Nanotechnology is an area that has been growing over the years, being possible nowadays to find numerous materials constructed at nanoscale. In addition, many applications have been attributed to ...these "new" materials. In this review is presented a brief overview of nanoparticles used for the immobilization of enzymes. Considering the extensive universe of immobilization in nanoparticles, some were chosen to be exposed here, such as chitosan, graphene, silica, polymers, magnetic, nanoflowers, among others. Advantages, disadvantages and limitations of nanoimmobilization also be discussed. Some applications of nanoimmobilized enzymes are presented, like as biodiesel, flavor synthesis ester and biosensors. The purpose of this paper is to provide an overview of what is being studied in relation to nanoparticles for enzymes immobilization, and some discussions about them, aimed at assisting researchers in future studies and reviews.
Advantages, drawbacks and trends in nanomaterials for enzyme immobilization.
•Lipase immobilization rate on octyl supports is reduced at high ionic strength.•Octyl-lipase is more rapidly inhibited by D-pNPP than covalent preparations.•Octyl-lipase inhibition by D-pNPP did not ...depend on ionic strength.•Octyl-lipase activity is less depended on ionic strength.•Lipase activity is not only depended on catalytic Ser exposition.
The lipases from Thermomyces lanuginosus and Pseudomonas cepacia have been immobilized on octyl and cyanogen bromide (CNBr) agarose beads. The immobilization on octyl-agarose is slowed with increasing ionic strength, while the immobilization on CNBr is not significantly affected by the ionic strength. The inhibition of the immobilized preparations with diethyl p-nitrophenylphosphate (D-pNPP) was analyzed. The inhibition was more rapid using octyl-lipase preparations than using covalent preparations, and the covalent preparations were much more sensitive to the reaction medium. The addition of detergent increased the inhibition rate of the covalent preparation while an increase on the ionic strength produced a slowdown of the inhibition rate by D-pNPP for both lipases. The effect of the medium on the activity versus fully soluble substrate (methyl mandelate) was in the same direction. The octyl preparations presented a slight decrease in activity when comparing the results using different concentrations of sodium phosphate buffer (between 0.025 and 1M), while the CNBr preparations suffered drastic drops in its activity at high ionic strength.
The results confirm that the lipases immobilized on octyl agarose presented their open form stabilized while the covalent preparation maintains a closing/opening equilibrium that may be modulated by altering the medium.
•Lipase PS has been immobilized on Accurel in less than 1h with a 70% increase in activity.•Irreversible inhibition with D-pNPP suggested the involvement of the open form of the lipase in the ...immobilization process.•The new biocatalyst is much more active that the commercial one in hydrolysis and synthetic reactions.•The resolution of (±)-1,3,4-tri-O-benzyl-myo-inositol (1) has been optimized using RSM.•The new biocatalyst is more efficient in kinetic resolution of 1 than Novozym 435.
Lipase from Burkholderia cepacia (PS) has been immobilized on Accurel MP 1000, and their performance has been compared to that of the most widely used immobilized commercial preparation of this enzyme. The maximum loading was 18mgprotein/g support, and its thermal and solvent stability was much higher than that of the commercial. PS preparations were used in hydrolysis of triacetin and methyl mandelate, in the esterification of oleic acid and ethanol and in the kinetic resolution of 1,3,4-tri-O-benzyl-myo-inositol (DL-1) using vinylacetate as activated acyl donor. For all reactions studied, PS on Accurel was more active than PS-IM. The conversion in the kinetic resolution of racemic DL-1 was optimized using response surface methodology. Optimal conditions were determined to be 2.0mg/mL of substrate, temperature of 40°C, 2.0mL of vinyl acetate and without water addition. Under these conditions, maximum loaded Accurel-PS preparation permitted to improve the activity in this kinetic resolution compared to the PS commercial preparation by a 55-fold factor, and compared to Novozym 435 (the most active described in literature for this reaction) by a 23-fold factor. The conversion attained was 49.9%±0.3 of conversion and ee of 99% after 24h. The reusability studies showed maintenance of conversion and ee during eight cycles.
One of the major challenges regarding the enzymatic production of biodiesel is the development of more robust, active, and stable immobilized biocatalysts. Thus, the present work aims to develop new ...enzymatic biocatalysts for use in esterification and transesterification reactions. New polymer supports based on poly(styrene‐co‐divinylbenzene) and poly(methyl methacrylate‐co‐divinylbenzene) platforms were synthesized, incorporating distinct functional compounds into the polymer chains (1‐octene, cardanol, and vinyl benzoate). Two distinct polymerization strategies were adopted for support syntheses: (i) Combined suspension and emulsion polymerization process (core‐shell particles); and (ii) Two‐step polymerization reaction comprising a suspension polymerization in presence of porogenic agents, followed by vacuum application (porous and nonporous particles). The obtained particles were employed for immobilization of lipase B from Candida antarctica. The addition of functional compounds resulted in particles with distinct textural properties. Moreover, particles with 91 m2.g−1 (P(MMA‐co‐DVB)/P(MMA‐co‐DVB)) were produced through combined suspension and emulsion polymerization, whilst particles with 154 m2.g−1 (PMMA‐co‐DVB‐co‐VB) were produced through suspension polymerization performed in presence of n‐heptane. Moreover, highly active biocatalysts were produced, leading to esterification conversions above 80%. Thus, based on the performance in esterification and transesterification reactions, the new functional matrices resulted in highly active biocatalysts with good potential for use in biodiesel industry.
Different core–shell polymeric supports, exhibiting different morphologies and composition, were produced through simultaneous suspension and emulsion polymerization, using styrene (S) and ...divinylbenzene (DVB) as co-monomers. Supports composed of polystyrene in both the core and the shell (PS/PS) and the new poly(styrene- co -divinylbenzene) support (PS- co -DVB/PS- co -DVB) were used for the immobilization of three different lipases (from Rhizomucor miehie (RML), from Themomyces lanuginosus (TLL) and the form B from Candida antarctica , (CALB)) and of the phospholipase Lecitase Ultra (LU). The features of the new biocatalysts were evaluated and compared to the properties of commercial biocatalysts (Novozym 435 (CALB), Lipozyme RM IM and Lipozyme TL IM) and biocatalysts prepared by enzyme immobilization onto commercial octyl-agarose, a support reported as very suitable for lipase immobilization. It was shown that protein loading and stability of the biocatalysts prepared with the core–shell supports were higher than the ones obtained with commercial octyl-agarose or the commercial lipase preparations. Besides, it was shown that the biocatalysts prepared with the core–shell supports also presented higher activities than commercial biocatalysts when employing different substrates, encouraging the use of the produced core–shell supports for immobilization of lipases and the development of new applications.
Alfalfa (Medicago sativa L.) leaves and roots grown in the presence and absence of 150ppm polycyclic aromatic hydrocarbons (PAHs) for 40days are washed with distilled water, dried with filter paper ...and immersed in n-hexane for 30s to remove adhered surface PAHs. Subsequently, the samples are mounted on slides with 50% glycerol and viewed under a fluorescence microscope (Leica DM 5000B model with Leica Filter cube A: UV excitation range, excitation filter BP340–380nm, dichromatic mirror 400nm and suppression filter LP 425nm). Images are recorded with a digital camera (Leica DFC 500) under UV and visible light. The location of PAHs is evidenced by the detection of blue autofluorescence, typical of the PAH studied, under UV light (*). This is the first report of PAHs in alfalfa tissues detected by fluorescence microscopy and intense fluorescence in the glandular secreting trichomes (GSTs) of plants grown in contaminated soil. These trichomes, with as as-yet-unknown function, may be sites of PAH conjugation and degradation in alfalfa.
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•First study to detect the presence of PAHs in aerial and root tissues of Medicago sativa L. using fluorescence microscopy.•First study to propose a physiological function of PAH conjugation and/or degradation trichomes (GSTs) of M. sativa L.•Simple, rapid and effective method for detecting PAHs, organic pollutants, derived from oil and oil products in M. sativa L.
Green technologies, such as phytoremediation, are effective for removing organic pollutants derived from oil and oil products, including polycyclic aromatic hydrocarbons (PAHs). Given the increasing popularity of these sustainable remediation techniques, methods based on fluorescence microscopy and multiphoton microscopy for the environmental monitoring of such pollutants have emerged in recent decades as effective tools for phytoremediation studies aimed at understanding the fate of these contaminants in plants. However, little is known about the cellular and molecular mechanisms involved in PAH uptake, responses and degradation by plants. Thus, the present study aimed to detect the location of pyrene, anthracene and phenanthrene using fluorescence microscopy techniques in shoots and roots of Medicago sativa L. (alfalfa) plants grown in artificially contaminated soil (150ppm PAHs) for 40days. Leaflet and root samples were then collected and observed under a fluorescence microscope to detect the presence of PAHs in various tissues. One important finding of the present study was intense fluorescence in the glandular secreting trichomes (GSTs) of plants grown in contaminated soil. These trichomes, with a previously unknown function, may be sites of PAH conjugation and degradation.
This work aimed to evaluate the phytoremediation capacity of the alfalfa cultivar Crioula in soils contaminated with polycyclic aromatic hydrocarbons (PAHs), primary pollutants with mutagenic and ...carcinogenic potential. Alfalfa was grown from seed for 40 days on soil amended with anthracene, pyrene, and phenanthrene. Soil and plant tissue was collected for biometric assay, dry mass analysis, and PAH analysis by liquid chromatography. Increased total PAH concentration was associated with decreases in plant biomass, height, and internode length. The Crioula cultivar had a satisfactory phytoremediation effect, reducing total PAH concentration (300 ppm) in the experimental soil by 85% in 20 days, and by more than 95% in 40 days. The PAH showed a tendency to be removed in the temporal order: phenanthrene before pyrene before anthracene, and the removal ratio was influenced by the initial soil concentration of each PAH.
New nanoparticles are synthesized through emulsion polymerization, using distinct comonomers (styrene, divinylbenzene, glycidyl methacrylate and pentafluorostyrene). Then, for the first time, two ...strategies are adopted to functionalize such nanoparticles using benzylamine and thiophenol: (i) after the manufacture of the nanoparticles; and (ii) in situ during the polymerization reaction. Afterwards, the functionalized nanoparticles are used as nanosupports for immobilization of lipase B from Candida antarctica and the performance of the novel nanobiocatalysts are evaluated. It is shown that the nanoparticles exhibit different properties (specific areas ranging from 34 m2 g−1 to 324 m2 g−1; and contact angles ranging from 29° to 126°), indicating that both procedures can be used to adjust the properties of the polymer supports. Moreover, the nanobiocatalysts are applied successfully in hydrolysis and esterification reactions, exhibiting higher activities than the non‐functionalized biocatalysts. It is also observed that more hydrophilic supports result in more active biocatalysts in hydrolysis (27 ± 1 U g−1) and intermediate hydrophobic matrices conduct to more active biocatalysts in esterification reactions (1564 ± 50 U g−1). It is shown that highly hydrophobic surfaces may cause a significant decrease in the activity of such biocatalysts, probably due to distortions on the enzyme active center and to more intense chemical partitioning effects.
New nanoparticles are synthesized through emulsion polymerization and then functionalized. Afterward, the particles are employed as nanosupports for the synthesis of nanobiocatalysts. High hydrophobic surfaces cause a decrease in the biocatalyst performance, probably due to distortions on the enzyme active center and more intense partitioning effects. These effects are nonexistent or less intense on hydrophilic and less hydrophobic matrices.
Chiral myo‐inositol derivatives play key roles in cell‐signaling processes. Despite the relevance of these compounds, few syntheses of them rely on enantioselective catalytic reactions. Even fewer ...reports describe the use of desymmetrization of myo‐inositol derivatives. In fact, most routes involve resolution by derivatization. Thus, a symmetrical partially protected myo‐inositol derivative, 1,3‐di‐O‐benzyl‐myo‐inositol (1), was used as a substrate in fast lipase‐catalyzed desymmetrization reactions. Among the lipases tested, both Lipozyme RM‐IM and Lipozyme TL‐IM were effective in catalyzing the formation of the chiral acetate l‐(+)‐6‐O‐acetyl‐1,3‐di‐O‐benzyl‐myo‐inositol l‐(+)‐2 with high conversion (98–99 %) and ee (>99 %). Conversely, Novozyme 435 and Lipomod 34P as biocatalysts showed different regioselectivity, leading to the formation of the symmetrical 5‐O‐acetylated product. We were able to reuse TL‐IM lipase seven times without any noticeable decrease in the conversion. Acetate l‐(+)‐2 is a potential precursor of biologically active myo‐inositol derivatives and other relevant materials for cell biology studies.
Depending on the choice of lipase, 1,3‐di‐O‐benzyl‐myo‐inositol (1) either undergoes desymmetrization to form l‐(+)‐6‐O‐acetyl‐1,3‐di‐O‐benzyl‐myo‐inositol (with Lipozyme RM‐IM or Lipozyme TL‐IM), or is transformed into the meso 5‐O‐acetyl derivative 3 (with Novozym 435).
