New acrylic microspheres were synthesised by photopolymerisation where the succinimide functional group was incorporated during the microsphere preparation. An optical biosensor for urea based on reflectance transduction with a large linear response range to urea was successfully developed using this material. The biosensor utilized succinimide-modified acrylic microspheres immobilized with a Nile blue chromoionophore (ETH 5294) for optical detection and urease enzyme was immobilized on the surface of the microspheres via the succinimide groups. No leaching of the enzyme or chromoionophore was observed. Hydrolysis of the urea by urease changes the pH and leads to a color change of the immobilized chromoionophore. When the color change was monitored by reflectance spectrophotometry, the linear response range of the biosensor to urea was from 0.01 to 1,000 mM (R2 = 0.97) with a limit of detection of 9.97 μM. The biosensor response showed good reproducibility (relative standard deviation = 1.43%, n = 5) with no interference by major cations such as Na+, K+, NH4+ and Mg2+. The use of reflectance as a transduction method led to a large linear response range that is better than that of many urea biosensors based on other optical transduction methods.
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.
Enzymatic synthesis of maltooligosaccharides is hampered due to lack of stability of soluble enzyme. This limitation can be tackled by cross linked enzyme aggregates (CLEAs) immobilization approach. However, substrate diffusion is a major bottleneck in cross linking technology. Herein, CLEAs of maltogenic amylase from Bacillus lehensis G1 (Mag1) was developed with addition of porous agent (Mag1-p-CLEAs). Comparison of thermal, pH and kinetic analysis with CLEAs without porous agent (Mag1-CLEAs) and free Mag1 was performed. Mag1-p-CLEAs with porous structure prepared at 0.8% (w/v) of citrus pectin (porous agent), 0.25% (w/v) of chitosan (cross linker) and cross linked for 1.5 h yielded 91.20% activity. 80% of activity is retained after 30 min of incubation at 40 °C and showed longer half-life than free Mag1 and Mag1-CLEAs. Mag1-p-CLEAs also showed pH stability at acidic and alkaline pH. The 1.68-fold increase in Vmax value in comparison to Mag1-CLEAs showed that the presence of pores of Mag1-p-CLEAs enhanced the beta-cyclodextrin accessibility. The increase in high catalytic efficiency (Kcat/Km) value, 1.90-fold and 1.05-fold showed that it also has better catalytic efficiency than free Mag1 and Mag1-CLEAs, respectively. Mag1-p-CLEAs not only improved substrate diffusibility of CLEAs, but also leads to higher thermal and pH stability of Mag1.
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.
A simple visual ethanol biosensor based on alcohol oxidase (AOX) immobilised onto polyaniline (PANI) film for halal verification of fermented beverage samples is described. This biosensor responds to ethanol via a colour change from green to blue, due to the enzymatic reaction of ethanol that produces acetaldehyde and hydrogen peroxide, when the latter oxidizes the PANI film. The procedure to obtain this biosensor consists of the immobilization of AOX onto PANI film by adsorption. For the immobilisation, an AOX solution is deposited on the PANI film and left at room temperature until dried (30 min). The biosensor was constructed as a dip stick for visual and simple use. The colour changes of the films have been scanned and analysed using image analysis software (i.e., ImageJ) to study the characteristics of the biosensor's response toward ethanol. The biosensor has a linear response in an ethanol concentration range of 0.01%-0.8%, with a correlation coefficient (r) of 0.996. The limit detection of the biosensor was 0.001%, with reproducibility (RSD) of 1.6% and a life time up to seven weeks when stored at 4 °C. The biosensor provides accurate results for ethanol determination in fermented drinks and was in good agreement with the standard method (gas chromatography) results. Thus, the biosensor could be used as a simple visual method for ethanol determination in fermented beverage samples that can be useful for Muslim community for halal verification.
An optical biosensor based on glutamate dehydrogenase (GLDH) immobilized in a chitosan film for the determination of ammonium in water samples is described. The biosensor film was deposited on a glass slide via a spin-coating method. The ammonium was measured based on beta-nicotinamide adenine dinucleotide (NADH) oxidation in the presence of alpha-ketoglutaric acid at a wavelength of 340 nm. The biosensor showed optimum activity at pH 8. The optimum chitosan concentrations and enzyme loading were found to be at 2% (w/v) and 0.08 mg, respectively. Optimum concentrations of NADH and alpha-ketoglutaric acid both were obtained at 0.15 mM. A linear response of the biosensor was obtained in the ammonium concentration range of 0.005 to 0.5 mM with a detection limit of 0.005 mM. The reproducibility of the biosensor was good, with an observed relative standard deviation of 5.9% (n=8). The biosensor was found to be stable for at least 1 month when stored dry at 4 degrees C.
Cellulose and its forms are widely used in biomedical applications due to their biocompatibility, biodegradability and lack of cytotoxicity. It provides ample opportunities for the functionalization of supported magnetic nanohybrids (CSMNs). Because of the abundance of surface hydroxyl groups, they are surface tunable in either homogeneous or heterogeneous solvents and thus act as a substrate or template for the CSMNs' development. The present review emphasizes on the synthesis of various CSMNs, their physicomagnetic properties, and potential applications such as stimuli-responsive drug delivery systems, MRI, enzyme encapsulation, nucleic acid extraction, wound healing and tissue engineering. The impact of CSMNs on cytotoxicity, magnetic hyperthermia, and folate-conjugates is highlighted in particular, based on their structures, cell viability, and stability. Finally, the review also discussed the challenges and prospects of CSMNs' development. This review is expected to provide CSMNs' development roadmap in the context of 21st-century demands for biomedical therapeutics.
The concept of de novo metabolic engineering through novel synthetic pathways offers new directions for multi-step enzymatic synthesis of complex molecules. This has been complemented by recent progress in performing enzymatic reactions using immobilized enzyme microreactors (IEMR). This work is concerned with the construction of de novo designed enzyme pathways in a microreactor synthesizing chiral molecules. An interesting compound, commonly used as the building block in several pharmaceutical syntheses, is a single diastereoisomer of 2-amino-1,3,4-butanetriol (ABT). This chiral amino alcohol can be synthesized from simple achiral substrates using two enzymes, transketolase (TK) and transaminase (TAm). Here we describe the development of an IEMR using His6-tagged TK and TAm immobilized onto Ni-NTA agarose beads and packed into tubes to enable multi-step enzyme reactions. The kinetic parameters of both enzymes were first determined using single IEMRs evaluated by a kinetic model developed for packed bed reactors. The Km(app) for both enzymes appeared to be flow rate dependent, while the turnover number kcat was reduced 3 fold compared to solution-phase TK and TAm reactions. For the multi-step enzyme reaction, single IEMRs were cascaded in series, whereby the first enzyme, TK, catalyzed a model reaction of lithium-hydroxypyruvate (HPA) and glycolaldehyde (GA) to L-erythrulose (ERY), and the second unit of the IEMR with immobilized TAm converted ERY into ABT using (S)-α-methylbenzylamine (MBA) as amine donor. With initial 60mM (HPA and GA each) and 6mM (MBA) substrate concentration mixture, the coupled reaction reached approximately 83% conversion in 20 min at the lowest flow rate. The ability to synthesize a chiral pharmaceutical intermediate, ABT in relatively short time proves this IEMR system as a powerful tool for construction and evaluation of de novo pathways as well as for determination of enzyme kinetics.
The contribution of chitosan/nanocellulose (CS-NC) to the enzymatic activity of Candida rugosa lipase covalently bound on the surface of CS-NC (CRL/CS-NC) was investigated. Cellulosic material from oil palm frond leaves (OPFL) were bleached, alkaline treated and acid hydrolyzed to obtain the purified NC and used as nano-fillers in CS. XRD, Raman spectroscopy and optical fluorescence microscopic analyses revealed existence of strong hydrogen bonds between CS and the NC nanofillers. The CRLs were successfully conjugated to the surface of the CS-NC supports via imine bonds that occurred through a Schiff's based mechanism. Process parameters for the immobilization of CRL were assessed for factors temperature, concentration of glutaraldehyde and pH, to afford the highest enzyme activity to achieve maximum conversion of butyl butyrate within 3h of incubation. Conversion as high as 88% was reached under an optimized condition of 25°C, 0.3% glutaraldehyde concentration and buffer at pH7. Thermal stability of CRL/CS-NCs was 1.5-fold greater than that of free CRL, with biocatalysts reusability for up to 8 successive esterification cycles. This research provides a promising approach for expanding the use of NC from OPFL for enhancing enzyme activity in favour of an alternative eco-friendly means to synthesize butyl butyrate.
Immobilized Candida antarctica lipase B-catalyzed esterification of xylitol and two fatty acids (capric and caproic acid) were studied in a solvent-free system. The Taguchi orthogonal array method based on three-level-four-variables with nine experiments was applied for the analysis and optimization of the reaction parameters including time, substrate molar ratio, amount of enzyme, and amount of molecular sieve. The obtained conversion was higher in the esterification of xylitol and capric acid with longer chain length. The optimum conditions derived via the Taguchi approach for the synthesis of xylitol caprate and xylitol caproate were reaction time, 29 and 18h; substrate molar ratio, 0.3 and 1.0; enzyme amount, 0.20 and 0.05g, and molecular sieve amount of 0.03g, respectively. The good correlation between the predicted conversions (74.18% and 61.23%) and the actual values (74.05% and 60.5%) shows that the model derived from the Taguchi orthogonal array can be used for optimization and better understanding of the effect of reaction parameters on the enzymatic synthesis of xylitol esters in a solvent-free system.
The conversion of aldehydes to valuable alkanes via cyanobacterial aldehyde deformylating oxygenase is of great interest. The availability of fossil reserves that keep on decreasing due to human exploitation is worrying, and even more troubling is the combustion emission from the fuel, which contributes to the environmental crisis and health issues. Hence, it is crucial to use a renewable and eco-friendly alternative that yields compound with the closest features as conventional petroleum-based fuel, and that can be used in biofuels production. Cyanobacterial aldehyde deformylating oxygenase (ADO) is a metal-dependent enzyme with an α-helical structure that contains di‑iron at the active site. The substrate enters the active site of every ADO through a hydrophobic channel. This enzyme exhibits catalytic activity toward converting Cn aldehyde to Cn-1 alkane and formate as a co-product. These cyanobacterial enzymes are small and easy to manipulate. Currently, ADOs are broadly studied and engineered for improving their enzymatic activity and substrate specificity for better alkane production. This review provides a summary of recent progress in the study of the structure and function of ADO, structural-based engineering of the enzyme, and highlight its potential in producing biofuels.
Recent environmental problems and societal concerns associated with the disposal of petroleum based plastics throughout the world have triggered renewed efforts to develop new biodegradable products compatible with our environment. This article describes the preparation, characterization and biodegradation study of poly(lactic acid)/layered double hydroxide (PLA/LDH) nanocomposites from PLA and stearate-Zn(3)Al LDH. A solution casting method was used to prepare PLA/stearate-Zn(3)Al LDH nanocomposites. The anionic clay Zn(3)Al LDH was firstly prepared by co-precipitation method from a nitrate salt solution at pH 7.0 and then modified by stearate anions through an ion exchange reaction. This modification increased the basal spacing of the synthetic clay from 8.83 Å to 40.10 Å. The morphology and properties of the prepared PLA/stearate-Zn(3)Al LDH nanocomposites were studied by X-ray diffraction (XRD), transmission electron microscope (TEM), scanning electron microscope (SEM), thermogravimetric analysis (TGA), tensile tests as well as biodegradation studies. From the XRD analysis and TEM observation, the stearate-Zn(3)Al LDH lost its ordered stacking-structure and was greatly exfoliated in the PLA matrix. Tensile test results of PLA/stearate-Zn(3)Al LDH nanocomposites showed that the presence of around 1.0-3.0 wt % of the stearate-Zn(3)Al LDH in the PLA drastically improved its elongation at break. The biodegradation studies demonstrated a significant biodegradation rate improvement of PLA in the presence of stearate-Zn(3)Al LDH nanolayers. This effect can be caused by the catalytic role of the stearate groups in the biodegradation mechanism leading to much faster disintegration of nanocomposites than pure PLA.
D-serine has been implicated as a brain messenger, promoting not only neuronal signalling but also synaptic plasticity. Thus, a sensitive tool for D-serine monitoring in brain is required to understand the mechanisms of D-serine release from glia cells. A biosensor for direct fixed potential amperometric monitoring of D-serine incorporating mammalian D-amino acid oxidase (DAAO) immobilized on a Nafion coated poly-ortho-phenylenediamine (PPD) modified Pt-Ir disk electrode was therefore developed. The combined layers of PPD and Nafion enhanced the enzyme activity and biosensor efficiency by approximately 2-fold compared with each individual layer. A steady state response time (t(90%)) of 0.7+/-0.1s (n=8) and limit of detection 20+/-1 nM (n=8) were obtained. Cylindrical geometry showed lower sensitivity compared to disk geometry (61+/-7 microA cm(-2) mM(-1), (n=4), R(2)=0.999). Interference by ascorbic acid (AA), the main interference species in the central nervous system and other neurochemical electroactive molecules was negligible. Implantation of the electrode and microinjection of D-serine into rat brain striatal extracellular fluid demonstrated that the electrode was capable of detecting D-serine in brain tissue in vivo.
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.
Type I pullulanase from Anoxybacillus sp. SK3-4 (PulASK) is an unusual debranching enzyme that specifically hydrolyzes starch α-1,6 linkages at long branches producing oligosaccharides (≥G8), but is nonreactive against short branches; thus, incapable of producing reducing sugars (G1-G7). We report on the effects of both single and co-immobilization of PulASK on product specificity. PulASK was purified and immobilized through covalent attachment to three epoxides (ReliZyme EP403/M, Immobead IB-150P, and Immobead IB-150A) and an amino-epoxide (ReliZyme HFA403/M) activated supports. Following immobilization, all PulASK derivatives were active on both short and long branches in starch producing reducing sugars (predominantly maltotriose) and oligosaccharides (≥G8), respectively, a feature that is absent in the free enzyme. This study also demonstrated that co-immobilization of PulASK and α-amylase from Anoxybacillus sp. SK3-4 (TASKA) on ReliZyme HFA403/M significantly changed the product specificity compared to the free enzymes alone or individually immobilized enzymes. In conclusion, individual or co-immobilization caused changes in the product specificity, presumably due to changes in the enzyme binding pocket caused by the influence of carrier surface properties (hydrophobic or hydrophilic) and the lengths of the spacer arms.
Azelaic acid (AzA) and its derivatives have been known to be effective in the treatment of acne and various cutaneous hyperpigmentary disorders. The esterification of azelaic acid with lauryl alcohol (LA) to produce dilaurylazelate using immobilized lipase B from Candida antarctica (Novozym 435) is reported. Response surface methodology was selected to optimize the reaction conditions. A well-fitting quadratic polynomial regression model for the acid conversion was established with regards to several parameters, including reaction time and temperature, enzyme amount, and substrate molar ratios. The regression equation obtained by the central composite design of RSM predicted that the optimal reaction conditions included a reaction time of 360 min, 0.14 g of enzyme, a reaction temperature of 46 °C, and a molar ratio of substrates of 1:4.1. The results from the model were in good agreement with the experimental data and were within the experimental range (R² of 0.9732).The inhibition zone can be seen at dilaurylazelate ester with diameter 9.0±0.1 mm activities against Staphylococcus epidermidis S273. The normal fibroblasts cell line (3T3) was used to assess the cytotoxicity activity of AzA and AzA derivative, which is dilaurylazelate ester. The comparison of the IC50 (50% inhibition of cell viability) value for AzA and AzA derivative was demonstrated. The IC50 value for AzA was 85.28 μg/mL, whereas the IC50 value for AzA derivative was more than 100 μg/mL. The 3T3 cell was still able to survive without any sign of toxicity from the AzA derivative; thus, it was proven to be non-toxic in this MTT assay when compared with AzA.
The influence of water activity and water content was investigated with farnesyl laurate synthesis catalyzed by Lipozyme RM IM. Lipozyme RM IM activity depended strongly on initial water activity value. The best results were achieved for a reaction medium with an initial water activity of 0.11 since it gives the best conversion value of 96.80%. The rate constants obtained in the kinetics study using Ping-Pong-Bi-Bi and Ordered-Bi-Bi mechanisms with dead-end complex inhibition of lauric acid were compared. The corresponding parameters were found to obey the Ordered-Bi-Bi mechanism with dead-end complex inhibition of lauric acid. Kinetic parameters were calculated based on this model as follows: V (max) = 5.80 mmol l(-1) min(-1) g enzyme(-1), K (m,A) = 0.70 mmol l(-1) g enzyme(-1), K (m,B) = 115.48 mmol l(-1) g enzyme(-1), K (i) = 11.25 mmol l(-1) g enzyme(-1). The optimum conditions for the esterification of farnesol with lauric acid in a continuous packed bed reactor were found as the following: 18.18 cm packed bed height and 0.9 ml/min substrate flow rate. The optimum molar conversion of lauric acid to farnesyl laurate was 98.07 ± 0.82%. The effect of mass transfer in the packed bed reactor has also been studied using two models for cases of reaction limited and mass transfer limited. A very good agreement between the mass transfer limited model and the experimental data obtained indicating that the esterification in a packed bed reactor was mass transfer limited.
The study reports the preparation of a composite consisting of magnetite coated with nanosilica extracted from oil palm leaves (OPL) ash as nanosupports for immobilization of Candida rugosa lipase (CRL) and its application for the synthesis of butyl butyrate. Results of immobilization parameters showed that ∼ 80% of CRL (84.5 mg) initially offered was immobilized onto the surface of the nanosupports to yield a maximum protein loading and specific activity of 67.5 ± 0.72 mg/g and 320.8 ± 0.42 U/g of support, respectively. Surface topography, morphology as well as information on surface composition obtained by Raman spectroscopy, atomic force microscopy, field emission scanning electron microscopy and transmission electron microscopy showed that CRL was successfully immobilized onto the nanosupports, affirming its biocompatibility. Under optimal conditions (3.5 mg/mL protein loading, at 45 ℃, 3 h and molar ratio 2:1 (1-butanol:n-butyric acid) the CRL/Gl-A-SiO2-MNPs gave a maximum yield of 94 ± 0.24% butyl butyrate as compared to 84 ± 0.32% in the lyophilized CRL. CRL/Gl-A-SiO2-MNPs showed an extended operational stability, retaining 50% of its initial activity after 17 consecutive esterification cycles. The results indicated that OPL derived nanosilica coated on magnetite can potentially be employed as carrier for lipase immobilization in replacement of the non-renewable conventionalsilica sources.
Highly oriented ZnO nanorod (NR) arrays were fabricated on a seeded substrate through a hydrothermal route. The prepared ZnO nanorods were used as an amperometric enzyme electrode, in which glucose oxidase (GOx) was immobilised through physical adsorption. The modified electrode was designated as Nafion/GOx/ZnO NRs/ITO. The morphology and structural properties of the fabricated ZnO nanorods were analysed using field-emission scanning electron microscope and X-ray diffractometer. The electrochemical properties of the fabricated biosensor were studied by cyclic voltammetry and amperometry. Electrolyte pH, electrolyte temperature and enzyme concentration used for immobilisation were the examined parameters influencing enzyme activity and biosensor performance. The immobilised enzyme electrode showed good GOx retention activity. The amount of electroactive GOx was 7.82 × 10-8 mol/cm2, which was relatively higher than previously reported values. The Nafion/GOx/ZnO NRs/ITO electrode also displayed a linear response to glucose ranging from 0.05 mM to 1 mM, with a sensitivity of 48.75 µA/mM and a low Michaelis-Menten constant of 0.34 mM. Thus, the modified electrode can be used as a highly sensitive third-generation glucose biosensor with high resistance against interfering species, such as ascorbic acid, uric acid and L-cysteine. The applicability of the modified electrode was tested using human blood samples. Results were comparable with those obtained using a standard glucometer, indicating the excellent performance of the modified electrode.
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.