Denaturant Induced Equilibrium Unfolding and Conformational Transitional Studies of Germinated Fenugreek ß-Amylase Revealed Molten Globule like State at Low pH.

BACKGROUND: ß-Amylase (EC 3.2.1.2) is a maltogenic enzyme, which releases ß-maltose from the non-reducing end of the substrates. The enzyme plays important roles for the production of vaccine, maltiol and maltose rich syrups. Apart from these applications the enzyme protects cells from abiotic as well as oxidative damage. The enzyme is ßwell characterized in ßplants and microbes and crystal structures of ß-amylases ßhave been ßobtained from sweet potato, soybean and Bacillus cereus. OBJECTIVE: Find out correlation between structural and functional stability induced by change in pH, temperature and chaotropes. METHODS: Activity, intrinsic fluorescence, extrinsic fluorescence, near- and far- ultraviolet circular dichroism spectroscopic measurements were performed. RESULTS: Peaks about 208 nm and 222 nm obtained by near-ultraviolet circular dichroism correspond to α-helix whereas peak at 215 nm shows presence of ß-sheet. At pH 2.0, absence of tertiary structures, exposed of hydrophobic regions and presence of substantial secondary structures, revealed the existence of molten globule like state. Temperature induced denaturation studies showed that the enzyme was stable up to 75 ºC and the process was found to be irreversible in nature. Chaotropes dependent equilibrium unfolding studies revealed that at low concentration of chaotropes, ellipticity and intrinsic fluorescence ßintensity were ßdecreased ßwhereas ßenzymatic activity remained unchanged, which revealed fenugreek ß-amylase is multi-domains enzyme and catalytic ßdomain ßis more ßstable compare to non-catalytic domain. Moreover, the transition was sigmoidal and non-coincidental. CONCLUSION: Results indicate the probable existence of intermediate states that might perform significant role in physiological process and biotechnological applications.

Nanoparticles decorated carbon nanotubes as novel matrix: A comparative study of influences of immobilization on the catalytic properties of Lensculinarisß-galactosidase (Lcß-gal).

In the present study, Multiwalled carbon nanotubes (MWCNT) decorated with two different nanoparticles namely tungsten disulfide (WS2) and tin oxide (SnO2), nanocomposites (NCs) were synthesized via hydrothermal method. Spectroscopic studies showed that both synthesized NCs possess nearly same functional groups but MWCNT-SnO2 NCs are rich in O-functional group. Microscopic studies revealed that both NCs have different morphological microstructure. Lens culinaris ß-galactosidase (Lcß-gal) was immobilized using glutaraldehyde cross-linker resulted in immobilization efficiency of 91.5% and 88% with MWCNT-WS2 and MWCNT-SnO2 NCs, respectively. Remarkable increase in rate of hydrolysis of whey lactose has been observed with both NCs i.e. Lcß-gal immobilized MWCNT-WS2 hydrolyzes the 97% whey lactose in 1.5 h while MWCNT-SnO2 showed maximum 92% of whey hydrolysis in 2 h at optimum conditions. Both nanobiocatalyst could serve as a promising candidates for dairy industries and would offer a potential platform for enzyme based biosensor fabrication.

Immobilization of fenugreek ß-amylase onto functionalized tungsten disulfide nanoparticles using response surface methodology: Its characterization and interaction with maltose and sucrose.

In this communication, fenugreek ß-amylase was immobilized onto functionalized tungsten disulfide nanoparticles through cross-linker glutaraldehyde and successful immobilization was confirmed by SEM, AFM and FTIR spectroscopy. To make the process economical and efficient, optimization of independent variables was carried out using Box-Behnken design of response surface methodology. Approximately similar predicted (85.6%) and experimental (84.2%) immobilization efficiency revealed that the model is suitable for design of space. Optimum temperature was calculated to be 60 °C. After immobilization, an increased Km (2.12 times) and a decreased Vmax (0.58 times), indicated inaccessibility of active site residues to the substrate. The immobilized enzyme retained 77% relative activity after 10 uses whereas 40% residual activity was obtained after 120 days. An increased half-life with concomitantly decreased kinetic rate constant revealed that the immobilized enzyme is more stable at a higher temperature and the process followed first-order kinetics (R2 > 0.93). The limit of detection for maltose and sucrose fluorescence biosensor was found to be 0.052 and 0.096 mM, respectively. Thermodynamic parameters such as changes in Gibbs free energy (ΔG < 0), enthalpy (ΔH > 0) and entropy (ΔS >0) revealed that the process is spontaneous and endothermic, driven by hydrophobic interactions. Thermo-stability data at higher temperature for the immobilized enzyme makes it a suitable candidate for industrial applications in the production of maltose in food and pharmaceutical industries. Furthermore, fluorescence biosensor could be used to detect and quantify maltose and sucrose to maintain the quality of industrial products.

Immobilization of fenugreek ß-amylase onto functionalized graphene quantum dots (GQDs) using Box-Behnken design: Its biochemical, thermodynamic and kinetic studies.

ß-Amylase was immobilized onto GQDs using 3-aminopropyltriethoxysilane and glutaraldehyde. Optimization was carried out by Box-Behnken design and binding was confirmed by SEM, AFM, FTIR and fluorescence microscopy. Predicted optimum immobilization efficiency (88.64%) was very close to actual (87.98%), which confirmed the success of the immobilization process. The immobilized enzyme showed maximum activity at pH 5.0 and 57 °C, whereas Km and Vmax were found to be 6.40 mg/mL and 714.28 µmol/min/mg, respectively. The enzyme retained 75% activity after 12 uses at 30 °C. Increased values of ΔG° ΔH°, half-life and activation energy of the enzyme inactivation (ΔEd) revealed that thermo-stability increases after immobilization and the process followed first-order kinetics (r2 > 0.96). The activation energy of catalysis (ΔEa) and ΔEd for immobilized enzyme were 22.58 and 158.99 ± 1.10 kJ/mol, respectively which revealed that denaturation of the enzyme requires a higher amount of energy rather than catalysis. Thermodynamic and fluorescence spectroscopic studies revealed that the process is non-spontaneous (ΔG > 0) and endothermic (ΔH > 0) and occurred through protein unfolding rather than aggregation (ΔS > 0). Thus increase in thermo-stability of immobilized fenugreek ß-amylase and non-toxic nature of GQDs could be exploited for maltose production in beverage, food and pharmaceutical industries.

Carbon nanotubes molybdenum disulfide 3D nanocomposite as novel nanoscaffolds to immobilize Lens culinaris ß-galactosidase (Lsbgal): Robust stability, reusability, and effective bioconversion of lactose in whey.

Multiwalled carbon nanotubes molybdenum disulfide 3D nanocomposite (MWCNT-MoS2 NC) was successfully synthesized via eco-friendly hydrothermal method. The microstructural characterization of synthesized nanocomposite was carried out using different spectroscopic and microscopic techniques. Nanocomposite was activated using glutaraldehyde chemistry and used as a platform to immobilize Lens culinaris ß-galactosidase (Lsbgal) which resulted in 93% of immobilization efficiency. Attachment of Lsbgal onto nanocomposite was confirmed by AFM, FE-SEM, FTIR, and CLSM. The nanobiocatalyst showed broadening in operational pH and temperature working range. Remarkable increase in thermal stability was observed as compared to soluble enzyme. Nanobiocatalyst showed outstanding increase in storage stability, retained 92% of residual activity over a period of 8 months. This offers good reusability as it retained ∼50% residual activity up to 21 reuses and exhibited higher rate of lactose hydrolysis in whey. MWCNT-MoS2 NC conjugated to biomolecules can serve as a potential platform for fabrication of lactose biosensor.

Biochemical and thermodynamic characterization of de novo synthesized ß-amylase from fenugreek.

ß-Amylase has been de novo synthesized from germinating fenugreek seeds. Enzyme has been isolated and purified from 36 h germinated seeds with 226-fold purification and specific activity of 763 U/mg. Homogeneity of the purified ß-amylase has been confirmed with size-exclusion chromatography, SDS-PAGE and MALDI MS/MS analysis. The isoelectric point, optimum pH and temperature of the enzyme were found to be pH 5.2, 5.7 and 57 °C, respectively. The enzyme was specific for soluble starch with Km and Vmax of 2.4 mg/mL and 833.3 U/mg, respectively. Maltose was found to be competitive inhibitor of the enzyme with inhibition constant (Ki) of 14 mM. However, metallic ions like Ag+ and Hg2+ were found to be non-competitive inhibitors of the enzyme. Thermodynamic parameters like Gibbs free energy (ΔG), enthalpy (ΔH) and entropy (ΔS) changes have further revealed that thermal denaturation of the enzyme has followed first-order with the enzyme unfolding rather an aggregation with the process being irreversible. The activation energy of ß-amylase during thermal activation and denaturation were 27.5 kJ/mol and 145.23 kJ/mol, respectively at R2 > 0.92. Thus, the enzyme was stable even at higher temperature with ability of undergoing catalysis making it commercially exploitable, particularly in food and pharmaceutical industries.