In this study, laccase was immobilized on nylon 6,6/Fe(3+) composite (NFC) nanofibrous membrane and used for the detoxification of 3,3'-dimethoxybenzidine (DMOB). The average size and tensile strength of the NFC membrane were found to be 60-80 nm (diameter) and 2.70 MPa, respectively. The FTIR results confirm that the amine (N-H) group of laccase was attached with Fe(3+) particles and the carbonyl (C=O) group of NFC membrane via hydrogen bonding. The half-life of the laccase-NFC membrane storage stability was increased from 6 to 11 weeks and the reusability was significantly extended up to 43 cycles against ABTS oxidation. Enhanced electro-oxidation of DMOB by laccase was observed at 0.33 V and the catalytic current was found to be 30 µA. The DMOB-treated mouse fibroblast 3T3-L1 preadipocytes showed maximum (97 %) cell inhibition at 75 µM L(-1) within 24 h. The cytotoxicity of DMOB was significantly decreased to 78 % after laccase treatment. This study suggests that laccase-NFC membrane might be a good candidate for emerging pollutant detoxification.
Lipid is the general name given to fats and oils, which are the basic components of cooking oils, shortening, ghee, margarine, and other edible fats. The chosen term depends on the physical state at ambient temperature; fats are solids and oils are liquids. The chemical properties of the lipids, including degree of saturation, fatty acid chain length, and acylglycerol molecule composition are the basic determinants of physical characteristics such as melting point, cloud point, solid fat content, and thermal behavior. This review will discuss the major lipid modification strategies, hydrogenation, and chemical and enzymatic interesterification, describing the catalysts used mechanisms, kinetics, and impacts on the health-related properties of the final products. Enzymatic interesterification will be emphasized as method that produces a final product with good taste, zero trans fatty acids, and a low number of calories, requires less contact with chemicals, and is cost efficient.
α-Amylase from Anoxybacillus sp. SK3-4 (ASKA) is a thermostable enzyme that produces a high level of maltose from starches. A truncated ASKA (TASKA) variant with improved expression and purification efficiency was characterized in an earlier study. In this work, TASKA was purified and immobilized through covalent attachment on three epoxide (ReliZyme EP403/M, Immobead IB-150P, and Immobead IB-150A) and an amino-epoxide (ReliZyme HFA403/M) activated supports. Several parameters affecting immobilization were analyzed, including the pH, temperature, and quantity (mg) of enzyme added per gram of support. The influence of the carrier surface properties, pore sizes, and lengths of spacer arms (functional groups) on biocatalyst performances were studied. Free and immobilized TASKAs were stable at pH 6.0-9.0 and active at pH 8.0. The enzyme showed optimal activity and considerable stability at 60 °C. Immobilized TASKA retained 50% of its initial activity after 5-12 cycles of reuse. Upon degradation of starches and amylose, only immobilized TASKA on ReliZyme HFA403/M has comparable hydrolytic ability with the free enzyme. To the best of our knowledge, this is the first report of an immobilization study of an α-amylase from Anoxybacillus spp. and the first report of α-amylase immobilization using ReliZyme and Immobeads as supports.
This study aimed to work out a simple and high-yield procedure for the immobilization of horseradish peroxidase on silver nanoparticle. Ultraviolet-visible (UV-vis) and Fourier-transform infrared spectroscopy and transmission electron microscopy were used to characterize silver nanoparticles. Horseradish peroxidase was immobilized on β-cyclodextrin-capped silver nanoparticles via glutaraldehyde cross-linking. Single-cell gel electrophoresis (Comet assay) was also performed to confirm the genotoxicity of silver nanoparticles. To decrease toxicity, silver nanoparticles were capped with β-cyclodextrin. A comparative stability study of soluble and immobilized enzyme preparations was investigated against pH, temperature, and chaotropic agent, urea. The results showed that the cross-linked peroxidase was significantly more stable as compared to the soluble counterpart. The immobilized enzyme exhibited stable enzyme activities after repeated uses.
The nanoenvironment of nanobiocatalysts, such as local hydrophobicity, pH and charge density, plays a significant role in optimizing the enzymatic selectivity and specificity. In this study, Kluyveromyces lactis β-galactosidase (Gal) was assembled onto polystyrene nanofibers (PSNFs) to form PSNF-Gal nanobiocatalysts. We proposed that local hydrophobicity on the nanofiber surface could expel water molecules so that the transgalactosylation would be preferable over hydrolysis during the bioconversion of lactose, thus improve the galacto-oligosaccharides (GOS) yield. PSNFs were fabricated by electro-spinning and the operational parameters were optimized to obtain the nanofibers with uniform size and ordered alignment. The resulting nanofibers were functionalized for enzyme immobilization through a chemical oxidation method. The functionalized PSNF improved the enzyme adsorption capacity up to 3100mg/g nanofiber as well as enhanced the enzyme stability with 80% of its original activity. Importantly, the functionalized PSNF-Gal significantly improved the GOS yield and the production rate was up to 110g/l/h in comparison with 37g/l/h by free β-galactosidase. Our research findings demonstrate that the localized nanoenvironment of the PSNF-Gal nanobiocatalysts favour transgalactosylation over hydrolysis in lactose bioconversion.
Functional nanomaterials have been pursued to assemble nanobiocatalysts since they can provide unique hierarchical nanostructures and localized nanoenvironments for enhancing enzyme specificity, stability and selectivity. Functionalized dendrimer-like hierarchically porous silica nanoparticles (HPSNs) was fabricated for assembling β-galactosidase nanobiocatalysts for bioconversion of lactose to galacto-oligosaccharides (GOS). The nanocarrier was functionalized with amino (NH2) and carboxyl (COOH) groups to facilitate enzyme binding, benchmarking with non-functionalized HPSNs. Successful conjugation of the functional groups was confirmed by FTIR, TGA and zeta potential analysis. HPSNs-NH2 showed 1.8-fold and 1.1-fold higher β-galactosidase adsorption than HPSNs-COOH and HPSNs carriers, respectively, with the highest enzyme adsorption capacity of 328mg/g nanocarrier at an initial enzyme concentration of 8mg/ml. The HPSNs-NH2 and β-galactosidase assembly (HPSNs-NH2-Gal) demonstrated to maintain the highest activity at all tested enzyme concentrations and exhibited activity up to 10 continuous cycles. Importantly, HPSNs-NH2-Gal was simply recycled through centrifugation, overcoming the challenging problems of separating the nanocarrier from the reaction medium. HPSNs-NH2-Gal had distinguished catalytic reaction profiles by favoring transgalactosylation, enhancing GOS production of up to 122g/l in comparison with 56g/l by free β-galactosidase. Furthermore, it generated up to 46g/l GOS at a lower initial lactose concentration while the free counterpart had negligible GOS production as hydrolysis was overwhelmingly dominant in the reaction system. Our research findings show the amino-functionalized HPSNs can selectively promote the enzyme activity of β-galactosidase for transgalactosylation, which is beneficial for GOS production.
Organophosphorus (OP) compounds are one of the most hazardous chemicals used as insecticides/pesticide in agricultural practices. A large variety of OP compounds are hydrolyzed by organophosphorus hydrolases (OPH; EC 3.1.8.1). Therefore, OPHs are among the most suitable candidates which could be used in designing enzyme-based sensors for detecting OP compounds. In the present work, a novel nanobiosensor for the detection of paraoxon was designed and fabricated. More specifically, OPH was covalently embedded onto chitosan and the enzyme-chitosan bioconjugate was then immobilized on negatively charged gold nanoparticles (AuNPs) electrostatically. The enzyme was immobilized on AuNPs without chitosan as well to compare the two systems in terms of detection limit and enzyme stability under different pH and temperature conditions. Coumarin 1, a competitive inhibitor of the enzyme, was used as a fluorogenic probe. The emission of coumarin 1 was effectively quenched by the immobilized Au-NPs when bound to the developed nanobioconjugates. However, in the presence of paraoxon, coumarin 1 left the nanobioconjugate leading to enhanced fluorescence intensity. Moreover, compared to the immobilized enzyme without chitosan, the chitosan-immobilized enzyme was found to possess decreased Km value by over 50%, increased Vmax and Kcat values by around 15% and 74%, respectively. Higher stability within a wider range of pH (2-12) and temperature (25-90°C) was also achieved. The method worked in the 0 to 1050 nM concentration ranges, and had a detection limit as low as 5 × 10(-11) M.
Cross-linked enzyme aggregate (CLEA) is easily prepared from crude enzyme and has many advantages to the environment and it is considered as an economic method in the context of industrial biocatalysis compared to free enzyme. In this work, a highly active and stable CLEA-lipase from cocoa pod husk (CPH) which is a by-product after removal of cocoa beans, were assayed for their hydrolytic activity and characterized under the optimum condition successfully. Face centered central composite design (FCCCD) under response surface methodology (RSM) was used to get the optimal conditions of the three significant factors (concentration of ammonium sulfate, concentration of glutaraldehyde and concentration of additive) to achieve higher enzyme activity of CLEA. From 20 runs, the highest activity recorded was around 9.407U (83% recovered activity) under the condition of using 20% saturated ammonium sulfate, 60mM glutaraldehyde as cross-linker and 0.17mM bovine serum albumin as feeder. Moreover, the optimal reaction temperature and pH value in enzymatic reaction for both crude enzyme and immobilized were found to be 45°C at pH 8 and 60°C at pH 8.2, respectively. A systematic study of the stability of CLEA and crude enzyme was taken with regards to temperature (25-60°C) and pH (5-10) value and in both factors, CLEA-lipase showed more stability than free lipase. The Km value of CLEA was higher compared to free enzyme (0.55mM vs. 0.08mM). The CLEA retained more than 60% of the initial activity after six cycles of reuse compared to free enzyme. The high stability and recyclability of CLEA-lipase from CPH make it efficient for different industrial applications.
A novel concept for the synthesis of a stable polymer hybrid matrix bead was developed in this study. The beads were further applied for enzyme immobilization to produce stable and active biocatalysts with low enzyme leakage, and high immobilization efficiency, enzyme activity, and recyclability. The immobilization conditions, including PEI concentration, activation time and pH of the PEI solution were investigated and optimized. All formulated beads were characterized for its functionalized groups, composition, surface morphology and thermal stability. Compared with the free β-glucosidase, the immobilized β-glucosidase on the hybrid matrix bead was able to tolerate broader range of pH values and higher reaction temperature up to 60 °C. The immobilized β-glucosidase was then used to hydrolyse pretreated macroalgae cellulosic residue (MCR) for the production of reducing sugar and a hydrolysis yield of 73.4% was obtained. After repeated twelve runs, immobilized β-glucosidase retained about 75% of its initial activity.
In view of several disadvantages as well as adverse effects associated with the use of chemical processes for producing esters, alternative techniques such as the utilization of enzymes on multi-walled carbon nanotubes (MWCNTs), have been suggested. In this study, the oxidative MWCNTs prepared using a mixture of HNO3 and H2SO4 (1:3 v/v) were used as a supportive material for the immobilization of Candida rugosa lipase (CRL) through physical adsorption process. The resulting CRL-MWCNTs biocatalysts were utilized for synthesizing geranyl propionate, an important ester for flavoring agent as well as in fragrances. Enzymatic esterification of geraniol with propionic acid was carried out using heptane as a solvent and the efficiency of CRL-MWCNTs as a biocatalyst was compared with the free CRL, considering the incubation time, temperature, molar ratio of acid:alcohol, presence of desiccant as well as its reusability. It was found that the CRL-MWCNTs resulted in a 2-fold improvement in the percentage of conversion of geranyl propionate when compared with the free CRL, demonstrating the highest yield of geranyl propionate at 6h at 55°C, molar ratio acid: alcohol of 1:5 and with the presence of 1.0g desiccant. It was evident that the CRL-MWCNTs biocatalyst could be reused for up to 6 times before a 50% reduction in catalytic efficiency was observed. Hence, it appears that the facile physical adsorption of CRL onto F-MWCNTs has improved the activity and stability of CRL as well as served as an alternative method for the synthesis of geranyl propionate.
The current demands of sustainable green methodologies have increased the use of enzymatic technology in industrial processes. Employment of enzyme as biocatalysts offers the benefits of mild reaction conditions, biodegradability and catalytic efficiency. The harsh conditions of industrial processes, however, increase propensity of enzyme destabilization, shortening their industrial lifespan. Consequently, the technology of enzyme immobilization provides an effective means to circumvent these concerns by enhancing enzyme catalytic properties and also simplify downstream processing and improve operational stability. There are several techniques used to immobilize the enzymes onto supports which range from reversible physical adsorption and ionic linkages, to the irreversible stable covalent bonds. Such techniques produce immobilized enzymes of varying stability due to changes in the surface microenvironment and degree of multipoint attachment. Hence, it is mandatory to obtain information about the structure of the enzyme protein following interaction with the support surface as well as interactions of the enzymes with other proteins. Characterization technologies at the nanoscale level to study enzymes immobilized on surfaces are crucial to obtain valuable qualitative and quantitative information, including morphological visualization of the immobilized enzymes. These technologies are pertinent to assess efficacy of an immobilization technique and development of future enzyme immobilization strategies.
Nanobiocatalysis is a new frontier of emerging nanosized material support in enzyme immobilization application. This paper is about a comprehensive review on cellulose nanofibers (CNF), including their structure, surface modification, chemical coupling for enzyme immobilization, and potential applications. The CNF surface consists of mainly -OH functional group that can be directly interacted weakly with enzyme, and its binding can be improved by surface modification and interaction of chemical coupling that forms a strong and stable covalent immobilization of enzyme. The knowledge of covalent interaction for enzyme immobilization is important to provide more efficient interaction between CNF support and enzyme molecule. Enzyme immobilization onto CNF is having potential for improving enzymatic performance and production yield, as well as contributing toward green technology and sustainable sources.
The nanobiocatalyst (NBC) is an emerging innovation that synergistically integrates advanced nanotechnology with biotechnology and promises exciting advantages for improving enzyme activity, stability, capability and engineering performances in bioprocessing applications. NBCs are fabricated by immobilizing enzymes with functional nanomaterials as enzyme carriers or containers. In this paper, we review the recent developments of novel nanocarriers/nanocontainers with advanced hierarchical porous structures for retaining enzymes, such as nanofibres (NFs), mesoporous nanocarriers and nanocages. Strategies for immobilizing enzymes onto nanocarriers made from polymers, silicas, carbons and metals by physical adsorption, covalent binding, cross-linking or specific ligand spacers are discussed. The resulting NBCs are critically evaluated in terms of their bioprocessing performances. Excellent performances are demonstrated through enhanced NBC catalytic activity and stability due to conformational changes upon immobilization and localized nanoenvironments, and NBC reutilization by assembling magnetic nanoparticles into NBCs to defray the high operational costs associated with enzyme production and nanocarrier synthesis. We also highlight several challenges associated with the NBC-driven bioprocess applications, including the maturation of large-scale nanocarrier synthesis, design and development of bioreactors to accommodate NBCs, and long-term operations of NBCs. We suggest these challenges are to be addressed through joint collaboration of chemists, engineers and material scientists. Finally, we have demonstrated the great potential of NBCs in manufacturing bioprocesses in the near future through successful laboratory trials of NBCs in carbohydrate hydrolysis, biofuel production and biotransformation.
Integrating polypyrrole-cellulose nanocrystal-based composites with glucose oxidase (GOx) as a new sensing regime was investigated. Polypyrrole-cellulose nanocrystal (PPy-CNC)-based composite as a novel immobilization membrane with unique physicochemical properties was found to enhance biosensor performance. Field emission scanning electron microscopy (FESEM) images showed that fibers were nanosized and porous, which is appropriate for accommodating enzymes and increasing electron transfer kinetics. The voltammetric results showed that the native structure and biocatalytic activity of GOx immobilized on the PPy-CNC nanocomposite remained and exhibited a high sensitivity (ca. 0.73 μA·mM(-1)), with a high dynamic response ranging from 1.0 to 20 mM glucose. The modified glucose biosensor exhibits a limit of detection (LOD) of (50 ± 10) µM and also excludes interfering species, such as ascorbic acid, uric acid, and cholesterol, which makes this sensor suitable for glucose determination in real samples. This sensor displays an acceptable reproducibility and stability over time. The current response was maintained over 95% of the initial value after 17 days, and the current difference measurement obtained using different electrodes provided a relative standard deviation (RSD) of 4.47%.
This paper demonstrates Pseudomonas cepacia lipase catalyzed hydrolysis of p-nitrophenyl palmitate under irradiation of light with wavelengths of 250-750 nm. The reaction follows Michaelis-Menten Kinetics and the light irradiation increases the overall rate of hydrolysis. Using Lineweaver-Burk plot K M and V max values for the reaction in presence of light are found to be 39.07 and 66.67 mM/min/g, respectively; while for the same reaction under dark condition, the values are 7.08 and 10.21 mM/min/g. The linear form of enzyme dependent rate of reaction confirms that no mass-transfer limitations are present and the reaction is a kinetically controlled enzymatic reaction.
A new silica-gel nanospheres (SiO2NPs) composition was formulated, followed by biochemical surface functionalization to examine its potential in urea biosensor development. The SiO2NPs were basically synthesized based on sol-gel chemistry using a modified Stober method. The SiO2NPs surfaces were modified with amine (-NH2) functional groups for urease immobilization in the presence of glutaric acid (GA) cross-linker. The chromoionophore pH-sensitive dye ETH 5294 was physically adsorbed on the functionalized SiO2NPs as pH transducer. The immobilized urease determined urea concentration reflectometrically based on the colour change of the immobilized chromoionophore as a result of the enzymatic hydrolysis of urea. The pH changes on the biosensor due to the catalytic enzyme reaction of immobilized urease were found to correlate with the urea concentrations over a linear response range of 50-500 mM (R2 = 0.96) with a detection limit of 10 mM urea. The biosensor response time was 9 min with reproducibility of less than 10% relative standard deviation (RSD). This optical urea biosensor did not show interferences by Na+, K+, Mg2+ and NH4+ ions. The biosensor performance has been validated using urine samples in comparison with a non-enzymatic method based on the use of p-dimethylaminobenzaldehyde (DMAB) reagent and demonstrated a good correlation between the two different methods (R2 = 0.996 and regression slope of 1.0307). The SiO2NPs-based reflectometric urea biosensor showed improved dynamic linear response range when compared to other nanoparticle-based optical urea biosensors.
The aim of this work was the synthesis of a novel hydroxyl-fatty acid derivative of kojic acid rich in kojic acid monoricinoleate (KMR) which can be widely used in the cosmetic and food industry. The synthesis of KMR was carried out by lipase-catalysed esterification of ricinoleic and kojic acids in solvent-free system. Three immobilized lipases were tested and the best KMR yields were attained with Lipozyme TL IM and Novozym 435. Since Lipozyme TL IM is the cheapest, it was selected to optimize the reaction conditions. The optimal reaction conditions were 80 °C for the temperature, 1:1 for the alcohol/acid molar ratio, 600 rpm for stirring speed and 7.8% for the catalyst concentration. Under these conditions, the reaction was scaled up in a 5×10⁻³ m³ stirred tank reactor. ¹H-¹³C HMBC-NMR showed that the primary hydroxyl group of kojic acid was regioselectively esterified. The KMR has more lipophilicity than kojic acid and showed antioxidant activity that improves the oxidation stability of biodiesel.
A pilot-scale production of lipase using palm oil mill effluent (POME) as a fermentation basal medium was carried out, and parameters for immobilization of the produced lipase were optimized. Lipase production in a 300-L bioreactor was performed using two proposed strategies, constant power per volume (P/V) and constant tip speed. Moreover, lipase immobilization on different materials was also investigated. Lipase production was performed using liquid-state bioconversion of POME as the medium and Candida cylindracea as the inoculum. The fermentation medium was composed of 1% total suspended solids (TSS) of POME, 0.5% (w/v) peptone, 0.7% (v/v) Tween-80, and 2.2% inoculum. The medium composition was decided on the basis of the medium optimization results of a previous study. The fermentation was carried out for 48 h at 30°C and pH 6. The maximum lipase production was 5.72U/mL and 21.34 U/mL, obtained from the scale-up strategies of constant tip speed and P/V, respectively. Four accessible support materials were screened for their potential use in immobilization. The most suitable support material was found to be activated carbon, with a maximum immobilization of 94%.
In this work, lipase from Candida rugosa was immobilized onto chitosan/graphene oxide beads. This was to provide an enzyme-immobilizing carrier with excellent enzyme immobilization activity for an enzyme group requiring hydrophilicity on the immobilizing carrier. In addition, this work involved a process for the preparation of an enzymatically active product insoluble in a reaction medium consisting of lauric acid and oleyl alcohol as reactants and hexane as a solvent. This product enabled the stability of the enzyme under the working conditions and allowed the enzyme to be readily isolated from the support. In particular, this meant that an enzymatic reaction could be stopped by the simple mechanical separation of the "insoluble" enzyme from the reaction medium. Chitosan was incorporated with graphene oxide because the latter was able to enhance the physical strength of the chitosan beads by its superior mechanical integrity and low thermal conductivity. The X-ray diffraction pattern showed that the graphene oxide was successfully embedded within the structure of the chitosan. Further, the lipase incorporation on the beads was confirmed by a thermo-gravimetric analysis. The lipase immobilization on the beads involved the functionalization with coupling agents, N-hydroxysulfosuccinimide sodium (NHS) and 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC), and it possessed a high enzyme activity of 64 U. The overall esterification conversion of the prepared product was 78% at 60 °C, and it attained conversions of 98% and 88% with commercially available lipozyme and novozyme, respectively, under similar experimental conditions.
Here, we focused on a simple enzymatic epoxidation of alkenes using lipase and phenylacetic acid. The immobilised Candida antarctica lipase B, Novozym 435 was used to catalyse the formation of peroxy acid instantly from hydrogen peroxide (H2O2) and phenylacetic acid. The peroxy phenylacetic acid generated was then utilised directly for in situ oxidation of alkenes. A variety of alkenes were oxidised with this system, resulting in 75-99% yield of the respective epoxides. On the other hand, the phenylacetic acid was recovered from the reaction media and reused for more epoxidation. Interestingly, the waste phenylacetic acid had the ability to be reused for epoxidation of the 1-nonene to 1-nonene oxide, giving an excellent yield of 90%.