Covalent Binding Method Research Articles

Nanoparticle-based Biosensors for Detection of Heavy Metal Ions

Heavy metal pollution is one of the most serious environmental problems in the world. Many efforts have been made to develop biosensors for monitoring heavy metals in the environment. Development of nanoparticle-based biosensors is the most effective way to solve this problem. This review presents the latest technology of nanoparticle-based biosensors for environment monitoring to detect heavy metal ions, which are magnetic chitosan biosensor, colorimetric biosensor, and electrochemical biosensor. Magnetic chitosan biosensor acts as a nanoabsorbent, which can easily detect and extract poisonous heavy metal ions such as lead ions and copper ions. There are several methods to prepare the chitosan based on the nanoparticle, which are cross-linking, co-precipitation, multi-cyanoguanidine, and covalent binding method. In colorimetric biosensor, gold and silver nanoparticles are commonly used to detect the lead and mercury ions. In addition, this biosensor is very sensitive, fast and selective to detect metal ions based on the color change of the solution mixture. Meanwhile, electrochemical biosensor is widely used to detect heavy metal ions due to a simple and rapid process, easy, convenient and inexpensive. This biosensor is focused on the surface area, which leads to significant improvement in the performance of devices in terms of sensitivity. The wide surface area can affect the performance of the biosensor due to a limited space for operation of electrode. Therefore, reduced graphene oxide is a suitable material for making the electrochemical biosensor due to a wide surface area, good conductivity and high mechanical strength. In conclusion, these three technologies have their own advantages in making a very useful biosensor in the detection of heavy metal ions.

Ultrasound‐assisted conjugation of Moringa proteins with gallic acid: characterisation of structural and techno‐functional properties

SummaryIn this study, ultrasound‐assisted covalent binding method, free radical‐induced method, was used to prepare Moringa protein isolate (MPI) – gallic acid conjugates (MPI‐GAC) to examine the improvement of the sonication application. Additionally, the influence of polyphenol conjugation on functional and structural attributes of MPI was assessed thru spectral analysis, scanning electron microscopy, antioxidant activity, emulsification, foaming, and water and oil holding properties. The enhanced value of grafted polyphenol species was observed after introducing ultrasound treatment during free radical mediation process. The structural changes in protein by gallic acid conjugation were confirmed by using FT‐IR which illustrated the modification in MPI‐GAC with modified peak width of the amide‐I band. Scanning electron microscopy also showed a significant difference in the structure of MPI and MPI‐GAC. The grafting of gallic acid onto MPI directed under ultrasound‐assisted conditions conferred distinctive functional characteristics, containing elevated emulsification capacity (51.4 mg mL−1) along with enhanced water and oil holding capacity by 2.07 and 5.11 g g−1, respectively. Furthermore, conjugation also increased the protein solubility 89.3–97.6% and significant trend (68.04–88.06%) of antioxidant capacity was observed by increasing the concentration. The findings of this research work offer new insights on the effective fabrication of MPI‐GAC with multifaceted functionalities, thus broadening the scope and possibilities of high‐value Moringa seed protein.

Nanobiocatalysis: Approach and Applications in Drug Development and Sensing.

Enzymes play a pivotal role in the human body, but their potential is not limited to just that. Scientists have successfully modified these enzymes as nanobiocatalysts or nanozymes for industrial or commercial use, either in the food, medicine, biotech or even textile industries. These nanobiocatalysts and nanozymes offer several advantages over enzymes, like better stability, improved shelf-life, increased percentage yield, and reuse potential, which is very difficult with normal enzymes. The various techniques of NBC synthesis using immobilization techniques like adsorption, covalent binding, affinity immobilization, and entrapment methods are briefly discussed. The enzymes are either entrapped or adsorbed on the nanocarrier matrices, which can be nanofibers, nanoporous carriers, or nanocontainers as nanobiocatalysts. We also highlight the challenges the nanobiocatalyst overcomes in the industrial production of some drugs like sitagliptin, montelukast, pregabalin, and atorvastatin. Also, the inactivation of an organophosphate or opioid poisoning treating agent, SSOPOX nanohybrid, is discussed in this paper. Nanozymes are intrinsic enzyme-like compounds, and they also show wide application in themselves. Their GQD/AGNP nanohybrid shows antibacterial potential; they can also be utilized in optical sensing to detect small molecules, ions, nucleic acids, proteins, and cancer cells. In this paper, various applications of these NBCs have been discussed, and their potential applications with examples are also mentioned. Nanoenzymes can address targeted drug delivery via the controlled release of drugs to increase the efficacy of anticancer drugs that minimize damage to healthy tissue or cells.

The effect of commercial β-glucosidase and its immobilization on quality of red wine production

To find a feasible strategy for commercial β-glucosidase to enhance red wine quality, immobilized enzyme technology were considered. The preferred epoxy resin using the covalent binding method was ES-103B, and its optimal immobilization conditions were pH 4.9, 25 h immobilization time and 3 mg/g enzyme concentration. Meanwhile, this immobilized β-glucosidase still contained weak esterase activity just like commercial β-glucosidase. Free commercial β-glucosidase had different effects on the fermentation of 11 clones from three grape varieties (Syrah, Merlot and Cabernet Sauvignon) to produce wine. For example, the free enzyme increased the concentrations of β-damascenone, 1-hexanol, phenylethyl alcohol, and three ethyl esters (ethyl hexanoate, ethyl octanoate and ethyl decanoate) in a Cabernet Sauvignon (CS169) wine, but total anthocyanins level was reduced, affecting the color depth (a* value). Notably, during the alcoholic fermentation stage of CS169, immobilized β-glucosidase did not significantly decrease polyphenolic levels and color depth (a* value). What's more, using immobilized β-glucosidase under this treatment condition resulted in the highest levels of higher alcohols, ethyl esters, and acetate. Moreover, β-damascenone content increased by more than 25.35%, as well as phenethyl alcohol and benzaldehyde of benzenic compounds significantly increased by over 72.23% and two-fold, respectively. As a result, CS169 wine possessed more complex aroma and highest sensory score. Therefore, enologists can use immobilized β-glucosidase to develop various strategies that enhance wine quality and produce different wine styles.

Ultrasound-assisted preparation of protein–polyphenol conjugates and their structural and functional characteristics

Herein, ultrasound-assisted conventional covalent binding methods (alkali treatment, free radical mediation, and an enzymatic method) were used to prepare soybean protein isolate (SPI)-(−)-epigallocatechin gallate (EGCG) conjugates to investigate the enhancement effect of the ultrasound synergistic treatment. In addition, the influence of EGCG grafting on the structure and properties of SPI was evaluated via reactive group analysis, spectral analysis, surface hydrophobicity measurements, emulsification property assessment, and α-glucosidase inhibition analysis. The obtained results revealed that the enzymatic method produced the highest polyphenol grafting content among the conventional techniques. Meanwhile, ultrasound treatment increased the amount of grafted polyphenol species during the alkali treatment and free radical mediation procedure, decreased the grafting efficiency in the enzymatic method, and maximized the grafting efficiency during the alkali treatment. In addition, reactive group and spectral analyses demonstrated that EGCG formed C–N and C–S bonds with SPI and decreased the α-helix content in the protein structure, thereby increasing the molecular flexibility of SPI. It also produced hydrogen bonds and hydrophobic interactions, as demonstrated by the results of molecular docking. Furthermore, the EGCG grafting of SPI conducted under the ultrasound-assisted conditions endowed SPI with unique functional characteristics, including good emulsification and antioxidant properties and high α-glucosidase inhibitory activity, while the ultrasound-assisted alkali treatment resulted in the optimal functional properties. The results of this study provide new insights into the effective preparation of SPI–EGCG complexes with multiple functionalities, thereby expanding the scope of high-value SPI utilization.

Immobilization of Pseudomonas fluorescens Lipase on Hollow Poly(o-phenylenediamine) Microspheres and Its Application in the Preparation of Citronellyl Acetate

In order to address the challenges associated with free lipase in organic solvents, including aggregation, poor stability, and low catalytic efficiency, this study developed two types of poly(o-phenylenediamine) microspheres (solid and hollow) as supports for immobilizing lipase. The immobilization process utilized an adsorption method, with the poly(o-phenylenediamine) hollow microspheres being identified as the optimal support in a 2:5 enzyme-to-support ratio. On this basis, the lipase was immobilized by the covalent binding method. The immobilization conditions consisted of treating the support with 2% glutaraldehyde and immobilization at 40 °C for 2 h in pH 7.0 buffer. The specific activity of the immobilized enzyme was 5.3 times higher than that of the free enzyme. Covalent-binding immobilized lipase was also used for the preparation of citronellyl acetate by transesterification reaction, and, in optimized reaction conditions where the amount of immobilized enzyme was 0.1 g/mL, the reaction temperature was 50 °C and the shaking speed was 200 r/min during the reaction. Under these conditions, the citronellyl acetate yields can exceed 99% after 2 h. Furthermore, the stability of the immobilized lipase was investigated, and the residual activity of the immobilized enzyme was 95% after seven repetitions, while that of the free enzyme was only 70%. After 56 days of storage at room temperature, the immobilized enzyme retained 60% of its original viability, while the free enzyme retained only 31%.

Effect of Glutaraldehyde Multipoint Covalent Treatments on Immobilized Lipase for Hydrolysis of Acidified Oil.

Immobilized lipase is a green and sustainable catalyst for hydrolysis of acidified oil. Glutaraldehyde is widely used for lipase immobilization while the appropriate strategy optimizes the catalytic performance of lipase. In this research, lipase from Candida rugosa (CRL) was immobilized on spherical silica (SiO2) by glutaraldehyde multipoint covalent treatments, including covalent binding method and adsorption-crosslinking method. The enzymatic stability properties and performance in hydrolysis of refined oil and acidified oil were studied. We confirmed that the residual activity decreased while the stability increased because of the influence on secondary structure of lipase after multipoint covalent treatments. In the comparison of different immobilization strategies in multipoint covalent treatment, SiO2-CRL (covalent binding method) showed lower loading capacity than SiO2-CRL (adsorption-crosslinking method), resulting in low activity. However, SiO2-CRL (covalent binding method) showed better reusability and stability. Immobilized lipase via covalent binding method was more potential in the application of catalytic hydrolysis of acidified oils.

Tunable metal-organic frameworks assist in catalyzing DNAzymes with amplification platforms for biomedical applications.

Various binding modes of tunable metal organic frameworks (MOFs) and functional DNAzymes (Dzs) synergistically catalyze the emergence of abundant functional nanoplatforms. Given their serial variability in formation, structural designability, and functional controllability, Dzs@MOFs tend to be excellent building blocks for the precise "intelligent" manufacture of functional materials. To present a clear outline of this new field, this review systematically summarizes the progress of Dz integration into MOFs (MOFs@Dzs) through different methods, including various surface infiltration, pore encapsulation, covalent binding, and biomimetic mineralization methods. Atomic-level and time-resolved catalytic mechanisms for biosensing and imaging are made possible by the complex interplay of the distinct molecular structure of Dzs@MOF, conformational flexibility, and dynamic regulation of metal ions. Exploiting the precision of DNAzymes, MOFs@Dzs constructed a combined nanotherapy platform to guide intracellular drug synthesis, photodynamic therapy, catalytic therapy, and immunotherapy to enhance gene therapy in different ways, solving the problems of intracellular delivery inefficiency and insufficient supply of cofactors. MOFs@Dzs nanostructures have become excellent candidates for biosensing, bioimaging, amplification delivery, and targeted cancer gene therapy while emphasizing major advancements and seminal endeavors in the fields of biosensing (nucleic acid, protein, enzyme activity, small molecules, and cancer cells), biological imaging, and targeted cancer gene delivery and gene therapy. Overall, based on the results demonstrated to date, we discuss the challenges that the emerging MOFs@Dzs might encounter in practical future applications and briefly look forward to their bright prospects in other fields.

Adsorption-degradation of malachite green using alkali-modified biochar immobilized laccase under multi-methods

• Laccase was immobilized onto A-MB via multi-immobilization methods. • The adsorption of MG on A-MB followed the pseudo-second-order equation. • The adsorption of MG on A-MB followed the Langmuir isotherm model. • A-MB possessed a relatively high q max (757.58 mg/g) for MG. • The maximum removal rate of immobilized laccases was high than 97.70 %. Malachite green (MG) is an environmental persistent pollutant. The excellent adsorption capacity and enzyme carrier potential of alkali modified biochar (A-MB) are potential solutions for the treatment of MG. In this paper, laccase was immobilized onto A-MB via multi-immobilization methods. The batch adsorption equilibrium experiments were conducted to study the adsorption behavior of MG on carrier A-MB. The results showed that the adsorption of MG on A-MB followed the pseudo-second-order equation and the Langmuir isotherm model. In addition, A-MB possessed a relatively high q max (757.58 mg/g) for MG. The comparative experiment was set up to compare the removal rate of MG on free laccase (FL) and immobilized laccase (adsorption method: A/lac@A-MB; adsorption-crosslinking method: A-C/lac@A-MB; covalent binding method: CB/lac@A-MB), the maximum removal rate of FL was 89.68 %, but the maximum removal rate of A/lac@A-MB, A-C/lac@A-MB and CB/lac@A-MB was 97.70 %, 98.77 % and 97.86 %, respectively.

Optimization and characterization of immobilized laccase on titanium dioxide nanostructure and its application in removal of Remazol Brilliant Blue R

The objective of this study was to determine optimum condition for immobilizing laccase onto porous TiO 2 nanostructure, and to characterize the immobilized enzyme. XRD and FTIR analyses revealed that laccase was successfully immobilized. The maximum recovery activity of 85.30 ± 0.89% was achieved when immobilization conditions were: 1.5 mg/mL laccase concentration, pH 7.0, and 18 h contact time. Shifting of optimum temperature and pH were observed after immobilization. Kinetics revealed that the immobilized form had lower substrate specificity and catalytic rate compared to the free enzyme. Storage stability of the immobilized form were improved compared to the free enzyme. Interestingly, the immobilized laccase could preserve 31.84 ± 1.86% of enzyme activity after keeping the enzyme at room temperature continuously for 14 days. Maximum RBBR removal of 76.09 ± 0.47% was achieved at pH 5.0, and repeatability of the immobilized enzyme in decolorization was performed for 3 cycles with 40% decrease in efficiency. The immobilized laccase can be removed easily from the solution using forceps without further separation processes. • Combination of laccase enzyme and nanostructure was applied to produce the nanobiocatalyst. • Laccase was successfully immobilized on nano TiO 2 using covalent binding method. • Stability, durability, and reusability of the immobilized laccase were improved. • Maximum RBBR decolorization of the immobilized laccase was 76.09 ± 0.47% at pH 5.0. • After completion of the reaction, the immobilized laccase can be easily separated.

Construction of graphene quantum dots-decorated EGFR cell membrane chromatography for screening active components from Peucedanum praeruptorum Dunn.

A novel stability-enhanced graphene quantum dot (GQD)-decorated epidermal growth factor receptor (EGFR) cell membrane chromatography was constructed to study the potential application of GQDs in bioaffinity chromatography, and to screen active components acting on EGFR from traditional Chinese medicine (TCM). The carboxyl groups on the surface of GQDs reacted with the amino groups of the amino-silica gel (SiO2-NH2) to form a covalent bond, thereby preparing the GQD-decorated silica gel (SiO2-GQDs). The EGFR cell membrane was further immobilized on the SiO2-GQDs through the same covalent binding method to obtain the GQD-decorated cell membrane stationary phase (SiO2-GQDs-CMSP). In this way, the cell membrane was firmly immobilized on the decorated silica carrier. The life span and stability of the GQD-decorated cell membrane chromatographic (SiO2-GQDs-CMC) column were both enhanced, and the optimal immobilization conditions of the EGFR cell membrane were also determined. This model was then verified by establishing a SiO2-GQDs-CMC online liquid chromatography-ion trap-time-of-flight (LC-IT-TOF) system to screen possible active components in Peucedanum praeruptorum Dunn. As a result, praeruptorin B (Pra-B) was screened out, and its inhibitory effect against EGFR cell growth was evaluated by the cell counting kit-8 (CCK-8) assay. Molecular docking assay was also conducted to further estimate the interaction between Pra-B and EGFR. Overall, this research indicated that GQDs may be a promising nanomaterial to be used in prolonging the life span of the CMC column, and Pra-B could be a potential EGFR inhibitor so as to treat cancer.

Enhanced paraquat removal from contaminated water using cell-immobilized biochar

The efficient performance of cell-immobilized biochar in removing paraquat (PQ) from contaminated water is described in this work. Pseudomonas putida was immobilized onto coconut fiber-derived biochar (BC) using the adsorption method and the covalent binding method. The FTIR spectrum analysis revealed that the main adsorption mechanism of bacteria and BC relates to the interaction between cell surface proteins and the functional group on biochar. The pseudo-second-order model could explain the kinetics of simultaneous adsorption and biodegradation of cell immobilization on BC. Cell-immobilized biochar using the covalent bonding method and adsorption method exhibited superior PQ removal capacity with 30–35% increases in efficiency compared with simple biochar. After 48 h using cell-immobilized biochar, the PQ was transformed to 4,4-bipyridyl and malic acid as detected by GC/MS. From the PQ kinetic removal rate and bacterial retention, the covalent binding method with 16.79 mg/g paraquat adsorption capacity and 0.626 g/mg/h kinetic rate constant is considered as the best cell-immobilization method for the immobilization technique. The cell immobilization on biochar using both the adsorption method and the covalent binding method can support bacteria to degrade PQ into small molecular substances in water treatment processes.

Development of Chitosan Functionalized Magnetic Nanoparticles with Bioactive Compounds.

In this study, magnetic maghemite nanoparticles, which belong to the group of metal oxides, were functionalized with chitosan, a non-toxic, hydrophilic, biocompatible, biodegradable biopolymer with anti-bacterial effects. This was done using different synthesis methods, and a comparison of the properties of the synthesized chitosan functionalized maghemite nanoparticles was conducted. Characterization was performed using scanning electron microscopy (SEM) and vibrating sample magnetometry (VSM). Characterizations of size distribution were performed using dynamic light scattering (DLS) measurements and laser granulometry. A chitosan functionalization layer was confirmed using potentiometric titration on variously synthesized chitosan functionalized maghemite nanoparticles, which is important for further immobilization of bioactive compounds. Furthermore, after activation of chitosan functionalized maghemite nanoparticles with glutaraldehyde (GA) or pentaethylenehexamine (PEHA), immobilization studies of enzyme cholesterol oxidase (ChOx) and horseradish peroxidase (HRP) were conducted. Factors influencing the immobilization of enzymes, such as type and concentration of activating reagent, mass ratio between carrier and enzyme, immobilization time and enzyme concentration, were investigated. Briefly, microparticles made using the chitosan suspension cross-linking process (MC2) proved to be the most suitable for obtaining the highest activity of immobilized enzyme, and nanoparticles functionalized with chitosan using the covalent binding method (MC3) could compete with MC2 for their applications.

Layer-by-layer immobilizing of polydopamine-assisted ε-polylysine and gum Arabic on titanium: Tailoring of antibacterial and osteogenic properties.

Bacterial infection has become a crucial reason that give rise to failure of medical implants in clinical applications. In this regard, various antibacterial surface modifications of implants have been developed in recent years. However, it remains a challenge to enable the implant surfaces with both suitable antibacterial and osteogenic properties. In this work, ε-polylysine and gum Arabic multilayer composite films were immobilized layer by layer (LBL) on anodized titanium with the assistance of polydopamine for the first time. In vitro antibacterial results showed that the bacteria numbers decreased with an increase in the loading amount of ε-polylysine. Furthermore, long-term antibacterial property up to 3weeks against both gram-positive Staphylococcus aureus (S. aureus) and gram-negative Escherichia coli (E. coli) was obtained combined with the merits of covalent binding and LBL methods. Meanwhile, the cell proliferation and osteogenic differentiation of BMSCs on titanium dioxide nanotubes (TNTs) modified with composite films was significantly improved. Remarkably, a facile method to optimize anti-infective and osteogenic properties of medical titanium has been developed, and it was demonstrated that the ε-polylysine and gum Arabic multilayer composite films with assistance of polydopamine were able to endow the orthopedic implant materials both improved antibacterial property and excellent biocompatibility, which is of profound significance for practical application of titanium-based implants.

Microfluidic biofuel cell based on cholesterol oxidase/laccase enzymes.

In this research two electrodes were developed, a bioanode where the enzyme oxidase was immobilized on Sigracet GDL 39 by means of the covalent binding method and a biocathode with laccase enzyme immobilized on Toray carbon paper by the adsorption method. The evaluation of these electrodes was carried out in a microfluidic fuel cell using cholesterol as fuel and oxygen as oxidant. The electrical conductivity of the electrodes was measured by the kelvin method. Microfluidic fuel cell (mFFC) was constructed using Poly-(methyl methacrylate) (PMMA) for the fuel and oxidant channels; the cathode electrode was incorporated as a wall to obtain the major O2 quantity for the reduction reaction. The evaluation of the microfluidic cell was carried out using different solutions of cholesterol all in phosphate buffer solutions at pH 7.5, where it was possible to obtain 1.38 mW/cm2 of maximum power density and 0.75V of open circuit potential using 500mg/dL of cholesterol.

Extraction and immobilization of Proteolytic Enzyme from local Yemeni Bean Seeds (Dolichos Lablab L.)

The aim of this study is to prepare immobilized protease with high hydrolytic activity forbiotechnological applications. Local Bean Seeds (Dolichos Lablab L.) were used for the extraction of acidprotease using tris-buffer, pH 4.5 as enzyme solvent. Free acidic protease was immobilized on entrapment in calcium alginate gel (in situ activated) by covalent binding method. Their activity and immobilization efficiency for hemoglobin hydrolysis was investigated. Temperature and pH maxima of the immobilized protease showed no changes before and after immobilization. The immobilized protease exhibited good thermal stability and re-usability.

Ameliorating the activity and stability of β galactosidase by tailoring potential nanobiocatalyst on functionalized nanographene: Headway to lactose hydrolysis

The ever-growing occurrence of intolerance to dairy products has led to an enormous requirement for immobilization of β galactosidase. In this report, we have shown the immobilization of β galactosidase onto polyaniline doped magnetic graphene (Fe3O4@PANI-GO) nanocomposite. A comparative analysis has been carried out for the integration via physical adsorption and covalent binding methods onto functionalized graphene nanocomposite for analytical application based on lactose reduction. The higher immobilization yield was exhibited in the case of the covalently bound enzyme with ƞ (effectiveness factor) value of 1.49. Characterization of functionalized graphene was studied by scanning and transmission electron microscope, and Fourier-transform infrared spectroscopy studies. Shift of pH optima was observed for covalently bound enzyme from pH 4.5 to 5. The immobilized β galactosidase displayed temperature optima similar to the soluble enzyme at 50 °C. Covalently bound enzyme retained 80% activity even after its 10th successive reuse. The immobilized enzyme displayed higher lactose reduction from milk than the free enzyme. Our conclusions have framed the way for the establishment of graphene-based nanobiosensor to upgrade the quality of milk and its products for easy assimilation by the lactose intolerant patients.

Laccase immobilization on amino-functionalized magnetic metal organic framework for phenolic compound removal

An amino-functionalized magnetic metal organic framework (MOF), Fe3O4-NH2@MIL-101(Cr), was employed for laccase immobilization for the first time. The immobilized laccase was synthesized by the adsorption and covalent binding method, thus exhibited high activity recovery, large immobilization capacity and good tolerance to low pH and high temperature conditions. The excellent stability enabled the immobilized laccase to retain 89% of its initial activity after storage for 28 days. When the ambient temperature reached 85 °C, the immobilized laccase showed 49.1% residual activity even after 6 h preservation. The stability of laccase in organic solvents such as methanol was also greatly improved. Application of the immobilized laccase for 2,4-dichlorophenol removal was also investigated. The adsorption by Fe3O4-NH2@MIL-101(Cr) contributed to a quick removal in the first hour, and the removal efficiency reached 87% eventually. When the reaction was completed, the immobilized laccase could be separated from the solution by a magnet. The results introduced a novel support for laccase immobilization, and the immobilized laccase had great potential in wastewater treatment.

Virus-like organosilica nanoparticles for lipase immobilization: Characterization and biocatalytic applications

Hydrophobic virus-like organosilica nanoparticles (VOSNs) with a spherical core formed by mesoporous organosilica surrounded by epitaxial perpendicular nanotubes were successfully synthesized for the first time. These hydrophobic VOSNs were used for the immobilization of the lipase B from Candida antarctica (CALB) via covalent binding method, where glutaraldehyde was used as a spacer to form covalent bonds connect between the enzyme and support. Under optimized immobilization conditions, the specific hydrolytic activity which determined via the hydrolysis of p-NPP was 23200 U/gsupport, and the maximum CALB loading was 115.6 mg/gsupport. Compared to virus-like silica nanoparticles (VSNs), the hydrophobic VOSNs protected the CALB active conformation from a hazardous external environment due to stronger hydrophobic interactions between the VOSNs and CALB molecule. Thus, compared with CALB@VSNs and free lipase, CALB@VOSNs exhibited better pH, thermal stability, organic solvents tolerance and storage stability. The CALB@VOSNs was used to catalyze the esterification reaction of levulinic acid (LA) and n-lauryl alcohol, which was optimized by a response surface method. Under the optimal conditions (temperature, 51.2 °C; LA: n-lauryl alcohol, 1:11.5; time, 22.6 h), the CALB@VOSNs still retained 75.7% of the LA conversion, whereas the N435 only retained 29.8% of the conversion after 9 cycles. These results showed that hydrophobicity VOSNs could be an efficient support to prepare immobilized enzymes as well as to improve the properties of enzymes.

Enhancement stability and catalytic activity of immobilized α-amylase using bioactive phospho-silicate glass as a novel inorganic support

α-Amylase enzyme was immobilized on bioactive phospho-silicate glass (PS-glass) as a novel inorganic support by physical adsorption and covalent binding methods using glutaraldehyde and poly glutaraldehyde as a spacer. Fourier transform infrared spectroscopy (FT-IR) and scanning electron microscopy (SEM) studies confirmed the glass-enzyme linkage. Dissolution of PS-glass in acidic and neutral pH is higher than that of alkaline pH. Some immobilization variables were optimized using statistical factorial design (Central Composite Design). Optimized immobilization variables enhanced the immobilization yield (IY) from 27.9 to 79.9% (2.9-fold). It was found that the immobilized enzyme had higher optimum temperature, higher half-life time (t1/2), lower activation energy (Ea), lower deactivation constant rate (kd) and higher decimal reduction time (D-values) within the temperature range of 40–60°C. Differential scanning calorimetry analysis (DSC) confirmed the thermalstability of the immobilized enzyme. The immobilized enzyme was stable at a wide pH range (5.0-8.0). Kinetic studies of starch hydrolysis demonstrated that immobilized enzyme had lower Michaelis constant (Km), maximum velocity (Vmax) and catalytic efficiency (Vmax/Km) values. The storage stability and reusability of the immobilized enzyme were found to be about 74.7 and 62.5% of its initial activity after 28days and 11cycles, respectively. Enhanced α-amylase stabilities upon immobilization make it suitable for industrial application.