Nanoclays for Lipase Immobilization: Biocatalyst Characterization and Activity in Polyester Synthesis.
ABSTRACT: The immobilization of Candida antarctica lipase B (CALB) was performed by physical adsorption on both neat and organo-modified forms of sepiolite and montmorillonite. The influence of different parameters, e.g., solvent, enzyme loading, cross-linking, and type of clay support, on immobilization efficiency and catalyst hydrolytic activity has been investigated. The highest hydrolytic activities were obtained for CALB immobilized on organo-modified clay minerals, highlighting the beneficial effect of organo-modification. The esterification activity of these CALB/organoclay catalysts was also tested in the ring-opening polymerization of ε-caprolactone. The polymerization kinetics observed for clay-immobilized catalysts confirmed that CALB adsorbed on organo-modified montmorillonite (CALB/MMTMOD) was the highest-performing catalytic system.
Project description:The photo-oxidative degradation processes of bio-based PA11 nanocomposites containing montmorillonite (MMT) and the organo-modified Cloisite®30B were investigated to discriminate the influence of organo-modified components on the polymer durability. Indeed, despite the extensive studies reported, there are still ambiguous points to be clarified from the chemical point of view. To this aim, UV-aged materials were analyzed by Size Exclusion Chromatography (SEC), Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) and Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). This enabled determining changes in both chemical structure and Molar Masses (MMs) induced by light, heat and oxygen exposure. The addition of organo-modified nanoclays strongly affected the PA11 light durability, triggering the macromolecular chains scission due to the typical ?H, Norrish I and II mechanisms. However, the main contribution in boosting the photo-oxidative degradation is induced by iron impurities contained into the clays. Conversely, thermo-oxidation process performed at 215 °C was unambiguously affected by the presence of the organo-modifiers, whose presence determined an enhancement of crosslinking reactions.
Project description:Fusion tags - amino acid sequences that are genetically coded to be expressed as attached moieties to a protein - have the potential to enhance the activity of native enzyme, enable specific purification of the enzyme, and promote simple and efficient immobilization of enzymes onto material supports. In this work, we demonstrate the effect of a Strep-tag II fusion tag on the properties of free and immobilized lipase B from Candida antarctica (CALB). The gene encoding the mature portion of CALB was codon-optimized and cloned in pASG-IBA2 plasmid for expression in E. coli. Purified recombinant Strep-tag II CALB was immobilized to Strep-Tactin based support through affinity binding, and the immobilized and free Strep-tag II CALB were compared to a commercial CALB. Following modification, the enzyme could be selectively purified from culture media with no observable non-specific binding. The catalytic efficiency of the purified fusion-tagged enzyme was significantly greater than that of the commercial CALB in its free form. Immobilization of the fusion-tagged enzyme to Strep-Tactin modified crosslinked agarose support yielded a catalytically active enzyme; however, the kcat of the immobilized enzyme was significantly reduced compared to the free tagged enzyme. This work indicates that a C-terminus Strep-tag II fusion tag may be employed to improve the catalytic efficiency of free CALB, but may not be suitable for immobilized applications that employ binding of the enzyme to a Strep-Tactin-modified support.
Project description:Direct C-O hydrogenolysis of bioglycerine to produce 1,3-propanediol selectively is a vital technology that can expand the scope of biodiesel industry and green chemical production from biomass. Herein we report sulphuric acid-activated montmorillonite clay supported platinum nanoparticles as highly effective solid acid catalysts for the selective production of 1,3-propanediol from glycerol. The catalytic performances of the catalysts were investigated in the hydrogenolysis of glycerol with a fixed bed reactor under ambient pressure. The results were found promising and showed that the activation of montmorillonite by sulphuric acid incorporated Brønsted acidity in the catalyst and significantly improved the selectivity to 1,3-propanediol. The catalytic performance of different platinum loaded catalysts was examined and 2?wt% Pt/S-MMT catalyst presented superior activity among others validating 62% 1,3-propanediol selectivity at 94% glycerol conversion. The catalytic activity of 2Pt/S-MMT was systematically investigated under varying reaction parameters including reaction temperature, hydrogen flow rate, glycerol concentration, weight hourly space velocity, and contact time to derive the optimum conditions for the reaction. The catalyst stability, reusability and structure-activity correlation were also elucidated. The high performance of the catalyst could be ascribed to well disperse Pt nanoparticles immobilized on acid-activated montmorillonite, wider pore-structure and appropriate acid sites of the catalyst.
Project description:Poly(lactic acid) (PLA), a well-known biodegradable and compostable polymer, was used in this study as a model system to determine if the addition of nanoclays affects its biodegradation in simulated composting conditions and whether the nanoclays impact the microbial population in a compost environment. Three different nanoclays were studied due to their different surface characteristics but similar chemistry: organo-modified montmorillonite (OMMT), Halloysite nanotubes (HNT), and Laponite® RD (LRD). Additionally, the organo-modifier of MMT, methyl, tallow, bis-2-hydroxyethyl, quaternary ammonium (QAC), was studied. PLA and PLA bio-nanocomposite (BNC) films were produced, characterized, and used for biodegradation evaluation with an in-house built direct measurement respirometer (DMR) following the analysis of evolved CO2 approach. A biofilm formation essay and scanning electron microscopy were used to evaluate microbial attachment on the surface of PLA and BNCs. The results obtained from four different biodegradation tests with PLA and its BNCs showed a significantly higher mineralization of the films containing nanoclay in comparison to the pristine PLA during the first three to four weeks of testing, mainly attributed to the reduction in the PLA lag time. The effect of the nanoclays on the initial molecular weight during processing played a crucial role in the evolution of CO2. PLA-LRD5 had the greatest microbial attachment on the surface as confirmed by the biofilm test and the SEM micrographs, while PLA-QAC0.4 had the lowest biofilm formation that may be attributed to the inhibitory effect also found during the biodegradation test when the QAC was tested by itself.
Project description:Montmorillonite is a type of nanoclay that originates from the clay fraction of the soil and is incorporated into polymers to form nanocomposites with enhanced mechanical strength, barrier, and flammability properties used for food packaging, automotive, and medical devices. However, with implementation in such consumer applications, the interaction of montmorillonite-based composites or derived byproducts with biological systems needs to be investigated.Herein we examined the potential of Cloisite Na+ (pristine) and Cloisite 30B (organically modified montmorillonite nanoclay) and their thermally degraded byproducts' to induce toxicity in model human lung epithelial cells. The experimental set-up mimicked biological exposure in manufacturing and disposal areas and employed cellular treatments with occupationally relevant doses of nanoclays previously characterized using spectroscopical and microscopical approaches. For nanoclay-cellular interactions and for cellular analyses respectively, biosensorial-based analytical platforms were used, with induced cellular changes being confirmed via live cell counts, viability assays, and cell imaging.Our analysis of byproducts' chemical and physical properties revealed both structural and functional changes. Real-time high throughput analyses of exposed cellular systems confirmed that nanoclay induced significant toxic effects, with Cloisite 30B showing time-dependent decreases in live cell count and cellular viability relative to control and pristine nanoclay, respectively. Byproducts produced less toxic effects; all treatments caused alterations in the cell morphology upon exposure.Our morphological, behavioral, and viability cellular changes show that nanoclays have the potential to produce toxic effects when used both in manufacturing or disposal environments.The reported toxicological mechanisms prove the extensibility of a biosensorial-based platform for cellular behavior analysis upon treatment with a variety of nanomaterials.
Project description:The design of interfaces that selectively react with molecules to transform them into compounds of industrial interest is an emerging area of research. An example of such reactions is the hydrolytic conversion of ester-based molecules to lipids and alcohols, which is of interest to the food, and pharmaceutical industries. In this study, a functional bio-interfaced layer was designed to hydrolyze 4-nitrophenyl acetate (pNPA) and Ricinus Communis (castor) oil rich in triglycerides using lipase b from Candida antarctica (CALB, EC 188.8.131.52). The attachment of CALB was performed via non-covalent immobilization over a polymer film of vertically aligned cylinders that resulted from the self-assembly of the di-block copolymer polystyrene-block-poly(4-vinyl pyridine) (PS-b-P4VP). This polymer-lipase model will serve as the groundwork for the design of further bioactive layers for separation applications requiring similar hydrolytic processes. Results from the fabricated functional bio-interfaced material include cylinders with featured pore size of 19?nm, d spacing of 34?nm, and ca. 40?nm of thickness. The polymer-enzyme layers were physically characterized using AFM, XPS, and FTIR. The immobilized enzyme was able to retain 91% of the initial enzymatic activity when using 4-nitrophenyl acetate (pNPA) and 78% when exposed to triglycerides from castor oil.
Project description:Sorption of organic molecules to mineral surfaces is an important control upon the aquatic carbon (C) cycle. Organo-mineral interactions are known to regulate the transport and burial of C within inland waters, yet the mechanisms that underlie these processes are poorly constrained. Streamwater contains a complex and dynamic mix of dissolved organic compounds that coexists with a range of organic and inorganic particles and microorganisms. To test how microbial metabolism and organo-mineral complexation alter amino acid and organic carbon fluxes we experimented with (13)C-labelled amino acids and two common clay minerals (kaolinite and montmorillonite). The addition of (13)C-labelled amino acids stimulated increased microbial activity. Amino acids were preferentially mineralized by the microbial community, concomitant with the leaching of other (non-labelled) dissolved organic molecules that were removed from solution by clay-mediated processes. We propose that microbial processes mediate the formation of organo-mineral particles in streamwater, with potential implications for the biochemical composition of organic matter transported through and buried within fluvial environments.
Project description:The enzymatic ring-opening polymerization of lactones is a method of increasing interest for the synthesis of biodegradable and biocompatible polymers. In the past it was shown that immobilization of Candida antarctica lipase B (CaLB) and the reaction medium play an important role in the polymerization ability especially of medium ring size lactones like ε-caprolactone (ε-CL). We investigated a route for the preparation of compartmentalized microgels based on poly(glycidol) in which CaLB was immobilized to increase its esterification ability. To find the ideal environment for CaLB, we investigated the acceptable water concentration and the accessibility for the monomer in model polymerizations in toluene and analyzed the obtained oligomers/polymers by NMR and SEC. We observed a sufficient accessibility for ε-CL to a toluene like hydrophobic phase imitating a hydrophobic microgel. Comparing free CaLB and Novozym® 435 we found that not the monomer concentration but rather the solubility of the enzyme, as well as the water concentration, strongly influences the equilibrium of esterification and hydrolysis. On the basis of these investigations, microgels of different polarity were prepared and successfully loaded with CaLB by physical entrapment. By comparison of immobilized and free CaLB, we demonstrated an effect of the hydrophobicity of the microenvironment of CaLB on its enzymatic activity.
Project description:In this study, an immobilization strategy for magnetic cross-linking enzyme aggregates of lipase B from Candida antarctica (CALB) was developed and investigated. Magnetic particles were prepared by conventional co-precipitation. The magnetic nanoparticles were modified with 3-aminopropyltriethoxysilane (APTES) to obtain surface amino-functionalized magnetic nanoparticles (APTES⁻Fe₃O₄) as immobilization materials. Glutaraldehyde was used as a crosslinker to covalently bind CALB to APTES⁻Fe₃O₄. The optimal conditions of immobilization of lipase and resolution of racemic 1-phenylethanol were investigated. Under optimal conditions, esters could be obtained with conversion of 50%, enantiomeric excess of product (eep) > 99%, enantiomeric excess of substrate (ees) > 99%, and enantiomeric ratio (E) > 1000. The magnetic CALB CLEAs were successfully used for enzymatic kinetic resolution of fifteen secondary alcohols. Compared with Novozym 435, the magnetic CALB CLEAs exhibited a better enantioselectivity for most substrates. The conversion was still greater than 49% after the magnetic CALB CLEAs had been reused 10 times in a 48 h reaction cycle; both ees and eep were close to 99%. Furthermore, there was little decrease in catalytic activity and enantioselectivity after being stored at -20 °C for 90 days.
Project description:Enzyme-catalyzed ring-opening polymerization of lactones is a method of increasing interest for the synthesis of polyesters. In the present work, we investigated which changes in the structure of Candida antarctica lipase B (CaLB) shift the catalytic equilibrium between esterification and hydrolysis towards polymerization. Therefore, we present two concepts: (i) removing the glycosylation of CaLB to increase the surface hydrophobicity; and (ii) introducing a hydrophobic lid adapted from Pseudomonas cepacia lipase (PsCL) to enhance the interaction of a growing polymer chain to the elongated lid helix. The deglycosylated CaLB (CaLB-degl) was successfully generated by site-saturation mutagenesis of asparagine 74. Furthermore, computational modeling showed that the introduction of a lid helix at position Ala148 was structurally feasible and the geometry of the active site remained intact. Via overlap extension PCR the lid was successfully inserted, and the variant was produced in large scale in Pichia pastoris with glycosylation (CaLB-lid) and without (CaLB-degl-lid). While the lid variants show a minor positive effect on the polymerization activity, CaLB-degl showed a clearly reduced hydrolytic and enhanced polymerization activity. Immobilization in a hydrophobic polyglycidol-based microgel intensified this effect such that a higher polymerization activity was achieved, compared to the "gold standard" Novozym® 435.