Glucoamylase immobilized on montmorillonite: influence of nature of binding on surface properties of clay-support and activity of enzyme

Glucoamylase was immobilized on acid activated montmorillonite clay via two different procedures namely adsorption and covalent binding. The immobilized enzymes were characterized by XRD, NMR and N2 adsorption measurements and the activity of immobilized glucoamylase for starch hydrolysis was determined in a batch reactor. XRD shows intercalation of enzyme into the clay matrix during both immobilization procedures. Intercalation occurs via the side chains of the amino acid residues, the entire polypeptide backbone being situated at the periphery of the clay matrix. 27Al NMR studies revealed the different nature of interaction of enzyme with the support for both immobilization techniques. N2 adsorption measurements indicated a sharp drop in surface area and pore volume for the covalently bound glucoamylase that suggested severe pore blockage. Activity studies were performed in a batch reactor. The adsorbed and covalently bound glucoamylase retained 49% and 66% activity of the free enzyme respectively. They showed enhanced pH and thermal stabilities. The immobilized enzymes also followed Michaelis–Menten kinetics. Km was greater than the free enzyme that was attributed to an effect of immobilization. The immobilized preparations demonstrated increased reusability as well as storage stability.

Enhanced pH and thermal stabilities of invertase immobilized on montmorillonite K-10

Invertase was adsorbed onto micro-porous acid-activated montmorillonite clay (K-10) by two procedures, namely adsorption and covalent binding. The immobilized enzymes were characterized by XRD, surface area measurements and 27Al NMR. XRD measurements revealed an expansion of clay layers due to immobilization which suggests that intercalation had taken place. Surface area measurements also support this observation. 27Al NMR showed that interaction of enzyme with tetrahedral and octahedral Al changes with the immobilization procedure. Sucrose hydrolysis was performed in a batch reactor. The immobilized enzymes showed enhanced pH and thermal stabilities. Optimum pH and temperature were found to increase upon immobilization. The effectiveness factor (η) and Michaelis constant (Km) suggest that diffusional resistances play a major role in the reaction. The immobilized invertase could be stored in buffer of pH 5 and 6 at 5 °C without any significant loss in activity for 20 days.

Acid activated montmorillonite: an efficient immobilization support for improving reusability, storage stability and operational stability of enzymes

Three enzymes, α-amylase, glucoamylase and invertase, were immobilized on acid activated montmorillonite K 10 via two independent techniques, adsorption and covalent binding. The immobilized enzymes were characterized by XRD, N2 adsorption measurements and 27Al MAS-NMR spectroscopy. The XRD patterns showed that all enzymes were intercalated into the clay inter-layer space. The entire protein backbone was situated at the periphery of the clay matrix. Intercalation occurred through the side chains of the amino acid residues. A decrease in surface area and pore volume upon immobilization supported this observation. The extent of intercalation was greater for the covalently bound systems. NMR data showed that tetrahedral Al species were involved during enzyme adsorption whereas octahedral Al was involved during covalent binding. The immobilized enzymes demonstrated enhanced storage stability. While the free enzymes lost all activity within a period of 10 days, the immobilized forms retained appreciable activity even after 30 days of storage. Reusability also improved upon immobilization. Here again, covalently bound enzymes exhibited better characteristics than their adsorbed counterparts. The immobilized enzymes could be successfully used continuously in the packed bed reactor for about 96 hours without much loss in activity. Immobilized glucoamylase demonstrated the best results.

Fixed bed reactor performance of invertase immobilized on montmorillonite

Invertase was immobilized on acid activated montmorillonite via two independent procedures, adsorption and covalent binding. The immobilized enzymes were characterized by XRD, NMR and N2 adsorption measurements and their activity was tested in a fixed bed reactor. XRD revealed that the enzyme was situated on the periphery of the clay and the side chains of different amino acid residues were involved in intercalation with the clay matrix. NMR demonstrated that tetrahedral Al was linked to the enzyme during adsorption and the octahedral Al was involved during covalent binding. Secondary interaction of the enzyme with Al was also observed. N2 adsorption studies showed that covalent binding of enzymes caused pore blockage since the highly polymeric species were located at the pore entrance. The fixed bed reactor proved to be efficient for the immobilized invertase. The optimum pH and pH stability improved upon immobilization. The kinetic parameters calculated also showed an enhanced efficiency of the immobilized systems. They could be used continuously for long period. Covalently bound invertase demonstrated greater operational stability.

Invertase immobilised on montmorillonite: reusability enhancement and reduction in leaching

Invertase was immobilised on microporous montmorillonite K-10 via adsorption and covalent binding. The immobilised enzymes were tested for sucrose hydrolysis activity in a batch reactor. Km for immobilised systems was greater than free enzyme. The immobilised forms could be reused for 15 continuous cycles without any loss in activity. After 25 cycles, 85% initial activity was retained. A study on leaching of enzymes showed that 100% enzyme was retained even after 15 cycles of reuse. Leaching increased with reaction temperature. Covalent binding resisted leaching even at temperatures of 70 °C.

Glucoamylase immobilized on montmorillonite: Synthesis, characterization and starch hydrolysis activity in a fixed bed reactor

Glucoamylase from Aspergillus Niger was immobilized on montmorillonite clay (K-10) by two procedures, adsorption and covalent binding. The immobilized enzymes were characterized using XRD, surface area measurements and 27Al MAS NMR and the activity of the immobilized enzymes for starch hydrolysis was tested in a fixed bed reactor (FBR). XRD shows that enzyme intercalates into the inter-lamellar space of the clay matrix with a layer expansion up to 2.25 nm. Covalently bound glucoamylase demonstrates a sharp decrease in surface area and pore volume that suggests binding of the enzyme at the pore entrance. NMR studies reveal the involvement of octahedral and tetrahedral Al during immobilization. The performance characteristics in FBR were evaluated. Effectiveness factor (η) for FBR is greater than unity demonstrating that activity of enzyme is more than that of the free enzyme. The Michaelis constant (Km) for covalently bound glucoamylase was lower than that for free enzyme, i.e., the affinity for substrate improves upon immobilization. This shows that diffusional effects are completely eliminated in the FBR. Both immobilized systems showed almost 100% initial activity after 96 h of continuous operation. Covalent binding demonstrated better operational stability.

Thermal characterisation of dye-intercalated K-10 montmorillonite ceramics using photoacoustic technique

Thermal diffusivity (TD) measurements were performed on some industrially important dyes – auramine O (AO), malachite green and methylene blue (MB) – adsorbed K-10 montmorillonites using photoacoustic method. The TD value for the dye-adsorbed clay mineral was observed to change with a variation in dye concentration. The contribution of the dye towards TD was also determined. The repeatedly adsorbed samples with MB and AO exhibited a lower TD than the single-adsorbed samples. TD values of sintered MB samples were also obtained experimentally. These sintered samples exhibit a higher TD, although they show a trend similar to that of non-sintered pellets. A variation in dye concentration and sintering temperature can be used for tuning the TD value of the clay mineral to the desired level.

Structural tuning of Montmorillonite clays by pillaring: Designing of shape selective solid acid catalysts and PANI - Montmorillonite nanocomposites

Green chemistry boots eco-friendly,natural clays as catalysts in the chemical as well as in the pharmaceutical industry.Industry demands thermal stability,mechanical strength etc for the catalyst and there the modification methods becomes important.Pillaring tunes clays as efficient catalytic templates for shape selective organic synthesis.Here pillared clays are used as promising alternatives for the environmentally hazardous homogeneous catalysts in some industrially important Friedel-Crafts alkylation reactions of arenes with lower alchohols and higher olefins.The layer structure is enhanced upon pillaring and allows the nanocomposite formation with polyaniline to develop today’s nanoscale diameter devices.Present work gives an entry of pillared clays to the world of conducting composite nanofibers.

Thermal characterisation of dye-intercalated K-10 montmorillonite ceramics using photoacoustic technique

Thermal diffusivity (TD) measurements were performed on some industrially important dyes – auramine O (AO), malachite green and methylene blue (MB) – adsorbed K-10 montmorillonites using photoacoustic method. The TD value for the dye-adsorbed clay mineral was observed to change with a variation in dye concentration. The contribution of the dye towards TD was also determined. The repeatedly adsorbed samples with MB and AO exhibited a lower TD than the single-adsorbed samples. TD values of sintered MB samples were also obtained experimentally. These sintered samples exhibit a higher TD, although they show a trend similar to that of non-sintered pellets. A variation in dye concentration and sintering temperature can be used for tuning the TD value of the clay mineral to the desired level

Continuous Production of Extracellular L-Glutaminase by Ca-Alginate-Immobilized Marine Beauveria bassiana BTMF S-10 in Packed-Bed Reactor

L-Glutamine amidohydrolase (L-glutaminase, EC 3.5.1.2) is a therapeutically and industrially important enzyme. Because it is a potent antileukemic agent and a flavor-enhancing agent used in the food industry, many researchers have focused their attention on L-glutaminase. In this article, we report the continuous production of extracellular L-glutaminase by the marine fungus Beauveria bassiana BTMF S-10 in a packed-bed reactor. Parameters influencing bead production and performance under batch mode were optimized in the order-support (Na-alginate) concentration, concentration of CaCl2 for bead preparation, curing time of beads, spore inoculum concentration, activation time, initial pH of enzyme production medium, temperature of incubation, and retention time. Parameters optimized under batch mode for L-glutaminase production were incorporated into the continuous production studies. Beads with 12 × 108 spores/g of beads were activated in a solution of 1% glutamine in seawater for 15 h, and the activated beads were packed into a packed-bed reactor. Enzyme production medium (pH 9.0) was pumped through the bed, and the effluent was collected from the top of the column. The effect of flow rate of the medium, substrate concentration, aeration, and bed height on continuous production of L-glutaminase was studied. Production was monitored for 5 h in each case, and the volumetric productivity was calculated. Under the optimized conditions for continuous production, the reactor gave a volumetric productivity of 4.048 U/(mL·h), which indicates that continuous production of the enzyme by Ca-alginate-immobilizedspores is well suited for B. bassiana and results in a higher yield of enzyme within a shorter time. The results indicate the scope of utilizing immobilized B. bassiana for continuous commercial production of L-glutaminase

Raman and FTIR studies of the structural aspects of Nasicon-type crystals; AFeTi(PO4)3 [A ¼ Ca, Cd]

Raman and FTIR spectra of CaFeTi(PO4)3 and CdFeTi(PO4)3 are recorded and analyzed. The observed bands are assigned in terms of vibrations of TiO6 octahedra and PO4 tetrahedra. The symmetry of TiO6 octrahedra and PO4 tetrahedra is lowered from their free ion symmetry. The presence of Fe3+ ion disrupts the Ti–O–P–O–Ti chain and leads to the distortion of TiO6 octrahedra and PO4 tetrahedra. The PO4 3 tetrahedra in both crystals are linearly distorted. The covalency bonding factor of PO4 3 polyanion of both the crystals are calculated from the Raman spectra and compared to that of other Nasicon-type systems. The numerical values of covalency bonding factor indicates that there is a reduction in redox energy and cell voltage and is attributed to strong covalency of PO4 3 polyanionin

FT-IR and FT-Raman study of Nasicon type phosphates, ASnFe(PO4)3 [A=Na2, Ca, Cd]

FT-Raman and FT-IR spectra of ASnFe(PO4)3 [A=Na2, Ca, Cd] were recorded and analyzed. The bands were assigned in terms of the vibrational group frequencies of SnO6 octahedral and PO4 tetrahedral. The spectral analysis shows that the symmetry of corner shared octahedral (SnO6) and the tetrahedral (PO4) are lowered from their free ion symmetry state. The presence of Fe3+ ions disrupts the S–N–O–S–N chain in the structure. This causes distortion of SnO6 and PO4 in the structure of all the compounds. Also it is seen that there are two distinct PO4 tetrahedra of different P–O bond lengths. One of these tetrahedra is linearly distorted in all the title compounds. The PO4 frequencies and bond lengths are calculated theoretically and are in agreement with the experimental values. The presence of PO4 polyanion in the structure can reduce the redox energy and hence reduce the metal oxygen covalency strength in the structure