Immobilized Enzyme System Research Articles

Functionalized ZnFe2O4@Mesoporous silica nano-support for lipase enzyme immobilization: Enhanced biocatalysis and antibacterial activity for food industry applications

Enzymes, as highly efficient biocatalysts, play a crucial role in diverse biotechnological applications. However, enzyme (re)purification and recovery challenges pose substantial obstacles, rendering them costly for industrial utilization. Addressing this, our study developed mesoporous zinc ferrite nanoparticles coated with amine-functionalized mesoporous silica structure (ZnFe2O4@MS) through the solvothermal method. These nanoparticles are an effective nano-support for immobilizing Candida rugosa Lipase (CRL), ensuring easy separability and recoverability through a magnetic field and providing a significant surface area for high mass transference capacity. Immobilizing the lipase on the nano-support (ZnFe2O4@MS/CRL) through covalent bonds on the surface and within the pores led to a notable increase in enzyme activity from 324 U/mg for free enzyme to 689 U/mg for immobilized enzyme. This indicates unchanged active sites post-immobilization, accompanied by enhanced catalytic activity. The immobilized lipases demonstrated prolonged stability at elevated temperatures, maintaining over 59% of initial catalytic activity through five cycles. Furthermore, ZnFe2O4@MS/CRL exhibited substantial antibacterial activity against Staphylococcus aureus, more than that observed in Gram-negative bacteria. The immobilized lipase was also effective in synthesizing banana flavor (isoamyl acetate) in n-hexane, achieving 64% esterification at 45 °C after 4 h. Overall, the study underscores the industrial promise of this immobilized enzyme system, emphasizing its potential for sustainable and cost-effective biotechnological processes.

Enhancing cellulose hydrolysis via cellulase immobilization on zeolitic imidazolate frameworks using physical adsorption.

This study investigates the immobilization of cellulase on zeolitic imidazolate frameworks (ZIFs) by physical adsorption, specifically the ZIF-8-NH2 and Fe3O4@ZIF-8-NH2, to enhance enzymatic hydrolysis efficiency. The immobilization process was thoroughly analyzed, including optimization of conditions and characterization of ZIF carriers and immobilized enzymes. The impacts on the catalytic activity of cellulase under various temperatures, pH levels, and storage conditions were examined. Additionally, the reusability of the immobilized enzyme was assessed. Results showed the cellulase immobilized on Fe3O4@ZIF-8-NH2 exhibited a high loading capacity of 339.64 mg/g, surpassing previous studies. Its relative enzymatic activity was found to be 71.39%. Additionally, this immobilized enzyme system demonstrates robust reusability, retaining 68.42% of its initial activity even after 10 cycles. These findings underscore the potential of Fe3O4@ZIF-8-NH2 as a highly efficient platform for cellulase immobilization, with promising implications for lignocellulosic biorefinery.

One-pot synthesis and biochemical characterization of the novel magnetic phospholipase D nanoflowers modified with polyphenols

Phospholipase D (PLD) can catalyze the hydrolytic cleavage of phosphodiester bonds and transphosphatidylation and has been widely employed in the chemical and pharmaceutical fields. To improve the stability and reusability of PLD, an immobilized enzyme system was developed using magnetic nanoparticles modified with polyphenols (dopamine and tannic acid) and metal hybrid nanoflowers to co-immobilize phospholipase D (PLD). This immobilization technique aimed to improve the enzymatic performance through increased surface area, reduced mass transfer limitations, and facilitated reuse. The hydrolytic and transphosphatidylation activities of PLD immobilized on polyphenol-modified magnetic nanoflowers (PDA-MNFs and TA-MNFs) were evaluated and compared to free PLD. PDA-MNFs and TA-MNFs showed significantly enhanced hydrolytic activity (19.9-fold and 21.7-fold increases, respectively) and transphosphatidylation activity (over 1.79-fold and 1.96-fold increases, respectively) compared to free PLD. Temperature and pH stability were also markedly improved for the immobilized PLD. After 10 reuse cycles, PDA-MNFs and TA-MNFs retained over 65 % of their initial transphosphatidylation activity. Therefore, this co-deposition and polymerization immobilization strategy using polyphenol-modified magnetic nanoflowers has the potential for efficiently increasing enzymatic performance and synthesizing industrially relevant compounds like phosphatidic acid and phosphatidylserine

Comparative study for enzymatic hydrolysis of sugarcane bagasse using free and nanoparticle immobilized holocellulolytic enzyme cocktail

This study aimed at a comparative analysis of enzymatic hydrolysis of sugarcane bagasse biomass by using a free holocellulase cocktail of an enzyme (Cellic CTec2) and immobilized enzyme system on IOMNP@SiO2-NH2 support to make the process cost-effective. The immobilization was substantiated by examining the nano biocatalysts using FTIR, TEM, and DLS analysis. IOMNP@SiO2 –NH2 and immobilized enzyme system have average particle sizes of 81.1, and 455.9 nm confirmed by DLS. Response surface methodology (RSM) based on the Box Behnken design was employed to optimize the saccharification process conditions. The free cellulase cocktail showed a maximum hydrolysis yield of 70.71 % at 12.5FPU/gm enzyme dose, while the immobilized enzyme at a dose of 20 FPU/gm achieved a conversion rate of 74.19 % under optimized parameters likes hydrolysis time, surfactant and substrate concentration. Pretreated sugarcane bagasse substrate was used to evaluate the reusability potential of magnetically immobilized cellulase enzyme compared to recovered free enzyme up to 5 cycles. In the second and third cycles, the conversion rates of cellulose to glucose were determined to be 68.21 %, and 52.6 %, respectively. The findings of this study demonstrate that the immobilized Cellic CTec2 enzyme cocktail on functionalized IOMNP is effective for hydrolyzing sugarcane bagasse which also provides the advantages of easy recovery and reusability of enzyme for multiple hydrolysis cycles.

A photo-enzyme coupled system for carbon dioxide conversion to solar fuel: The rate-matching and compatibility between photocatalysis and enzyme catalysis

Using reduced nicotinamide adenine dinucleotide (NADH) as cofactor, formate dehydrogenase (FDH) can reversely catalyze CO2 reduction to formic acid. However, the industrialized application of FDH is fairly limited due to its high cost and stoichiometric cofactor consumption. Enzyme immobilization and cofactor regeneration are vital solutions to these problems. Ascribed to the benefits of utilizing a cheap, environmentally friendly, and renewable energy source, photocatalytic NADH regeneration is naturally sustainable and promising. Nonetheless, the rate-matching and compatibility between photocatalysis and enzyme catalysis have not been well revealed. In this study, polyethyleneimine (PEI) modified hollow fiber membrane (HFM) as the enzyme support was in-situ integrated with a visible-light-driven NADH regeneration system using thiophene-modified macroporous graphitic carbon nitride (ATCN-CN) as the catalyst. First, NADH regeneration conditions were regulated to accommodate enzyme catalysis. Then, the photo-enzyme coupled system (PECS) for the sustainable synthesis of formic acid with triethanolamine (TEOA) or H2O as electron donor was constructed, and the influencing factors were investigated. At last, the PECS was compared with other methods as well as a system that used FDH immobilized on silica nanoparticles. The results show that the reaction pH, electron donor, and catalyst concentration have a significant impact on NADH regeneration. For the PECS with TEOA as electron donor, the optimal pH value and photocatalyst concentration for formic acid synthesis are 7.0 and 1.2 mg mL−1, respectively. With the initial addition of 1 mmol L−1 NAD+, a formic acid yield of 35.3% was obtained after 5 h, standing out among the reported values. H2O as a green and low-cost electron donor meets the desire for low-cost photocatalysis. Although the production of formic acid after 5 h was lower in the PECS with H2O as electron donor than in the TEOA system, it was still 14.6 times that of the immobilized enzyme system without cofactor regeneration, and the yield of formic acid could reach 14.8% and 98.3% with 1 or 0.1 mmol L−1 NAD+, validating that current PECS is feasible to a sustainable and green synthesis of solar fuel from CO2.

A kinetic non-steady state analysis of immobilized enzyme systems with external mass transfer resistance

<abstract><p>The goal of this paper is to utilize the homotopy perturbation method (HPM) and Laplace transform to provide an approximate analytical expression to the non-linear time-dependent reaction diffusion equation arising in a mathematical model of an immobilized enzyme system with external mass transfer resistance. This mathematical model is a non-steady, non-linear reaction diffusion equation based on Michaelis–Menten kinetics. Approximate analytical expressions are also provided for various geometries of the enzyme catalytic pellets, namely, planar, cylindrical, and spherical. Obtained semi-analytical expressions are proven to fit for all the parameters appearing in the system and for all the geometries of enzyme catalytic pellets. When comparing the numerical and approximate analytical solutions, satisfactory results are obtained. Also, approximate analytical expressions of the effectiveness factor (EF) of the immobilized system are presented, and the effect of parameters on the EF is also analyzed.</p></abstract>

Cellulase immobilization on zeolitic imidazolate frameworks for boosting cellulose hydrolysis at high solids loading

This study investigates the cellulase immobilization onto zeolitic imidazolate frameworks (ZIFs) to enable efficient cellulose hydrolysis at high solids loading. Different ZIF carriers (ZIF-8, ZIF-8-NH2, Fe3O4@ZIF-8, and Fe3O4@ZIF-8-NH2) were prepared and characterized. The resulting ZIF-immobilized cellulases were subsequently analyzed for their loading capacity and enzyme activity, as well as their biochemical properties such as temperature stability, pH stability, storage stability, enzyme leaching, and enzyme kinetics. Results revealed that the cellulase immobilized on Fe3O4@ZIF-8-NH2 exhibited an exceptional loading capacity of 359.89 mg/g and a relative enzyme activity of 69.39%. Remarkably, at a solid loading of 15% (w/w) and an enzyme dosage of 5 FPU/g cellulose, the Fe3O4@ZIF-8-NH2 immobilized cellulase demonstrated a 20.44% higher hydrolysis yield compared to the free cellulase. Furthermore, this immobilized enzyme system displayed robust reusability, retaining 71.03% of its initial activity even after 10 cycles of reuse. A proposed mechanism of enhanced enzymatic hydrolysis by ZIF-immobilized cellulases was also presented. These findings have significant implications for the future application of ZIF-immobilized cellulases in efficient and cost-effective lignocellulosic bioconversion.

Approximation of intra-particle reaction/diffusion effects of immobilized enzyme system following reverse Michaelis–Menten (rMM) mechanism: third degree polynomial and Akbari–Ganji methods

Two approximate analytical expressions based on third degree polynomial and Akbari–Ganji’s method (AGM) were derived for the reaction/diffusion controlled kinetics of an immobilized enzyme (IE) systems. The approximation methods predict substrate concentration profile and effectiveness factor (eta) in a porous spherical particle. The reaction is assumed to follow reverse Michaelis–Menten (rMM) kinetics. The approximate methods predictions were comparable to that of numerical solution (using the Matlab finite difference function, bvp4c) at wide range of {phi }^{2} and {y}_{o} especially at low {phi }^{2} and high {y}_{o} (polynomial equation) and low {phi }^{2} and low {y}_{o} (AGM equation). Although the approximate solution was derived for rMM kinetics, the results can be used to describe other important enzymatic reaction kinetics such as simple Michaelis–Menten (MM) kinetics and MM with competitive product inhibition kinetics. A necessary design equation should be satisfied when using polynomial or AGM approximation for different enzyme kinetic equations. In this work, two examples of enzymatic reactions of industrial interest were studied, namely glucose-fructose isomerization follows rMM kinetics and hydrolysis of lactose follows Michaelis–Menten (MM) equation with competitive product (galactose) inhibition. Predictions of the developed third degree polynomial and AGM approximation equations agree with that of numerical solution, the percentage relative error for the effectiveness factor was less than 11 in comparison with the numerical solution. Good agreement between approximate and numerical estimations demonstrates the validity of these approximation methods.

Çok Duvarlı Karbon Nanotüpler/Politiyofen Kompozit Kullanılarak Hazırlanan Amperometrik Glikoz Biyosensörü

In this study, multi-walled carbon nanotubes/polythiophene composite (MWCNTs/PTh) modified glassy carbon electrode was used for the amperometric detection of glucose. Glucose oxidase (GOx) was entrapped by a crosslinking agent on the MWCNTs/PTh composite film synthesized by electrochemical polymerization of thiophene onto MWCNTs. Characterization of composite film was achieved by cyclic voltammetry (CV), fourier transform infrared (FTIR) spectroscopy and scanning electron microscopy (SEM) techniques. The amperometric measurements of electrode was performed at +0.70V vs. SCE, which was the electrooxidation potential of enzymatically produced H2O2. The effects of thiophene amount in the composite, pH, temperature and substrate concentration were investigated on the response of enzyme electrode. Optimum pH was 7.0 at room temperature and the response time of enzyme electrode was 25 s. The upper limit of the linear working range was 4.85 mM glucose concentration. The limit of detection of sensor was calculated as 148 µM. The sensitivity of glucose biosensor was determined as 4.39 µA mM-1 cm-2. The value of apparent Michaelis-Menten constant (KMapp) was 1.68 mM according to the Lineweaver-Burk equation. The activation energy of this immobilized enzyme system was 88.92 kJ mol-1.

Dual-target affinity analysis and separation of α-amylase and α-glucosidase inhibitor from Morus alba leaves by magnetic bifunctional immobilized enzyme system.

Morus alba leaves is a natural product with great anti-diabetic potential. However, the therapeutic efficacy of natural product is usually achieved through the interaction of active compounds with specific targets. Among them, active compounds with multi-target therapeutic functions are more effective than single target enzyme. In this study, a bienzyme system was constructed by co-immobilizing α-amylase and α-glucosidase onto Fe3 O4 for affinity screening of dual-target active component in the complex extract from M. alba leaves. As a result, a potential active compound was selectively screened by ligand fishing, separated by HSCCC using a solvent system of ethyl acetate-n-butanol-water (3:2:5, v/v), and identified as rutin. In addition, the result of molecular docking showed that rutin could interact with the active center of α-amylase and α-glucosidase through multiple hydrogen bonds, van der Waals forces, etc. to play an inhibitory role. These results demonstrated the effectiveness of polydopamine magnetically immobilized bienzyme system for dual-target affinity screening of active substance. This study not only revealed the chemical basis of the antidiabetic activity of M. alba leaves from a dual-target perspective, but also promoted the progress of multi-target affinity screening.

Production of Siamenoside I and Mogroside IV from Siraitia grosvenorii Using Immobilized β-Glucosidase

Siraitia grosvenorii is a type of fruit used in traditional Chinese medicine. Previous studies have shown that the conversion of saponins was often carried out by chemical hydrolysis, which can be problematic because of the environmental hazards it may cause and the low yield it produces. Therefore, the purpose of this study is to establish a continuous bioreactor with immobilized enzymes to produce siamenoside I and mogroside IV. The results show that the immobilization process of β-glucosidase exhibited the best relative activity with a glutaraldehyde (GA) concentration of 1.5%, carrier activation time of 1 h and binding enzyme time of 12 h. After the immobilization through GA linkage, the highest relative activity of β-glucosidase was recorded through the reaction with the substrate at 60 °C and pH 5. Subsequently, the glass microspheres with immobilized β-glucosidase were filled into the reactor to maintain the optimal active environment, and the aqueous solution of Siraitia grosvenorii extract was introduced by controlling the flow rate. The highest concentration of siamenoside I and mogroside IV were obtained at a flow rate of 0.3 and 0.2 mL/min, respectively. By developing this immobilized enzyme system, siamenoside I and mogroside IV can be prepared in large quantities for industrial applications.

Deproteinization of Shrimp Shell Waste by Kurthia gibsonii Mb126 immobilized chitinase

This work was aimed at immobilization, characterization, and utilization of chitinase from Kurthia gibsonii Mb126. Immobilization of Kurthia gibsonii Mb126 chitinase on glutaraldehyde treated chitosan was carried out with immobilization yield of 106%. The optimal factors of the immobilization technique such as concentration of glutaraldehyde, chitinase concentration, and immobilization time were evaluated. After optimizing process parameters of immobilization (Glutaraldehyde concentration 4%, chitinase conc. 60mg, immobilization time 30min.), the specific activity of immobilized chitinase improved to 4.3-fold compared to the free form of chitinase. Temperature and pH optima of the immobilized chitinase and free enzyme were same i.e., 7.5 and 40°C respectively. The relative activity of immobilized chitinase remained 90% at 40°C, at 50°C, and at 60°C for 120 min. In the pH range from 5.5 to 8, the immobilized chitinase retained 100% activity. The results confirmed that the pH stability and thermal stability of chitinase increased by immobilizing chitinase on chitosan. The immobilized enzyme system maintained 90% of its efficiency even after 16 successive reaction cycles. The immobilized chitinase maintained 78% of its activity even after 20 months. Fermentation of prawn shell waste with immobilized chitinase indicated a high level of deproteinization. Deproteinization experiments were carried out with 5mL (0.4 mg/mL ) of immobilized and free chitinase on 300 mg/mL of prawn shell waste for 20 days without any additional supplements at 40°C and 6.5 pH. Protein content was reduced from 38.4 to 0.8% with immobilized chitinase. Results suggests the possibility of using immobilized enzymes to remove the prawn shell waste from the environment. To the best of our knowledge there was no such study about the deproteinization of prawn shell waste using immobilized chitinase till the date.

Mathematical Analysis of Reaction-Diffusion Equations Modeling the Michaelis-Menten Kinetics in a Micro-Disk Biosensor.

In this study, we have investigated the mathematical model of an immobilized enzyme system that follows the Michaelis–Menten (MM) kinetics for a micro-disk biosensor. The film reaction model under steady state conditions is transformed into a couple differential equations which are based on dimensionless concentration of hydrogen peroxide with enzyme reaction and substrate within the biosensor. The model is based on a reaction–diffusion equation which contains highly non-linear terms related to MM kinetics of the enzymatic reaction. Further, to calculate the effect of variations in parameters on the dimensionless concentration of substrate and hydrogen peroxide, we have strengthened the computational ability of neural network (NN) architecture by using a backpropagated Levenberg–Marquardt training (LMT) algorithm. NNs–LMT algorithm is a supervised machine learning for which the initial data set is generated by using MATLAB built in function known as “pdex4”. Furthermore, the data set is validated by the processing of the NNs–LMT algorithm to find the approximate solutions for different scenarios and cases of mathematical model of micro-disk biosensors. Absolute errors, curve fitting, error histograms, regression and complexity analysis further validate the accuracy and robustness of the technique.

Purification, characterisation and immobilisation of an acid-stable, raw-starch hydrolysing thiol β-amylase, over produced in the stem of Paederia foetida

A 200 KDa acid-stable thiol β-amylase, appreciably present in the stem of Paederia foetida (48,000 ± 5,500 Units (U)/100 g fresh wt.) was purified by (NH4)2SO4 precipitation, ion exchange chromatography, size exclusion chromatography and HPLC. The enzyme was optimally active in pH 6.0 at 60 °C, showed a specific activity of 3466 U/mg protein and displayed a Km of 3.5 ± 0.1 mg/ml (soluble starch). Activity was stable in the pH range of 3.0–8.0, retaining 94 ± 1% activity at pH 3. The enzyme was stable up to 55–57 °C, beyond which the activity fell sharply. Hg2+ and Ag+ (1 mM concentration) completely inhibited the enzyme activity. Enzyme was strongly inhibited by DTNB, PCMB, N-ethylmaleimide, iodoacetic acid, iodoacetamide, and was experimentally determined to be a thiol amylase (3 SH group/mole); the DTNB inhibition of activity being released by cysteine. The enzyme efficiently hydrolysed potato starch (DE = 51), corn starch (DE= 46), gelatinised cereal flours (wheat, rice and gram), amylopectin, raw starch and raw cereal flours. The enzyme was determined to be a β-amylase (maltose as the only product) by end product analysis and its inability to hydrolyse β-limit dextrin. Immobilisation of the enzyme (crude) on oxidised bagasse (dialdehyde cellulose) increased the temperature optima (by 10 °C) and thermo-stability (retaining 48 ± 2% and 38 ± 1% activity at 70 and 80 °C respectively). The immobilised enzyme system efficiently produced maltose from cereal and tuber starches, remaining 85 ± 1% and 80 ± 1% active after 10th and 20th cycles respectively.

Immobilization of Candida rugosa lipase on magnetic chitosan beads and application in flavor esters synthesis

In this work, magnetic chitosan (MCH) beads were synthesized by phase-inversion method, and grafted with polydopamine (PDA) and then used for direct immobilization of Candida rugosa lipase by Schiff base reaction. The amount of immobilized enzyme and the retained activity were found to be 47.3 mg/g and 72.8%, respectively, at pH 7.0, and at 25 °C. The apparent Km (9.7 mmol/L), and Vmax (384 U/mg) values of the immobilized lipase were significantly changed compared to the free lipase. The MCH@PDA-lipase was better thermal and storage stability at different temperatures than those of the free lipase. In hexane medium, the esterification reaction results showed that the maximum conversions of isoamylalcohol and isopentyl alcohol to isoamyl acetate and isopentyl acetate using the MCH@PDA-lipase were found to be 98.4 ± 1.3% and 73.7 ± 0.7%, respectively. These results showed that the MCH@PDA-lipase can be used as an operative immobilized enzyme system for many biotechnological applications.

Analytical Expression of Effectiveness Factor for Immobilized Enzymes System with Reversible Michaelis Menten Kinetics

The mathematical model of immobilized enzyme system in porous spherical particle is presented. This model is based on a non-stationary diffusion equation containing a nonlinear term related to Michaelis-Menten kinetics of enzymatic reaction. A general and closed form of an analytical expression pertaining to the substrate concentration profile and effectiveness factor are reported for all possible values of Thiele modules φ andα . However, we have employed New Homotopy Perturbation Method (NHPM) to solve the nonlinear reaction/diffusion equation in immobilized enzymes system. Therefore, analytical results were found to be in an appropriate agreement with simulation result.

Towards a Novel Computer-Aided Optimization of Microreactors: Techno-Economic Evaluation of an Immobilized Enzyme System

Immobilized multi-enzyme cascades are increasingly used in microfluidic devices. In particular, their application in continuous flow reactors shows great potential, utilizing the benefits of reusability and control of the reaction conditions. However, capitalizing on this potential is challenging and requires detailed knowledge of the investigated system. Here, we show the application of computational methods for optimization with multi-level reactor design (MLRD) methodology based on the underlying physical and chemical processes. We optimize a stereoselective reduction of a diketone catalyzed by ketoreductase (Gre2) and Nicotinamidadenindinukleotidphosphat (NADPH) cofactor regeneration with glucose dehydrogenase (GDH). Both enzymes are separately immobilized on magnetic beads forming a packed bed within the microreactor. We derive optimal reactor feed concentrations and enzyme ratios for enhanced performance and a basic economic model in order to maximize the techno-economic performance (TEP) for the first reduction of 5-nitrononane-2,8-dione.

Stabilization of β-Galactosidase on Modified Gold Nanoparticles: A Preliminary Biochemical Study to Obtain Lactose-Free Dairy Products for Lactose-Intolerant Individuals.

The unique chemical, optical, and electrical characteristics of nanoparticles make their utilization highly successful in every field of biological sciences as compared to their bulk counterpart. These properties arise as a result of their miniature size, which provides them an excellent surface area-to-volume ratio, inner structure, and shape, and hence increases their surface characteristics. Therefore, this study was undertaken to engineer gold nanoparticles (AuNPs) for improving their catalytic activity and stability in biotechnological processes. The characterization of AuNPs was performed by XRD, UV spectra, and TEM. The synthesized AuNPs were surface-modified by polyvinyl alcohol (PVA) for binding the enzyme in excellent yield. The developed immobilized enzyme system (PVA-AuNPs-β-galactosidase) displayed pH optima at pH 7.0 and temperature optima at 40 °C. Moreover, the stability of PVA-AuNPs-β-galactosidase was significantly enhanced at wider pH and temperature ranges and at higher galactose concentrations, in contrast to the free enzyme. β-galactosidase bound to PVA-modified AuNPs exhibited greater operational activity, even after its sixth reuse. The developed nanosystem may prove useful in producing lactose-free dairy products for lactose-intolerant patients.

A Comparative Analysis of Different Enzyme Immobilization Nanomaterials: Progress, Constraints and Recent Trends.

Immobilization techniques have been popularly used to preserve the operational stability of the enzymes for industrial applications. The three main components of an immobilized enzyme system are the enzyme, the matrix/support, and the technique of immobilization. So far, different supports have been developed to improve the efficiency of the immobilized enzymes. But in the recent decade, nanotechnology has been of considerable research interest in the field of immobilized enzyme carriers. The materials at the nano-scale, due to their unique physicochemical properties, including; specific surface area, mass transfer limitation, and effective enzyme loading, are considered interesting matrices for enzyme immobilization. This review describes techniques employed to immobilize enzymes, and provides an integrated focus on the most common nanoparticles for enzyme conjugation. Additionally, the pros and cons of nanoparticles as immobilization matrices are also discussed. Depending on the type of enzyme and its application, in this review, the researchers are directed to select an appropriate method and support for enzyme immobilization in terms of enzyme stability and functionality.

Evaluation of continuous ethyl ester synthesis from acid soybean oil using a dual immobilized enzyme system

The use of non-edible oils, waste oils, and animal fats have been investigated as a low-cost feedstock for biodiesel (fatty acid alkyl esters) synthesis. However, these alternative raw materials have some drawbacks due to the high content of free fatty acid (FFA). This work aimed to study the continuous enzymatic ethyl esters synthesis, in a solvent-free system, from the reaction of acid soybean oil (acid value, AV, of 0.5, 8.5, 50, and 90) and ethanol using a dual commercial immobilized lipase system (Novozym 435®, Lipozyme TL IM®, Lipozyme RM IM®). Initially, a dual lipase system was used in a single packed-bed reactor (PBR). The blend of Lipozyme TL IM and Novozym 435 had a positive synergistic effect on biodiesel synthesis: an increase of 76% in ethyl ester content was observed after 2 h using refined soybean oil. However, a reduction in the yield was observed in longer reaction times (25 h). Therefore, a continuous reaction system, operated with two different catalytic beds, was investigated. The use of two series reactors allowed high ethyl ester yield (>90%) and high acid conversion (>90%). The reaction using acid soybean oil (50 AV), and Lipozyme RM IM and Novozym 435 reached an ethyl ester content of 95% and 70% after 2 and 25 h, respectively. The results showed that using two different commercial immobilized lipases in intercalated packed-bed reactors enhanced biodiesel synthesis from acid soybean oil. The enzymatic system allows a high fatty acid conversion present in the acidic oil into ethyl ester.