Concept: Immobilized enzyme
In this work, a simple method for alcohol synthesis with high enantiomeric purity was proposed. For this, colloidal gold and silver surface modifications with 3-mercaptopropanoic acid and cysteamine were used to generate carboxyl and amine functionalized gold and silver nanoparticles of 15 and 45 nm, respectively. Alcohol dehydrogenase from Thermoanaerobium brockii (TbADH) and its cofactor (NADPH) were physical and covalent (through direct adsorption and using cross-linker) immobilized on nanoparticles' surface. In contrast to the physical and covalent immobilizations that led to a loss of 90% of the initial enzyme activity and 98% immobilization, the use of a cross-linker in immobilization process promoted a loss to 30% of the initial enzyme activity and >92% immobilization. The yield of NADPH immobilization was about 80%. The best results in terms of activity were obtained with Ag-citr nanoparticle functionalized with carboxyl groups (Ag-COOH), Au-COOH(CTAB), and Au-citr functionalized with amine groups and stabilized with CTAB (Au-NH2(CTAB)) nanoparticles treated with 0.7% and 1.0% glutaraldehyde. Enzyme conformation upon immobilization was studied using fluorescence and circular dichroism spectroscopies. Shift in ellipticity at 222 nm with about 4 to 7 nm and significant decreasing in fluorescence emission for all bioconjugates were observed by binding of TbADH to silver/gold nanoparticles. Emission redshifting of 5 nm only for Ag-COOH-TbADH bioconjugate demonstrated change in the microenvironment of TbADH. Enzyme immobilization on glutaraldehyde-treated Au-NH2(CTAB) nanoparticles promotes an additional stabilization preserving about 50% of enzyme activity after 15 days storage. Nanoparticles attached-TbADH-NADPH systems were used for enantioselective (ee > 99%) synthesis of (S)-7-hydroxy-2-tetralol.
The magnetic chitosan nanocomposites have been studied intensively and been used practically in various biomedical and biological applications including enzyme immobilization. However, the loading capacity and the remained activity of immobilized enzyme based on existing approaches are not satisfied. Simpler and more effective immobilization strategies are needed. Here we report a simple catechol modified protocol for preparing a novel catechol-chitosan (CCS)-iron oxide nanoparticles (IONPs) composites carrying adhesive moieties with strong surface affinity. The ω-transaminase (ω-TA) was immobilized onto this magnetic composite via nucleophilic reactions between catechol and ω-TA. Under optimal conditions, 87.5% of the available ω-TA was immobilized on the composite, yielding an enzyme loading capacity as high as 681.7 mg/g. Furthermore, the valuation of enzyme activity showed that ω-TA immobilized on CCS-IONPs displayed enhanced pH and thermal stability compared to free enzyme. Importantly, the immobilized ω-TA retained more than 50% of its initial activity after 15 repeated reaction cycles using magnetic separation and 61.5% of its initial activity after storage at 4°C in phosphate buffered saline (PBS) for 15 days. The results suggested that such adhesive magnetic composites may provide an improved platform technology for bio-macromolecules immobilized.
Lipases are promising enzymes that catalyze the hydrolysis of triacylglycerol ester bonds at the oil/water interface. Apart from allowing biocatalyst reuse, immobilization can also affect enzyme structure consequently influencing its activity, selectivity, and stability. The lipase from Penicillium sp. section Gracilenta (CBMAI 1583) was successfully immobilized on supports bearing butyl, phenyl, octyl, octadecyl, and divinylbenzyl hydrophobic moieties wherein lipases were adsorbed through the highly hydrophobic opened active site. The highest activity in aqueous medium was observed for the enzyme adsorbed on octyl support, with a 150% hyperactivation regarding the soluble enzyme activity, and the highest adsorption strength was verified with the most hydrophobic support (octadecyl Sepabeads), requiring 5% Triton X-100 to desorb the enzyme from the support. Most of the derivatives presented improved properties such as higher stability to pH, temperature, and organic solvents than the covalently immobilized CNBr derivative (prepared under very mild experimental conditions and thus a reference mimicking free-enzyme behavior). A 30.8- and 46.3-fold thermostabilization was achieved in aqueous medium, respectively, by the octyl Sepharose and Toyopearl butyl derivatives at 60 °C, in relation to the CNBr derivative. The octyl- and phenyl-agarose derivatives retained 50% activity after four and seven cycles of p-nitrophenyl palmitate hydrolysis, respectively. Different derivatives exhibited different properties regarding their properties for fish oil hydrolysis in aqueous medium and ethanolysis in anhydrous medium. The most active derivative in ethanolysis of fish oil was the enzyme adsorbed on a surface covered by divinylbenzyl moieties and it was 50-fold more active than the enzyme adsorbed on octadecyl support. Despite having identical mechanisms of immobilization, different hydrophobic supports seem to promote different shapes of the adsorbed open active site of the lipase and hence different functional properties.
The enzymatic performance of trypsin in hydrolysis of N-α-benzoyl-DL-arginine-4-nitroanilide (BAPNA) was improved by adsorption on Santa Barbara Amorphous (SBA)-15 mesoporous silica. The optimal immobilization conditions were screened and the properties of immobilized enzyme have also been studied. Under the optimal conditions, the immobilized trypsin displays maximum specific activity (49.8 μmol/min/g). The results also indicate that the immobilized trypsin exhibits better storage stability.
Immobilization of lysozyme-CLEA onto electrospun chitosan nanofiber for effective antibacterial applications.
- International journal of biological macromolecules
- Published almost 6 years ago
Chitosan (CS) nanofibers with a diameter of 150-200nm were fabricated from a mixed chitosan/poly (vinyl alcohol) (PVA) solution by the electrospinning method. The nascent CS/PVA nanofibers were treated with 0.5M NaOH solution to make stable CS nanofibers by removing PVA under aqueous conditions. Hen egg-white lysozyme was immobilized on electrospun CS nanofibers via cross-linked enzyme aggregates (CLEAs) and used for effective and continuous antibacterial applications. The maximum amount of lysozyme immobilized on the CS nanofibers was determined to be 62.3mg/g of nanofibers under the optimum conditions. The immobilized lysozyme-CLEA retained more than 75.4% of its initial activity after 80 days of storage at room temperature, while the free lysozyme lost all of its activity under the same conditions. In addition, the immobilized lysozyme-CLEA retained more than 76% of its activity after 100 consecutive uses. Finally, the durability of the lysozyme-CLEA immobilized CS nanofibers showed bacteriostasis ratios of 82.4%, 79.8%, 83.4%, and 84.1% after 10 cycles against 4 pathogenic bacteria, viz. Staphylococcus aureus, Bacillus subtilis, Shigella flexneri, and Psedomonas aeruginosa, respectively. These results demonstrated that lysozyme-CLEA immobilized CS nanofibers could be used as a promising material for enhanced and continuous antibacterial applications.
The Aspergillus niger NRC1ami pectinase was evaluated according to its hydrolysis efficiency of dry untreated orange peels (UOP), HCl-treated orange peels and NaOH-treated orange peels (HOP and NOP). Pectinase was entrapped in polyvinyl alcohol (PVA) sponge and the optimum pH and temperature of the free and immobilized enzymes were shifted from 4, 40°C to 6, 50°C respectively. The study of pH stability of free and immobilized pectinase showed that the immobilization process protected the enzyme strongly from severe alkaline pHs. The immobilization process improved the enzyme thermal stability to great instant. The unique feature of the immobilization process is its ability to solve the orange juice haze problem completely. Immobilized enzyme was reused 12 times in orange juice clarification with 9% activity loss from the original activity. Maximum reaction rate (V(max)) and Michaelis-Menten constant (K(m)) of the partially purified form were significantly changed after immobilization.
Chitosan grafted with polymethyl methacrylate (PMMA-g-CS) was prepared via a free-radicals polymerization technique as a carrier for enzyme immobilization. α-Chymotrypsin (CT), as an enzyme model in this study, was immobilized onto the prepared PMMA-g-CS via covalent bonding. Calcium alginate (CA) beads were developed for encapsulating PMMA-g-CS-CT to produce PMMA-g-CS-CT/CA composite beads. Morphology and size of PMMA-g-CS particles were investigated by TEM and found to be in the nanoscale. The structure and surface morphology of the beads before and after immobilization process were characterized by FT-IR and SEM, respectively. Both the bound CT content and relative activity of immobilized enzyme were measured. A higher retained activity (about 97.7%) obtained for the immobilized CT at pH 9 for 24h. The results indicated that immobilized CT maintained excellent performance even after 25 reuses and retained 75% from its original activity after 60 days of storage at 25°C.
The application of monoliths for realization of solid-phase biocatalytic processes was dramatically extended since the beginning of new century. Different enzyme immobilization techniques regarding these modern stationary phases have been developed, adapted, and optimized within last decade. The choice of enzyme immobilization method depends on material nature and monolith manufacturing. The present review collected, analyzed, and discussed the accessible published data on existing approaches and specialties of preparation of flow-through enzyme reactors based on monoliths.
Commercial inulinase from Aspergillus niger was immobilized in montmorillonite and then treated in pressurized propane and liquefied petroleum gas (LPG). Firstly, the effects of system pressure, exposure time, and depressurization rate, using propane and LPG, on enzymatic activity were evaluated through central composite design 2(3). Residual activities of 145.1 and 148.5 % were observed for LPG (30 bar, 6 h, and depressurization rate of 20 bar min(-1)) and propane (270 bar, 1 h, and depressurization rate of 100 bar min(-1)), respectively. The catalysts treated at these conditions in both fluids were then used for the production of fructooligosaccharides (FOS) using sucrose and inulin as substrates in aqueous and organic systems. The main objective of this step was to evaluate the yield and productivity in FOS, using alternatives for enhancing enzyme activity by means of pressurized fluids and also using low-cost supports for enzyme immobilization, aiming at obtaining a stable biocatalyst to be used for synthesis reactions. Yields of 18 % were achieved using sucrose as substrate in aqueous medium, showing the potential of this procedure, hence suggesting a further optimization step to increase the process yield.
In this study, it was the first report that the Brevibacterium lutescens CCZU12-1 was employed as a sulfur removing bacteria. Using dibenzothiophene (DBT) as the sole sulfur source, B. lutescens could selectively degrade DBT into 2-hydroxybiphenyl (2-HBP) via the “4S” pathway. In the basal salt medium (BSM) supplemented with 0.25 mM DBT and 0.5 g/L Tween-80, high desulfurization rate (100 %) was obtained by growth cells after 60 h. Furthermore, the n-octane-water (10:90, v/v) biphasic system was built for the biodesulfurization by resting cells. Moreover, a combination of magnetic nano Fe3O4 particles with calcium alginate immobilization was used for enhancing biodesulfurization. In this n-octane-water biphasic system, immobilized B. lutescens cells could be reused for not less than four times. Therefore, B. lutescens CCZU12-1 shows high potential in the biodesulfurization.