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.
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 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.
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.
The electrochemical biosensors based on poly(o-phenylenediamine) (PoPD) and acetylcholinesterase (AChE) and choline oxidase (ChO) enzymes were fabricated on carbon fibre (CF) substrate. The electropolymerized PoPD was used to reduce the interfering substances. The electrode assembly was completed by depositing functionalized carbon nano tubes (FCNTs) and Nafion (Naf). Amperometric detection of acetylcholine (ACh) and choline (Ch) were realized at an applied potential of +750 mV vs Ag/AgCl (saturated KCl). At pH 7.4, the final assembly, Naf-FCNTs/AChE-ChO((10:1))/PoPD/CF(Elip), was observed to have high sensitivity towards Ch (6.3±0.3 μA mM(-1)) and ACh (5.8±0.3 μA mM(-1)), linear range for Ch (K(M)=0.52±0.03 mM) and ACh (K(M)=0.59±0.07 mM), and for Ch the highest ascorbic acid blocking capacity (97.2±2 1mM AA). It had a response time of <5s and with 0.045 μM limit of detection. Studies on different ratio (ACh/Ch) revealed that 10:1, gave best overall response.
In the last few years, biodiesel has emerged as one of the most potential renewable energy to replace current petrol-derived diesel. It is a renewable, biodegradable and non-toxic fuel which can be easily produced through transesterification reaction. However, current commercial usage of refined vegetable oils for biodiesel production is impractical and uneconomical due to high feedstock cost and priority as food resources. Low-grade oil, typically waste cooking oil can be a better alternative; however, the high free fatty acids (FFA) content in waste cooking oil has become the main drawback for this potential feedstock. Therefore, this review paper is aimed to give an overview on the current status of biodiesel production and the potential of waste cooking oil as an alternative feedstock. Advantages and limitations of using homogeneous, heterogeneous and enzymatic transesterification on oil with high FFA (mostly waste cooking oil) are discussed in detail. It was found that using heterogeneous acid catalyst and enzyme are the best option to produce biodiesel from oil with high FFA as compared to the current commercial homogeneous base-catalyzed process. However, these heterogeneous acid and enzyme catalyze system still suffers from serious mass transfer limitation problems and therefore are not favorable for industrial application. Nevertheless, towards the end of this review paper, a few latest technological developments that have the potential to overcome the mass transfer limitation problem such as oscillatory flow reactor (OFR), ultrasonication, microwave reactor and co-solvent are reviewed. With proper research focus and development, waste cooking oil can indeed become the next ideal feedstock for biodiesel.
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.
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.
Immobilized Candida rugosa lipase was used for the synthesis of citronellyl laurate from citronellol and lauric acid. Screening of different types of support (Amberlite MB-1 and Celite) for immobilization of lipase and solvent (n-hexane, n-heptane, and iso-octane) and optimization of reaction conditions, such as catalyst loading, effect of substrates molar ratio and temperature, have been studied. The maximum enzyme activity was obtained at 310 K. The immobilized C. rugosa lipase onto Amberlite MB-1 support was found to be the best support with a conversion of 89% of citronellyl laurate ester in iso-octane compared to Celite 545. Deactivation of C. rugosa lipase at 313, 318 and 323 K were observed. Ordered bi bi mechanism with dead end complex of lauric acid was found to fit the initial rate data and the kinetic parameters were obtained by non-linear regression analysis.
Immobilization can be used to improve the stability of lipases and enhances lipase recovery and reusability, which increases its commercial value and industrial applications. Nevertheless, immobilization frequently causes conformational changes of the lipases, which decrease lipase catalytic activity. in the present work, we synthesized UIO-66 and grafted UIO-66 crystals with proline for immobilization of Candida rugosa lipase (CRL). As indicated by steady-state fluorescence microscopy, grafting of proline onto UIO-66 crystals induced beneficial conformational change in CRL. CRL immobilized on UIO-66/Pro (CRL@UIO-66/Pro) demonstrated higher enzyme activity and better recyclability than that immobilized on UIO-66 (CRL@UIO-66) in both hydrolysis (CRL@UIO-66/Pro: 0.34 U; CRL@UIO-66: 0.15 U) and transesterification (CRL@UIO-66/Pro: 0.93 U; CRL@UIO-66: 0.25 U) reactions. The higher values of kcat and kcat/Km of CRL@UIO-66/Pro also showed that it had better catalytic efficiency as compared to CRL@UIO-66. It is also worth noting that CRL@UIO-66/Pro (0.93 U) demonstrated a much higher transesterification activity as compared to free CRL (0.11 U), indicating that UIO-66/Pro has increased the solvent stability of CRL. Both CRL@UIO-66 and CRL@UIO-66/Pro were also used for the fabrication of biosensors for nitrofen with a wide linear range (0-100 μM), lower limit of detection, and good recovery rate.
Short-chain fructooligosaccharides (scFOSs) can be produced from the levan hydrolysis using levanase. Levanase from Bacillus lehensis G1 (rlevblg1) is an enzyme that specifically converts levan to scFOSs. However, the use of free levanase presents a lack of stability and reusability, thus hindering the synthesis of scFOSs for continuous reactions. Here, CLEAs for rlevblg1 were prepared and characterized. Cross-linked levanase aggregates using glutaraldehyde (CLLAs-ga) and bovine albumin serum (CLLAs-ga-bsa) showed the best activity recovery of 92.8% and 121.2%, respectively. The optimum temperature of CLLAs-ga and CLLAs-ga-bsa was increased to 35 °C and 40 °C, respectively, from its free rlevblg1 (30 °C). At high temperature (50 °C), the half-life of CLLAs-ga-bsa was higher than that of free rlevblg1 and CLLAs-ga. Both CLLAs exhibited higher stability at pH 9 and pH 10. Hyperactivation of CLLAs-ga-bsa was achieved with an effectiveness factor of more than 1 and with improved catalytic efficiency. After 3 h reaction, CLLAs-ga-bsa produced the highest total scFOSs yield of 35.4% and total sugar of 60.4% per gram levan. Finally, the reusability of CLLAs for 8 cycles with more than 50% activity retained makes them as a potential synthetic catalyst to be explored for scFOSs synthesis.
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.
Conjugated linoleic acid (CLA)-rich triacylglycerols (TAG) have received significant attention owing to their health promoting properties. In this study, CLA-rich TAG were successfully synthesized by an immobilized mutant lipase (MAS1-H108A)-catalyzed esterification of CLA-rich fatty acids and glycerol under vacuum. MAS1-H108A was first immobilized onto ECR1030 resin. Results showed that the lipase/support ratio of 41 mg/g was suitable for the immobilization and the thermostability of immobilized MAS1-H108A was greatly enhanced. Subsequently, the immobilized MAS1-H108A was employed for the synthesis of CLA-rich TAG and 95.21% TAG with 69.19% CLA was obtained under the optimized conditions. The TAG content (95.21%) obtained by immobilized MAS1-H108A is the reported highest value thus far, which was significantly higher than that (9.26%) obtained by Novozym 435 under the same conditions. Although the TAG content comparable to the results obtained in this study could also be obtained by Novozym 435, the used enzyme amount is approximately 5-fold of the immobilized MAS1-H108A. Additionally, the immobilized MAS1-H108A exhibited excellent recyclability during esterification retaining 95.11% of its initial activity after 10 batches. Overall, such immobilized mutant lipase with superior esterification activity and recyclability has the potential to be used in oils and fats industry.
Although laccase has been recognized as a wonder molecule and green enzyme, the use of low yielding fungal strains, poor production, purification, and low enzyme kinetics have hampered its large-scale application. Thus,this study aims to select high yielding fungal strains and optimize the production, purification, and kinetics of laccase of Aspergillus sp. HB_RZ4. The results obtained indicated that Aspergillus sp. HB_RZ4 produced a significantly large amount of laccase under meso-acidophilic shaking conditions in a medium containing glucose and yeast extract. A 25 μM CuSO4 was observed to enhance the enzyme yield. The enzyme was best purified on a Sephadex G-100 column. The purified enzyme resembled laccase of A. flavus. The kinetics of the purified enzyme revealed high substrate specificity and good velocity of reaction,using ABTS as a substrate. The enzyme was observed to be stable over various pH values and temperatures. The peptide structure of the purified enzyme was found to resemble laccase of A. kawachii IFO 4308. The fungus was observed to decolorize various dyes independent of the requirement of a laccase mediator system.Aspergillus sp. HB_RZ4 was observed to be a potent natural producer of laccase, and it decolorized the dyes even in the absence of a laccase mediator system. Thus, it can be used for bioremediation of effluent that contains non-textile dyes.
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 chemical route of producing geranyl propionate involves the use of toxic chemicals, liberation of unwanted by-products as well as problematic separation process. In view of such problems, the use of Rhizomucor miehei lipase (RML) covalently bound onto activated chitosan-graphene oxide (RML-CS/GO) support is suggested. Following analyses using Fourier transform infrared spectroscopy, field emission scanning electron microscopy, transmission electron microscopy, and thermogravimetry, properties of the RML-CS/GO were characterized. A response surface methodological approach using a 3-level-four-factor (incubation time, temperature, substrate molar ratio, and stirring rate) Box-Behnken design was used to optimize the experimental conditions to maximize the yield of geranyl propionate. Results revealed that 76 ± 0.02% of recovered protein had yielded 7.2 ± 0.04 mg g(-1) and 211 ± 0.3% U g(-1) of the maximum protein loading and esterification activity, respectively. The actual yield of geranyl propionate (49.46%) closely agreed with the predicted value (49.97%) under optimum reaction conditions (temperature: 37.67°C, incubation time: 10.20 hr, molar ratio (propionic acid:geraniol): 1:3.28, and stirring rate: 100.70 rpm) and hence, verifying the suitability of this approach. Since the method is performed under mild conditions, the RML-CS/GO biocatalyst may prove to be an environmentally benign alternative for producing satisfactory yield 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.
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 novel method for the rapid modification of fullerene for subsequent enzyme attachment to create a potentiometric biosensor is presented. Urease was immobilized onto the modified fullerene nanomaterial. The modified fullerene-immobilized urease (C60-urease) bioconjugate has been confirmed to catalyze the hydrolysis of urea in solution. The biomaterial was then deposited on a screen-printed electrode containing a non-plasticized poly(n-butyl acrylate) (PnBA) membrane entrapped with a hydrogen ionophore. This pH-selective membrane is intended to function as a potentiometric urea biosensor with the deposition of C60-urease on the PnBA membrane. Various parameters for fullerene modification and urease immobilization were investigated. The optimal pH and concentration of the phosphate buffer for the urea biosensor were 7.0 and 0.5 mM, respectively. The linear response range of the biosensor was from 2.31 × 10-3 M to 8.28 × 10-5 M. The biosensor's sensitivity was 59.67 ± 0.91 mV/decade, which is close to the theoretical value. Common cations such as Na+, K+, Ca2+, Mg2+ and NH4+ showed no obvious interference with the urea biosensor's response. The use of a fullerene-urease bio-conjugate and an acrylic membrane with good adhesion prevented the leaching of urease enzyme and thus increased the stability of the urea biosensor for up to 140 days.
This work reviews the stripping off, role of water molecules in activity, and flexibility of immobilized Candida antarctica lipase B (CALB). Employment of CALB in ring opening polyester synthesis emphasizing on a polylactide is discussed in detail. Execution of enzymes in place of inorganic catalysts is the most green alternative for sustainable and environment friendly synthesis of products on an industrial scale. Robust immobilization and consequently performance of enzyme is the essential objective of enzyme application in industry. Water bound to the surface of an enzyme (contact class of water molecules) is inevitable for enzyme performance; it controls enzyme dynamics via flexibility changes and has intensive influence on enzyme activity. The value of pH during immobilization of CALB plays a critical role in fixing the active conformation of an enzyme. Comprehensive selection of support and protocol can develop a robust immobilized enzyme thus enhancing its performance. Organic solvents with a log P value higher than four are more suitable for enzymatic catalysis as these solvents tend to strip away very little of the enzyme surface bound water molecules. Alternatively ionic liquid can work as a more promising reaction media. Covalent immobilization is an exclusively reliable technique to circumvent the leaching of enzymes and to enhance stability. Activated polystyrene nanoparticles can prove to be a practical and economical support for chemical immobilization of CALB. In order to reduce the E-factor for the synthesis of biodegradable polymers; enzymatic ring opening polyester synthesis (eROPS) of cyclic monomers is a more sensible route for polyester synthesis. Synergies obtained from ionic liquids and immobilized enzyme can be much effective eROPS.