Support For Immobilization Of Lipase Research Articles

Application of Spectrogel clay as a support for immobilization of lipase from Burkholderia cepacia

AbstractThe interest in maximizing the production of ethyl esters in a sustainable way and with lower energy costs has increased the use of immobilized enzymes as catalysts. This study aimed to apply the commercial clay Spectrogel® as a support for the immobilization of lipase from Burkholderia cepacia by adsorption and covalent bonding methods. The immobilizations were carried out using a 23 factorial design to study the effects of the activity offered (U g−1), pH, and the molar concentration of the enzyme solution buffer (mol L−1) on the enzyme activity obtained (U g−1). From the statistical analysis of the results, the best conditions for immobilization were pH 7.0 and 0.1 mol L−1 for both tested immobilization methods, with the best offered activity being 5709 and 7600 U g−1. The activities obtained were 1219.81 ± 7.51 and 1274.89 ± 14.99 U g−1 for adsorption and covalent bonding, respectively. The biocatalysts exhibited protein leaching of 33.55 ± 1.08% and 19.44 ± 2.43% when immobilized by adsorption and covalent bonding, respectively. The optimal activity temperature and thermal stability were obtained at 40°C. Additionally, the immobilization of lipase in Spectrogel® by both methods was efficient, showing higher thermal stability than the free enzyme. Thus, this work contributed scientifically to the development of a new and economical biocatalyst for ethyl ester production.

Zr-based MOF as a support for lipase immobilization to enhance enzymatic transesterification for biodiesel production

The development of novel biocatalysts is essential to promote the commercialization of biodiesel production by transesterification reaction. In this paper, Rhizopus oryzae lipase (ROL) was immobilized on an amino-functionalized zirconium-based metal organoskeleton by interfacial adsorption. The immobilization conditions were optimized and the enzymatic properties were tested, and the resulting novel biocatalysts exhibited higher stability and heat resistance. SEM, XRD and BET analyses were used to characterize the biocatalysts and carrier materials. The catalytic performance of ROL@UiO-66-NH2 in the production of biodiesel by transesterification reaction was explored, and the production process was optimized by response surface method. The results showed that the conversion rate of FAEE reached 82.05% at molar ratio of ethanol/oil of 15.43:1, reaction temperature of 50.28°C, reaction time of 120.9 min, DES addition of 48.08 wt%, biocatalyst addition of 3 wt%, and ultrasonic power of 90 W. In addition, ROL@UiO-66-NH2 demonstrated good recyclability, with the catalytic efficiency remaining at 71.87% after five cycles.

Alloyed Trimetallic Nanocomposite as an Efficient and Recyclable Solid Matrix for Ideonella sakaiensis Lipase Immobilization.

In this work, a trimetallic (Ni/Co/Zn) organic framework (tMOF), synthesized by a solvothermal method, was calcinated at 400 and 600 °C and the final products were used as a support for lipase immobilization. The material annealed at 400 °C (Ni-Co-Zn@400) had an improved surface area (66.01 m2/g) and pore volume (0.194 cm3/g), which showed the highest enzyme loading capacity (301 mg/g) with a specific activity of 0.196 U/mg, and could protect the enzyme against thermal denaturation at 65 °C. The optimal pH and temperature for the lipase were 8.0 and 45 °C but could tolerate pH levels 7.0-8.0 and temperatures 40-60 °C. Moreover, the immobilized enzyme (Ni-Co-Zn@Lipase, Ni-Co-Zn@400@Lipase, or Ni-Co-Zn@600@Lipase) could be recovered and reused for over seven cycles maintaining 80, 90, and 11% of its original activity and maintained a residual activity >90% after 40 storage days. The remarkable thermostability and storage stability of the immobilized lipase suggest that the rigid structure of the support acted as a protective shield against denaturation, while the improved pH tolerance toward the alkaline range indicates a shift in the ionization state attributed to unequal partitioning of hydroxyl and hydrogen ions within the microenvironment of the active site, suggesting that acidic residues may have been involved in forming an enzyme-support bond. The high enzyme loading capacity, specific activity, encouraging stability, and high recoverability of the tMOF@Lipase indicate that a multimetallic MOF could be a better platform for efficient enzyme immobilization.

Evaluation of mesoporous silica particles as a support for lipase immobilization in biodiesel production: Enhanced ethyl ester synthesis from algal oil

The utilization of novel materials in the biofuel production domain has been regarded as a significant advancement in catalyst development. Incorporating lipases in biodiesel manufacturing is often viewed as a cost-restrictive phase since the expenses associated with recovering these catalysts can render market expectations unfeasible. Within this investigation, mesoporous silica (MPS) particles were assessed as a supporting medium for immobilizing commercial lipase, subsequent to their utilization in ethyl ester synthesis. Initially, the production of mesoporous silica was achieved through the co-precipitation technique at a consistent pH and was subsequently chosen as an immobilization support for the enzyme. The characterization of the biocatalyst support materials produced was performed using various analytical methods including X-ray diffraction (XRD), scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), and BET analysis. The Novozym 435 lipase was then immobilized within the matrices, followed by the transesterification process of algal oil derived from Nannochloropsis. The resulting biodiesel samples demonstrated compliance with commercial standards, exhibiting an ester conversion rate exceeding 94.8 %. Additionally, properties such as density (0.93 g cm−3) and viscosity (5.01 mm2 s−1) satisfied the specifications mandated by ASTM for biofuel application. Experimental planning techniques showed that increasing lipase loading and using temperatures between 38 and 48 °C resulted in higher conversion rates of ethyl esters. This confirms the effectiveness of the enzymatic catalyst in generating primary fatty acid esters, even with residual lipid feedstocks.

Activated carbon from sugarcane bagasse as support for lipase immobilization by physical adsorption technique

Lipases are recognized as the most important group of catalysts in biotechnology. However, utilization of free enzymes is often hampered by the need for more operational stability, high cost, and non-reusability. Most of these obstacles can be solved by lipase immobilization. This work's objective was to evaluate the performance of the activated carbon obtained from sugarcane straw (SAC) as a support for lipase immobilization. Two lipases were immobilized by physical adsorption on SAC: Aspergillus niger 11T53A14 lipase and CalB (lipase B from Candida antarctica, Novozymes). Results revealed that the lipase had been anchored on the activated carbon with the lipase binding efficiency of 89 % (A. niger lipase) and 100 % (CalB) at the optimum experimental conditions (initial protein concentration 0.1 mg mL-1, 0.15 g of SAC, 25 °C, and 120 min). Langmuir isotherm fitted the adsorption equilibrium data of the lipases on SAC. SAC presents a high surface area and protein adsorption capacity. These results show that activated carbon synthesized from the sugarcane straw is a promising support for enzyme immobilization.

Polyethyleneimine-MOF composite as a support for immobilization of lipase with enhanced activity in kinetic resolution

Lipase PS from Pseudomonas cepacia was steadily immobilized on a new support prepared with metal-organic framework (MOF) and polyethyleneimine (PEI) through a PEI-boosted strategy (PS@MOF-5-PEI). Evaluated by the enantioselective transesterification reaction of racemic 1-(4-(trifluoromethyl) phenyl) ethanol (TFMP) with vinyl acetate, PS@MOF-5-PEI expressed more than 10 times of increase in specific activity (U/mg protein) compared with free lipase PS. PS@MOF-5-PEI had an enhanced enzyme-substrate affinity and catalytic efficiency than free lipase PS, as supported by a lower apparent Km value (7.3 vs 11.3 mM) and an increased Vmax/Km value (0.38 vs 0.05 min−1), respectively. Under the optimal conditions involving 45 °C, substrate ratio of 1:18, enzyme dosage of 20 mg and reaction time of 60 min, the ees of 99.8% and c of 50.1% for kinetic resolution of TFMP enantiomers were achieved with PS@MOF-5-PEI, which can be recycled for 10 times without significant loss in activity and with enantioselectivity well maintained.

Immobilization of Lipases Using Poly(vinyl) Alcohol.

Lipases are very versatile enzymes because they catalyze various hydrolysis and synthesis reactions in a chemo-, regio-, and stereoselective manner. From a practical point of view, immobilization allows the recovery and stabilization of the biocatalyst for its application in different types of bioreactors. Among the various support options for immobilizing lipases is polyvinyl alcohol (PVA), which, when functionalized or combined with other materials, provides different characteristics and properties to the biocatalyst. This review analyzes the multiple possibilities that PVA offers as a material to immobilize lipases when combined with alginate, chitosan, and hydroxypropylmethylcellulose (HPMC), incorporating magnetic properties together with the formation of fibers and microspheres. The articles analyzed in this review were selected using the Scopus database in a range of years from 1999 to 2023, finding a total of 42 articles. The need to expand knowledge in this area is due to the great versatility and scaling possibilities that PVA has as a support for lipase immobilization and its application in different bioreactor configurations.

Tailoring Lignin-Based Spherical Particles as a Support for Lipase Immobilization

Lignin-based spherical particles have recently gained popularity due to their characteristic and the usage of biopolymeric material. In this study, lignin-based spherical particles were prepared using choline chloride at different pH values, ranging from 2 to 10. Their dispersive, microstructural, and physicochemical properties were studied by a variety of techniques, including scanning electron microscopy, Fourier transform infrared spectroscopy, and zeta potential analysis. The best results were obtained for the particles prepared at pH 5 and 7, which had a spherical shape without a tendency to form aggregates and agglomerates. The lignin-based spherical particles were used for the immobilization of lipase, a model enzyme capable of catalyzing a wide range of transformations. It was shown that the highest relative activity of immobilized lipase was obtained after 24 h of immobilization at 30 °C and pH 7, using 100 mg of the support. Moreover, the immobilized lipase exhibited enhanced stability under harsh process conditions, and demonstrated high reusability, up to 87% after 10 cycles, depending on the support used. In the future, the described approach to enzyme immobilization based on lignin spheres may play a significant role in the catalytic synthesis of organic and fine chemicals, with high utility value.

Metal-organic framework based on iron and terephthalic acid as a multiporous support for lipase Burkholderia lata LBBIO-BL02 and its potential for biocatalysis

Metal-organic frameworks (MOFs) are versatile materials because they have a large internal surface area and tuneable pores, making them suitable for enzyme immobilization. In this study, we prepared a typical microporous Fe-BDC MOF through a thermal treatment to produce additional meso and macropores interconnected to each other, capable of immobilizing the Burkholderia lata LBBIO-BL02 (BLL) lipase by entrapment and physical adsorption. The immobilization efficiency (E) was 90%, and the activity retention (R) was 400% (pNPP hydrolysis). The immobilized lipase (BLL@BDC) also showed excellent activity in the hydrolysis of vegetable oils in aqueous medium, achieving up to 3,200 U g−1 for olive oil, as well as high stability in organic solvents, especially for polar ones, such as iso-propanol (101.5 ± 2.6%), ethanol (103.0 ± 6.0%) and acetone (107.7 ± 8.3%). The results indicate that the multiporous Fe-BDC MOF is a promising support for lipase immobilization and further application in biocatalysis performed in organic media.

Immobilization of Lipases on Modified Silica Clay for Bio-Diesel Production: The Effect of Surface Hydrophobicity on Performance

The hydrophobicity of a support plays a critical role in the catalytic efficiency of immobilized lipases. 3-aminopropyltriethoxysilane (APTES)-modified silica clay (A-SC) was coupled with silane coupling agents of different alkyl chains (methyl triethoxysilane, vinyl triethoxysilane, octyl triethoxysilane, and dodecyl triethoxysilane) to prepare a series of hydrophobic support for lipase immobilization. The lipases were immobilized onto the support by conducting glutaraldehyde cross-linking processes. The results showed that the activity of the immobilized biocatalyst increased with hydrophobicity. The hydrolytic activity of Lip-Glu-C12-SC (contact angle 119.8°) can reach 5900 U/g, which was about three times that of Lip-Glu-A-SC (contact angle 46.5°). The immobilized lipase was applied as a biocatalyst for biodiesel production. The results showed that the catalytic yield of biodiesel with highly hydrophobic Lip-Glu-C12-SC could be as high as 96%, which is about 30% higher than that of Lip-Glu-A-SC. After being recycled five times, the immobilized lipase still maintained good catalytic activity and stability. This study provides a good strategy to improve the efficiency of immobilized lipases, showing great potential for future industrial application on biodiesel production.

Tailoring the hydrophobicity of wrinkled silica nanoparticles and of the adsorption medium as a strategy for immobilizing lipase: An efficient catalyst for biofuel production

Hydrophobic wrinkled silica nanoparticles (WSNs) were obtained by surface functionalization with perfluorodecyltriethoxysilane (PDTES) by chemical vapour deposition (CVD). Surface functionalization was made to design a hydrophobic surface to immobilize lipase in its open active conformation by interfacial activation. Moreover, to modulate the closed/open form equilibrium, favouring the open conformation, n-hexane was added to the water/lipase solution, creating a micro-oily environment. Physicochemical characterization of supports was carried out by solid state 29Si nuclear Magnetic Resonance (NMR), the Brunauer–Emmett–Teller (BET) method, thermogravimetric (TG) analysis, contact angle (CA) measurement, scanning electron microscopy (SEM) and Fourier transform infrared (FT-IR) spectroscopy. Three different supports for physical immobilization of lipase were prepared, differing in the degree of hydrophobicity. The effect of the different hydrophobicity and of the addition of n-hexane on the adsorption of lipase was evaluated. The hyperactivation of the best biocatalyst was tested in the hydrolysis and transesterification of sunflower seed oil and compared to free lipase. The reaction yields were 87% and 75% respectively for hydrolysis, and 93% and 56% respectively for transesterification. The results suggest that both the hydrophobicity of the support and the addition of n-hexane favour the adsorption of lipase in the active conformation.

Magnetic COFs as satisfactory support for lipase immobilization and recovery to effectively achieve the production of biodiesel by maintenance of enzyme activity

BackgroundProduction of biodiesel from renewable sources such as inedible vegetable oils by enzymatic catalysis has been a hotspot but remains a challenge on the efficient use of an enzyme. COFs (Covalent Organic Frameworks) with large surface area and porosity can be applied as ideal support to avoid aggregation of lipase and methanol. However, the naturally low density limits its application. In this work, we reported a facile synthesis of core–shell magnetic COF composite (Fe3O4@COF-OMe) to immobilize RML (Rhizomucor miehei lipase), to achieve its utilization in biodiesel production.ResultThis strategy gives extrinsic magnetic property, and the magnetic COFs is much heavier and could disperse in water medium well, facilitating the attachment with the enzyme. The resultant biocomposite exhibited an excellent capacity of RML due to its high surface area and fast response to the external magnetic field, as well as good chemical stability. The core–shell magnetic COF-OMe structure not only achieved highly efficient immobilization and recovery processes but also maintained the activity of lipase to a great extent. RML@Fe3O4@COF-OMe performed well in practical applications, while free lipase did not. The biocomposite successfully achieved the production of biodiesel from Jatropha curcas Oil with a yield of about 70% in the optimized conditions.ConclusionMagnetic COFs (Fe3O4@COF-OMe) for RML immobilization greatly improved catalytic performance in template reaction and biodiesel preparation. The magneticity makes it easily recovered and separated from the system. This first successful attempt of COFs-based immobilized enzyme broadened the prospect of biodiesel production by COFs with some inspiration.

Enhancing bio-catalytic performance of lipase immobilized on ionic liquids modified magnetic polydopamine

In this study, imidazolium-based ionic liquid with [tf2N]− as the anion was successfully grafted to magnetic polydopamine nanoparticles (MPDA). The prepared materials were well characterized and used as supports for lipase immobilization. The immobilized lipase (PPL-ILs-MPDA) exhibited excellent activity and stability. The specific activity of PPL-ILs-MPDA was 2.15 and 1.49 folds higher than that of free PPL and PPL-MPDA. In addition, after 10 rounds of reuse, the residual activity of PPL-ILs-MPDA was 86.2 % higher than that of PPL-MPDA (75.4 %). Furthermore, the kinetic assay indicated that the affinity between PPL-ILs-MPDA and substrate had increased. Analysis of the secondary structure using circular dichroism was used to explain the mechanism underlying the improvement in the performance of PPL-ILs-MPDA. In addition, the immobilized lipase can be easily separated from the reaction system with a magnet. The observations regarding the development of new supports for lipase immobilization may provide new ideas regarding further studies in this field.

Pristine and Poly(Dimethylsiloxane) Modified Multi-Walled Carbon Nanotubes as Supports for Lipase Immobilization.

The presented study deals with the fabrication of highly stable and active nanobiocatalysts based on Candida antarctica lipase B (CALB) immobilization onto pristine and poly(dimethylsiloxane) modified MWCNTs. The MWCNTs/PDMS nanocomposites, containing 40 wt.% of the polymer with two molecular weights, were successfully synthesized via adsorption modification. The effect of PDMS chains length on the textural/structural properties of produced materials was studied by means of the nitrogen adsorption–desorption technique, Raman spectroscopy, and attenuated total reflectance Fourier transform infrared spectroscopy. P-MWCNTs and MWCNTs/PDMS nanocomposites were tested as supports for lipase immobilization. Successful deposition of the enzyme onto the surface of P-MWCNTs and MWCNTs/PDMS nanocomposite materials was confirmed mainly using ATR-FTIR spectroscopy. The immobilization efficiency, stability, and catalytic activity of the immobilized enzyme were studied, and the reusability of the produced biocatalytic systems was examined. The presented results demonstrate that the produced novel biocatalysts might be considered as promising materials for biocatalytic applications.

Agroindustrial Wastes as a Support for the Immobilization of Lipase from Thermomyces lanuginosus: Synthesis of Hexyl Laurate.

As a consequence of intense industrialization in the last few decades, the amount of agro-industrial wastes has increasing, where new forms of valorization are crucial. In this work, five residual biomasses from Maranhão (Brazil) were investigated as supports for immobilization of lipase from Thermomyces lanuginosus (TLL). The new biocatalysts BM-TLL (babaçu mesocarp) and RH-TLL (rice husk) showed immobilization efficiencies >98% and hydrolytic activities of 5.331 U g−1 and 4.608 U g−1, respectively, against 142 U g−1 by Lipozyme® TL IM. High esterification activities were also found, with 141.4 U g−1 and 396.4 U g−1 from BM-TLL and RH-TLL, respectively, against 113.5 U g−1 by TL IM. Results of porosimetry, SEM, and BET demonstrated BM and RH supports are mesoporous materials with large hydrophobic area, allowing a mixture of hydrophobic adsorption and confinement, resulting in hyperactivation of TLL. These biocatalysts were applied in the production of hexyl laurate, where RH-TLL was able to generate 94% conversion in 4 h. Desorption with Triton X-100 and NaCl confirmed that new biocatalysts were more efficient with 5 times less protein than commercial TL IM. All results demonstrated that residual biomass was able to produce robust and stable biocatalysts containing immobilized TLL with better results than commercial preparations.

Synthesis of Mesoporous Silica Nanowires and Their Application in Enzyme Immobilization

Hydrophobic mesoporous silica nanowires were synthesis and then employed as support for immobilization of lipase from Candida antarctica via covalent bonding (CALB@MSW). The parameters were optimized and the optimum conditions were as follows: GA concentration 5.5 wt.%, activation time 60 min and CALB concentration 4 mg/mL. Under these conditions, the protein loading and specific activity of CALB@MSW were 138.3 mg/gsupport and 41.1 U/mgsupport, respectively. Compared with free CALB, CALB@MSW showed better thermal stability and pH stability. The maximum yield of biodiesel catalytic by CALB@MSW was 93.4 %. After reused 8 times, CALB@MSW still remained 95.75 % initial activity showing better stability than free CALB.

BIOCATALYTIC PROCESSES OF LOW-TEMPERATURE SYNTHESIS OF ESTERS. MODULATION OF THE FUNCTIONAL PROPERTIES OF LIPASE BY THE CHOICE OF SOLVENT AND SUPPORT FOR IMMOBILIZATION

Biocatalytic processes for the synthesis of valuable products, such as different esters for various purposes, have been studied using heterogeneous biocatalysts prepared by immobilizing the recombinant lipase rPichia/lip on mesoporous silica (SiO2) and macroporous carbon aerogel (MCA). It was found that the functional properties of immobilized lipase, such as enzymatic activity, specificity toward pair of substrates (acid and alcohol), the molecules of which differ in the number of carbon atoms (C), as well as operational stability, depended on the method of adsorptive immobilization, the chemical nature of the support, and polarity of the organic solvent, i.e. logP. The functional properties of rPichia/lip have been shown to be modulated by the selection of an organic solvent and support for lipase immobilization.

Development of activated carbon from pupunha palm heart sheaths: Effect of synthesis conditions and its application in lipase immobilization

Activated carbon obtained from pupunha palm heart sheaths was used as a support for lipase immobilization. The effect of temperature, carbonization time, and impregnation ratio on the specific surface area (SBET) and the average pore diameter (Dp) of the carbon was evaluated. In addition, a detailed analysis of the lipase immobilization on activated carbon by the adsorption method was performed. The impregnation ratio and the carbonization temperature had a significant effect on all the response variables. The carbon from the best synthesis conditions presented a high SBET (1598 m2 g−1) and Dp (5.39 nm), which favored the lipase immobilization. The support produced showed a high immobilization capacity (576 mg g−1) of lipase with maximum hydrolytic activity at pH 4 (4.727 U/mg). The adjustments of the kinetic models suggest that the enzyme immobilization on carbon occurs predominantly by chemosorption. Based on the thermodynamic parameters, lipase adsorption on the matrix is a spontaneous and endothermic process.

Designing a Support for Lipase Immobilization Based On Magnetic, Hydrophobic, and Mesoporous Silica.

A mesoporous, magnetic, and hydrophobic material was designed step by step to act as a support for lipase immobilization. Its pore size (8.0 nm) is compatible with the size of lipase from Thermomyces lanuginosus (TLL), and its hydrophobic surface (contact angle of a water drop = 125°) was planned to interact with lipase on its interfacially activated form (open conformation). The presence of magnetite particles provides magnetic retrieval of the material and enables recyclability of the biocatalysts. Regarding immobilization parameters, the hydrophobic support was tested in comparison to the unmodified hydrophilic support in phosphate buffer solution (50 mmol L-1, pH 7.5) at 25 °C. Hydrophobicity was found to be critical for the amount of immobilized TLL (immobilization yield of 97% versus 36% for the hydrophilic support), whereas the hydrophilic support favors the native conformational state and substrate access to the enzyme's catalytic site (specific activity of 5.7 versus 4.7 U g-1 for the hydrophobic support, even when it has higher TLL content). Therefore, the hydrophobic support immobilizes higher amounts of TLL and the hydrophilic support keeps the enzyme hyperactivated. Last, due to the stronger interactions of TLL with hydrophobic surfaces, the hydrophobic support offers better preservation of enzyme activity in repeated cycles (76% of activity retained after three cycles versus 50% for the hydrophilic support).

Bacterial cellulose production from biodiesel–derived crude glycerol, magnetic functionalization, and its application as carrier for lipase immobilization

The present study aims towards the kinetic analysis of bacterial cellulose (BC) production by Gluconobacter xylinus from biodiesel–derived crude glycerol and its application as support for immobilization of lipase. Enhancement in strength of BC membrane and its magnetic functionalization were accomplished by the impregnation of iron oxide nanoparticles into the BC matrix. Fitting of experimental results to various substrate inhibition models revealed a reduction of substrate affinity (KS) and reaction rate (Vmax), and increase in substrate inhibition concentration of G. xylinus cells in presence of crude glycerol, in comparison to the pure form of glycerol. Improvement in mechanical properties of pristine BC and magnetic strength of functionalized BC membrane were confirmed by stress–strain curve and vibrating sample magnetometry analysis, respectively. This magnetic BC membrane provided suitable support for the immobilization of Candida rugosa lipase. The immobilized enzymes exhibited better activity at various temperatures, broader pH–flexibility, thermostability (retention of 48% of its activity after 180 min at 50 °C), and reusability (59% of its activity sustained after five consecutive runs). In comparison to free lipase, the immobilized lipase exhibited improved stability and activity, which could be applicable for industrial scale.