in N-methylpyrrolidone (NMP) as the solvent using phase-inversion method. The attenuated total reflectance (ATR)–FTIR spectrum showed characteristic peaks of the immobilized Cal-B on the matrix at peak 3184.03 cm−1 (–N–H bonds) and 1683.49 cm−1 (–CH deformation bonds). The Raman spectroscopy of the PES-based membranes before and after sulfonation reaction showed the deviations from the symmetrical structure of PES, with specific Raman shifts at 784.11 cm−1, 1150.95 cm−1, and 1588.9 cm−1. Cal-B was successfully immobilized and loaded onto SPES membrane. By Lowry assay, it was detected that 140.3 μg/cm2 enzyme was successfully loaded into the 17.3 cm2 of membrane. The value was one and a half times higher than PES (91.0 μg/cm2 in 17.3 cm2). However, the hydrolytic activity of Cal-B immobilized onto SPES membrane (17.0 p-NP/min/cm2) was five times lower than Cal-B immobilized onto PES membrane (80.4 p-NP/min/cm2).

Non-covalent and covalent immobilization of Candida antarctica lipase B on chemically modified multiwalled carbon nanotubes for a green acylation process in supercritical CO2

• Efficient immobilization of Candida antarctica B lipase on modified MWCNTs. • Enzyme loading reaching as high as 21 wt.% for non-covalent immobilization. • Active O-acylation of geraniol into geranyl acetate by CAL-B in supercritical CO 2 . • Reaction regioselectivity unchanged through immobilization. • Development of a fully green enzymatic process. Candida antarctica B lipase (CAL-B) was immobilized on purified and functionalized multiwalled carbon nanotubes (MWCNTs). Both immobilization routes, physical adsorption and covalent bonding, were investigated. MWCNT functionalization by a non-aggressive oxidation by potassium permanganate led to an interesting balance between the hydrophilic and the hydrophobic areas of the MWCNT surface; the former being responsible of the good dispersion of MWCNTs in water and the latter having a favorable affinity with CAL-B. The enzyme loadings reached were significant: around 16 wt. % and 21 wt.% for non-covalent and covalent immobilization, respectively. The enzymatic activity was studied with the reaction of O-acylation of geraniol into geranyl acetate by CAL-B in supercritical CO 2 . Even if a decay in synthesis of geranyl acetate was observed over cycling for both CAL-B@MWCNT catalysts, it was demonstrated that the regioselectivity of CAL-B was unchanged through immobilization on the MWCNT surface for both routes. Interestingly, it was shown that a fully green enzymatic process can be achieved with these prepared CAL-B@MWCNT biocatalyst. Such approach could be transferred to other support/enzyme systems for developing new eco-friendly synthesis processes.

Directly covalent immobilization of Candida antarctica lipase B on oxidized aspen powder by introducing poly‑lysines: An economical approach to improve enzyme performance.

In our previous study, we could achieve high soluble expression of Candida antarctica lipase B (CalB) in E. coli by fusion poly‑amino acid tags on CalB (pCalB). Herein, we are surprised to find that pCalB can be easily and directly covalent binding on a simply oxidized aspen powder (OAP) by the aid of poly‑lysine tags. Under the optimal conditions, 72.9 ± 3.6% of the total protein could be immobilized, and the activity recovery of immobilized pCalB (pCalB-OAP) was 98.9 ± 3.8%. The analysis of scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR) indicated that OAP was a suitable carrier for enzyme immobilization. The immobilized pCalB-OAP could exhibit excellent thermal stabilities, and it retained a residual activity of 58.4 ± 2.8% at 55 °C, whereas only 21.2 ± 2.2% of its initial activity for free pCalB was observed. And it could also display a nice tolerance for the changes of pH environment, compared with that of free pCalB. The results that pCalB-OAP could retained 73.6 ± 2.9% of their initial activity in (R, S)-NEMPAME hydrolysis after the tenth cycles, suggested that pCalB-OAP could be effectively recycled. The immobilization strategies established here were simple and inexpensive.

Immobilization of Candida antarctica Lipase B onto organically-modified SBA-15 for efficient production of soybean-based mono and diacylglycerols

In this study, SBA-15 was modified by a series of silane coupling reagents and later used to immobilize Candida antartica lipase B (CALB). The enzymatic properties of the immobilized CALB samples were studied. In addition, the catalytic performance in glycerolysis of soybean oil for diacylglycerols (DAG) production was also investigated. The highest enzymatic activity up to 6100.00 ± 246.41 U/g was observed from the propyl methacrylate group modified SBA-15 supported CALB. No loss of activity was observed from the propyl methacrylate group modified SBA-15 supported CALB, but a higher-than-initial activity was notably found from 3-aminopropyl group and n-octyl group modified SBA-15 supported CALB after a 4-h incubation in air at 70 °C. 1-isocyanatopropane group modified SBA-15 supported CALB exhibited selectivity for DAG production. DAG content up to 61.90 ± 2.38 wt% and a DAG/MAG ratio at 3.11 ± 0.08 was obtained after a 24-h reaction at 60 °C in a solvent-free system.

In situ immobilization of commercial lipase in rigid polyurethane foam and application in the esterification of geraniol and oleic acid

The aim of this work was to evaluate the immobilization of Candida antarcticalipase B (CalB) using polyurethane (PU) foam. The stability of the enzymatic derivative against temperature (40, 60 and 80 °C) together with the recycle capacity and storage stability (4 and 25°C) were evaluated. The application of the enzymatic derivative of CalB in esterification reactions was accomplished. Results indicated that the molar ratio between polyol and isocyanate monomers of 5:3 (v/v) was the best one for the immobilization. The immobilization process resulted in a 1363% increase in activity for the enzymatic derivative. The enzymatic derivative obtained kept its initial activity even after 21 h of exposure to high temperatures. The enzymatic activity of the complex was maintained during 30 days at low temperatures, and showed stability during 4 consecutive reuse cycles. The synthesis of geranyl oleate catalyzed by the immobilized derivative presented conversion of 87%. Taking into account the promising results obtained, the low cost of the immobilization support employed and the whole technique developed, this work comprise an innovative contribution.