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.
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.
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.
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.
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.
A new alcohol oxidase (AOX) enzyme-based formaldehyde biosensor based on acrylic microspheres has been developed. Hydrophobic poly(n-butyl acrylate-N-acryloxy-succinimide) [poly(nBA-NAS)] microspheres, an enzyme immobilization matrix, was synthesized using photopolymerization in an emulsion form. AOX-poly(nBA-NAS) microspheres were deposited on a pH transducer made from a layer of photocured and self-plasticized polyacrylate membrane with an entrapped pH ionophore coated on a Ag/AgCl screen printed electrode (SPE). Oxidation of formaldehyde by the immobilized AOX resulted in the production of protons, which can be determined via the pH transducer. Effects of buffer concentrations, pH and different amount of immobilization matrix towards the biosensor's analytical performance were investigated. The formaldehyde biosensor exhibited a dynamic linear response range to formaldehyde from 0.3-316.2 mM and a sensitivity of 59.41 ± 0.66 mV/decade (R(2) = 0.9776, n = 3). The lower detection limit of the biosensor was 0.3 mM, while reproducibility and repeatability were 3.16% RSD (relative standard deviation) and 1.11% RSD, respectively (n = 3). The use of acrylic microspheres in the potentiometric formaldehyde biosensor improved the biosensor's performance in terms of response time, linear response range and long term stability when compared with thick film immobilization methods.
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.
A tri-enzyme system consisting of choline kinase/choline oxidase/horseradish peroxidase was used in the rapid and specific determination of the biomarker for bacterial sepsis infection, secretory phospholipase Group 2-IIA (sPLA2-IIA). These enzymes were individually immobilized onto the acrylic microspheres via succinimide groups for the preparation of an electrochemical biosensor. The reaction of sPLA2-IIA with its substrate initiated a cascading enzymatic reaction in the tri-enzyme system that led to the final production of hydrogen peroxide, which presence was indicated by the redox characteristics of potassium ferricyanide, K₃Fe(CN)₆. An amperometric biosensor based on enzyme conjugated acrylic microspheres and gold nanoparticles composite coated onto a carbon-paste screen printed electrode (SPE) was fabricated and the current measurement was performed at a low potential of 0.20 V. This enzymatic biosensor gave a linear range 0.01-100 ng/mL (R² = 0.98304) with a detection limit recorded at 5 × 10-3 ng/mL towards sPLA2-IIA. Moreover, the biosensor showed good reproducibility (relative standard deviation (RSD) of 3.04% (n = 5). The biosensor response was reliable up to 25 days of storage at 4 °C. Analysis of human serum samples for sPLA2-IIA indicated that the biosensor has potential for rapid bacterial sepsis diagnosis in hospital emergency department.
An optical biosensor consisting of a chromoionophore (ETH5294) (CM) doped sol-gel film interfaced with another sol-gel film immobilized with acetylcholinesterase (AChE) was employed to detect the insecticide dichlorvos. The main advantage of this optical biosensor is the use of a sol-gel layer with immobilized CM that possesses lipophilic property. The highly lipophilic nature of the CM and its compatibility with the sol-gel matrix has prevented leaching, which is frequently a problem in optical sensor construction based on pH indicator dyes. The immobilization of the indicator and enzyme was simple and need no chemical modification. The CM layer is pH sensitive and detects the pH changes of the acetylcholine chloride (AChCl) substrate when hydrolyzed by AChE layer deposited above. In the absence of the AChE layer, the pH response of the CM layer is linear from pH 6 to 8 (R(2)=0.98, n=3) and it showed no leaching of the lipophilic chromoionophore. When the AChE layer is deposited on top, the optical biosensor responds to AChCl with a linear dynamic range of 40-90mM AChCl (R(2)=0.984, n=6). The response time of the biosensor is 12min. Based on the optimum incubation time of 15min, a linear calibration curve of dichlorvos against the percentage inhibition of AChE was obtained from 0.5 to 7mg/L of dichlorvos (17-85% inhibition, R(2)=0.991, n=9). The detection limit for dichlorvos was 0.5mg/L. The results of the analysis of 1.7-6.0mg/L of dichlorvos using this optical biosensor agreed well with a gas chromatography-mass spectrometry detection method.
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.
This work reports on a novel glucose biosensor based on co-immobilization of glucose oxidase (GOx) and horseradish peroxidase with polymerized multiporous nanofiber (MPNFs) of SnO2 onto glassy carbon electrode with chitosan. Multiporous nanofibers of SnO2 were synthesized by electrospinning method from the tin precursor which possesses high surface area good electrical conductivity, and the nanofibers were polymerized with polyaniline (PANI). GOx and HRP were then co-immobilized with the nanofibers on the surface of the glassy carbon electrode by using chitosan. The polymerized nanofibers play a significant role in facilitating the direct electron transfer between the electroactive center of the immobilized enzyme and the electrode surface. The morphology of the nanofiber and polymerized nanofiber has been evaluated by field emission scanning electron microscopy (FESEM). Cyclic Voltammetry and amperometry were employed to study and optimize the performance of the fabricated biosensor. The PANI/SnO2-NF/GOx-HRP/Ch/GC biosensor displayed a linear amperometric response towards the glucose concentration range from 5 to 100 μM with a detection limit of 1.8 μM (S/N = 3). Also, the anti-interference study and real sample analysis was investigated. Furthermore, the biosensor reported in this work exhibited excellent stability, reproducibility, and repeatability.
The discharge of an alarming number of recalcitrant pollutants from various industrial activities presents a serious threat to environmental sustainability and ecological integrity. Bioremediation has gained immense interest around the world due to its environmentally friendly and cost-effective nature. In contrast to physical and chemical methods, the use of microbial enzymes, particularly immobilized biocatalysts, has been demonstrated as a versatile approach for the sustainable mitigation of environmental pollution. Considerable attention is now devoted to developing novel enzyme engineering approaches and state-of-the-art bioreactor design for ameliorating the overall bio-catalysis and biodegradation performance of enzymes. This review discusses the contemporary and state of the art technical and scientific progress regarding applying oxidoreductase enzyme-based biocatalytic systems to remediate a vast number of pharmaceutically active compounds from water and wastewater bodies. A comprehensive insight into enzyme immobilization, the role of mediators, bioreactors designing, and transformation products of pharmaceuticals and their associated toxicity is provided. Additional studies are necessary to elucidate enzymatic degradation mechanisms, monitor the toxicity levels of the resulting degraded metabolites and optimize the entire bio-treatment strategy for technical and economical affordability.
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.
Electrochemical biosensors for phenolic compound determination were developed by immobilization of tyrosinase enzyme in a series of methacrylic-acrylic based biosensor membranes deposited directly using a photocuring method. By modifying the hydrophilicity of the membranes using different proportions of 2-hydroxyethyl methacrylate (HEMA) and butyl acrylate (nBA), we developed biosensor membranes of different hydrophilic characters. The differences in hydrophilicity of these membranes led to changes in the sensitivity of the biosensors towards different phenolic compounds. In general biosensors constructed from the methacrylic-acrylic based membranes showed the poorest response to catechol relative to other phenolic compounds, which is in contrast to many other biosensors based on tyrosinase. The decrease in hydrophilicity of the membrane also allowed better selectivity towards chlorophenols. However, phenol biosensors constructed from the more hydrophilic membrane materials demonstrated better analytical performance towards phenol compared with those made from less hydrophilic ones. For the detection of phenols, these biosensors with different membranes gave detection limits of 0.13-0.25 microM and linear response range from 6.2-54.2 microM phenol. The phenol biosensors also showed good phenol recovery from landfill leachate samples (82-117%).
Carbon nanomaterials, due to their catalytic activity and high surface area, have potential as cell immobilization supports to increase the production of xylanase. Recombinant Kluyveromyces lactis used for xylanase production was integrated into a polymeric gel network with carbon nanomaterials. Carbon nanomaterials were pretreated before cell immobilization with hydrochloric acid (HCl) treatment and glutaraldehyde (GA) crosslinking, which contributes to cell immobilization performance. Carbon nanotubes (CNTs) and graphene oxide (GO) were further screened using a Plackett-Burman experimental design. Cell loading and agar concentration were the most important factors in xylanase production with low cell leakage. Under optimized conditions, xylanase production was increased by more than 400% compared to free cells. Immobilized cell material containing such high cell densities may exhibit new and unexplored beneficial properties because the cells comprise a large fraction of the component. The use of carbon nanomaterials as a cell immobilization support along with the entrapment method successfully enhances the production of xylanase, providing a new route to improved bioprocessing, particularly for the production of enzymes. KEY POINTS: • Carbon nanomaterials (CNTs, GO) have potential as cell immobilization supports. • Entrapment in a polymeric gel network provides space for xylanase production. • Plackett-Burman design screen for the most important factor for cell immobilization.
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.
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%.