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.

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.

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.

Physiological responses of fenugreek seedlings and plants treated with cadmium.

The bioaccumulation efficiency of cadmium (Cd) by fenugreek (Trigonella foenum-graecum) was examined using different concentrations of CdCl2. The germination rate was similar to control except at 10 mM Cd. However, early seedling growth was quite sensitive to the metal from the lowest Cd level. Accordingly, amylase activity was reduced substantially on treatment of seeds with 0.5, 1, and 10 mM Cd. Cadmium also affected various other plant growth parameters. Its accumulation was markedly lower in shoots as compared to roots, reducing root biomass by almost 50 %. Plants treated with 1 and 5 mM Cd presented chlorosis due to a significant reduction in chlorophyll b especially. Furthermore, at Cd concentrations greater than 0.1 mM, plants showed several signs of oxidative stress; an enhancement in root hydrogen peroxide (H2O2) level and in shoot malondialdehyde (MDA) content was observed. Conversely, antioxidant enzyme activities (superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT)) increased in various plant parts. Likewise, total phenolic and flavonoid contents reached their highest values in the 0.5 mM Cd treatment, consistent with their roles in quenching low concentrations of reactive oxygen species (ROS). Consequently, maintaining oxidant and antioxidant balance may permit fenugreek to hyperaccumulate Cd and allow it to be employed in extremely Cd polluted soils for detoxification purposes.

Immobilisation of Fenugreek ß-amylase on chitosan/PVP blend and chitosan coated PVC beads: a comparative study.

A Box-Behnken design of Response Surface Methodology (RSM) was utilised for optimisation of parameters affecting immobilisation of Fenugreek ß-amylase on chitosan coated PVC (polyvinyl chloride) beads and beads made from chitosan/PVP (polyvinylpyrrolidone) blend, which resulted in 85.2% and 81% immobilisation efficiency, respectively. Immobilisation resulted in shift of pH optima while the optimum temperature remained unaffected. Enhancement in thermal stability of the enzyme was observed on conjugation with both the matrices. The immobilised enzyme appeared suitable for industrial applications due to the non-toxic nature of chosen matrices, ease of immobilisation procedure, enhanced stability and reusability with retention of 72% and 60% residual activity after 10 uses for the enzyme immobilised on chitosan coated PVC beads and on the beads of chitosan/PVP blend, respectively.

Functionalized graphene sheets as immobilization matrix for Fenugreek ß-amylase: enzyme kinetics and stability studies.

ß-Amylase finds application in food and pharmaceutical industries. Functionalized graphene sheets were customised as a matrix for covalent immobilization of Fenugreek ß-amylase using glutaraldehyde as a cross-linker. The factors affecting the process were optimized using Response Surface Methodology based Box-Behnken design of experiment which resulted in 84% immobilization efficiency. Scanning and Transmission Electron Microscopy (SEM, TEM) and Fourier Tansform Infrared (FTIR) spectroscopy were employed for the purpose of characterization of attachment of enzyme on the graphene. The enzyme kinetic studies were carried out for obtaining best catalytic performance and enhanced reusability. Optimum temperature remained unchanged, whereas optimum pH showed shift towards acidic range for immobilized enzyme. Increase in thermal stability of immobilized enzyme and non-toxic nature of functionalized graphene can be exploited for production of maltose in food and pharmaceutical industries.

Identification of active site residues of Fenugreek ß-amylase: chemical modification and in silico approach.

The amino acid sequence of Fenugreek ß-amylase is not available in protein data bank. Therefore, an attempt has been made to identify the catalytic amino acid residues of enzyme by employing studies of pH dependence of enzyme catalysis, chemical modification and bioinformatics. Treatment of purified Fenugreek ß-amylase with EDAC in presence of glycine methyl ester and sulfhydryl group specific reagents (IAA, NEM and p-CMB), followed a pseudo first-order kinetics and resulted in effective inactivation of enzyme. The reaction with EDAC in presence of NTEE (3-nitro-l-tyrosine ethylester) resulted into modification of two carboxyl groups per molecule of enzyme and presence of one accessible sulfhydryl group at the active site, per molecule of enzyme was ascertained by titration with DTNB. The above results were supported by the prevention of inactivation of enzyme in presence of substrate. Based on MALDI-TOF analysis of purified Fenugreek ß-amylase and MASCOT search, ß-amylase of Medicago sativa was found to be the best match. To further confirm the amino acid involved in catalysis, homology modelling of ß-amylase of M. sativa was performed. The sequence alignment, superimposition of template and target models, along with study of interactions involved in docking of sucrose and maltose at the active site, led to identification of Glu187, Glu381 and Cys344 as active site residues.

Β-amylase from starchless seeds of Trigonella foenum-graecum and its localization in germinating seeds.

Fenugreek (Trigonella foenum-graecum) seeds do not contain starch as carbohydrate reserve. Synthesis of starch is initiated after germination. A ß-amylase from ungerminated fenugreek seeds was purified to apparent electrophoretic homogeneity. The enzyme was purified 210 fold with specific activity of 732.59 units/mg. Mr of the denatured enzyme as determined from SDS-PAGE was 58 kD while that of native enzyme calculated from size exclusion chromatography was 56 kD. Furthermore, its identity was confirmed to be ß-amylase from MALDI-TOF analysis. The optimum pH and temperature was found to be 5.0 and 50°C, respectively. Starch was hydrolyzed at highest rate and enzyme showed a Km of 1.58 mg/mL with it. Antibodies against purified Fenugreek ß-amylase were generated in rabbits. These antibodies were used for localization of enzyme in the cotyledon during different stages of germination using fluorescence and confocal microscopy. Fenugreek ß-amylase was found to be the major starch degrading enzyme depending on the high amount of enzyme present as compared to α-amylase and also its localization at the periphery of amyloplasts. A new finding in terms of its association with protophloem was observed. Thus, this enzyme appears to be important for germination of seeds.

Arbuscular mycorrhizal symbiosis modulates antioxidant response in salt-stressed Trigonella foenum-graecum plants.

An experiment was conducted to evaluate the influence of Glomus intraradices colonization on the activity of antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT), peroxidase (PX), ascorbate peroxidase (APX), and glutathione reductase (GR)] and the accumulation of nonenzymatic antioxidants (ascorbic acid, α-tocopherol, glutathione, and carotenoids) in roots and leaves of fenugreek plants subjected to varying degrees of salinity (0, 50, 100, and 200 mM NaCl) at two time intervals (1 and 14 days after saline treatment, DAT). The antioxidative capacity was correlated with oxidative damage in the same tissue. Under salt stress, lipid peroxidation and H2O2 concentration increased with increasing severity and duration of salt stress (DoS). However, the extent of oxidative damage in mycorrhizal plants was less compared to nonmycorrhizal plants. The study reveals that mycorrhiza-mediated attenuation of oxidative stress in fenugreek plants is due to enhanced activity of antioxidant enzymes and higher concentrations of antioxidant molecules. However, the significant effect of G. intraradices colonization on individual antioxidant molecules and enzymes varied with plant tissue, salinity level, and DoS. The significant effect of G. intraradices colonization on antioxidative enzymes was more evident at 1DAT in both leaves and roots, while the concentrations of antioxidant molecules were significantly influenced at 14DAT. It is proposed that AM symbiosis can improve antioxidative defense systems of plants through higher SOD activity in M plants, facilitating rapid dismutation of O2 (-) to H2O2, and subsequent prevention of H2O2 build-up by higher activities of CAT, APX, and PX. The potential of G. intraradices to ameliorate oxidative stress generated in fenugreek plants by salinity was more evident at higher intensities of salt stress.

Cloning and functional characterization of a caffeic acid O-methyltransferase from Trigonella foenum-graecum L.

A cDNA encoding an O-methyltransferase (namely FGCOMT1) was identified from the medicinal plant Trigonella foenum-graecum L. The FGCOMT1 enzyme is a functional caffeic acid O-methyltransferase (COMT) and is localized in the cytosol. Kinetic analysis indicated that FGCOMT1 protein exhibited the highest catalyzing efficiency towards 5-hydroxy ferulic acid and caffeic acid as substrates, but did not possess the abilities to methylate either quercetin or tricetin in vitro. Furthermore, transformation of Arabidopsis loss-of-function Atomt1 mutant with a FGCOMT1 cDNA partially complements accumulation of sinapoyl derivatives but did not function to produce the major methylated flavonol isorhamnetin in seeds. The results from this study indicated that FGCOMT1 is a COMT with substrate preference to monomeric lignin precursors but is not involved in the flavonoid methylation in T. foenum-graecum L.

Cloning and functional characterization of a chalcone isomerase from Trigonella foenum-graecum L.

Flavonoids belong to a group of plant natural products with variable phenolic structures and play important roles in protection against biotic and abiotic stress. Fenugreek (Trigonella foenum-graecum L.) seeds and stems contain flavonol glycosides and isoflavone derivatives. Up to now, the molecular features of fenugreek flavonoid biosynthesis have not been characterized. Here we present cloning of a cDNA encoding a chalcone isomerase (namely TFGCHI-1) from the leaves of T. foenum-graecum which convert chalcones to flavanones in vitro. Transformation of Arabidopsis loss-of-function TT5 (CHI) mutant with a TFGCHI-1 cDNA complemented TT5 and produced higher levels of flavonol glycosides than wild-type Col-0.

The emergence of embryos from hard seeds is related to the structure of the cell walls of the micropylar endosperm, and not to endo-beta-mannanase activity.

BACKGROUND AND AIMS: Seeds of carob, Chinese senna, date and fenugreek are hard due to thickened endosperm cell walls containing mannan polymers. How the radicle is able penetrate these thickened walls to complete seed germination is not clearly understood. The objective of this study was to determine if radicle emergence is related to the production of endo-beta-mannanase to weaken the mannan-rich cell walls of the surrounding endosperm region, and/or if the endosperm structure itself is such that it is weaker in the region through which the radicle must penetrate. METHODS: Activity of endo-beta-mannanase in the endosperm and embryo was measured using a gel assay during and following germination, and the structure of the endosperm in juxtaposition to the radicle, and surrounding the cotyledons was determined using fixation, sectioning and light microscopy. KEY RESULTS: The activity of endo-beta-mannanase, the major enzyme responsible for galactomannan cell wall weakening increased in activity only after emergence of the radicle from the seed. Thickened cell walls were present in the lateral endosperm in the hard-seeded species studied, but there was little to no thickening in the micropylar endosperm except in date seeds. In this species, a ring of thin cells was visible in the micropylar endosperm and surrounding an operculum which was pushed open by the expanding radicle to complete germination. CONCLUSIONS: The micropylar endosperm presents a lower physical constraint to the completion of germination than the lateral endosperm, and hence its structure is predisposed to permit radicle protrusion.

Phenolics, their antioxidant and antimicrobial activity in dark germinated fenugreek sprouts in response to peptide and phytochemical elicitors.

The phenylpropanoid pathway (PPP) was stimulated in fenugreek sprouts through the pentose phosphate and shikimate pathway, by natural elicitors such as Fish Protein Hydrolysates (FPH), Lactoferrin (LF) and Oregano Extract (OE). Among treatments 0.5 ml/L FPH elicited fenugreek sprouts had the highest phenolic content of 0.75 mg/g FW on day 3 of germination which was approximately 25 % higher than control on the same day. The antioxidant activity estimated by beta-carotene assay was highest for LF and OE elicited sprouts on day 2 and 4, respectively with an antioxidant protection factor (APF) of 1.47 for both. In all treatments and control, higher antioxidant activity was observed during early germination, which correlates to higher phenolic content, suggesting that initially phenolics are antioxidant in nature. This increased activity also correlates with high guaiacol peroxidase (GPX) activity indicating that the polymerized phenolics required for lignification with growth have antioxidant function. The antioxidant activity as estimated by beta-carotene and 1,1,-diphenyl-2-picryl hydrazyl (DPPH) assays indicate that fenugreek sprout extract can quench the superoxide free radical and also possibly scavenge the hydrogen peroxide generated in the reaction mix. OE elicited the highest levo dihydroxy phenylalanine (L-DOPA) synthesis of 1.59 mg/g FW, followed by FPH with 1.56 mg/g FW and LF 1.5 mg/g FW all on day 2 which was 24.5%, 23 % and 20 % higher than control, respectively. Higher L-DOPA content was observed in the elicited fenugreek sprouts during early germination, correlating to high phenolics and antioxidant activity, suggesting that L-DOPA also contributes to the high antioxidant activity. The glucose-6-phosphate dehydrogenase (G6PDH) activity was higher during early germination (day 1-4) and gradually decreased during later stages (day 5-8) for all treatments and control. The early increase is possibly due to the carbohydrate mobilization from the cotyledons directed towards the high nutrient requirements of the growing sprout. As mobilization occurred, an allosteric feedback inhibition by sugar-phosphates is suggested, as lower G6PDH activity was observed on days 6-8. The elevated levels of GPX during early germination coincide with the higher phenolic synthesis; SOD activity and antioxidant activity suggests the elevated production and quenching of reactive oxygen species by elicitation. High antimicrobial activity against peptic ulcer-linked Helicobacter pylori was observed in the fenugreek sprout extract from control and LF treatments only. We hypothesized that in fenugreek sprouts, simple free phenolics that are less polymerized have more antimicrobial function.

Tobacco transgenic lines that express fenugreek galactomannan galactosyltransferase constitutively have structurally altered galactomannans in their seed endosperm cell walls.

Galactomannans [(1-->6)-alpha-D-galactose (Gal)-substituted (1-->4)-beta-D-mannans] are major cell wall storage polysaccharides in the endosperms of some seeds, notably the legumes. Their biosynthesis in developing legume seeds involves the functional interaction of two membrane-bound glycosyltransferases, mannan synthase (MS) and galactomannan galactosyltransferase (GMGT). MS catalyzes the elongation of the mannan backbone, whereas GMGT action determines the distribution and amount of Gal substitution. Fenugreek (Trigonella foenum-graecum) forms a galactomannan with a very high degree of Gal substitution (Man/Gal = 1.1), and its GMGT has been characterized. We now report that the endosperm cell walls of the tobacco (Nicotiana tabacum) seed are rich in a galactomannan with a very low degree of Gal substitution (Man/Gal about 20) and that its depositional time course is closely correlated with membrane-bound MS and GMGT activities. Furthermore, we demonstrate that seeds from transgenic tobacco lines that express fenugreek GMGT constitutively in membrane-bound form have endosperm galactomannans with increased average degrees of Gal substitution (Man/Gal about 10 in T(1) generation seeds and about 7.5 in T(2) generation seeds). Membrane-bound enzyme systems from transgenic seed endosperms form galactomannans in vitro that are more highly Gal substituted than those formed by controls under identical conditions. To our knowledge, this is the first report of structural manipulation of a plant cell wall polysaccharide in transgenic plants via a biosynthetic membrane-bound glycosyltransferase.

Transfer specificity of detergent-solubilized fenugreek galactomannan galactosyltransferase.

The current experimental model for galactomannan biosynthesis in membrane-bound enzyme systems from developing legume-seed endosperms involves functional interaction between a GDP-mannose (Man) mannan synthase and a UDP-galactose (Gal) galactosyltransferase. The transfer specificity of the galactosyltransferase to the elongating mannan chain is critical in regulating the distribution and the degree of Gal substitution of the mannan backbone of the primary biosynthetic product. Detergent solubilization of the galactosyltransferase of fenugreek (Trigonella foenum-graecum) with retention of activity permitted the partial purification of the enzyme and the cloning and sequencing of the corresponding cDNA with proof of functional identity. We now document the positional specificity of transfer of ((14)C)Gal from UDP-((14)C)Gal to manno-oligosaccharide acceptors, chain lengths 5 to 8, catalyzed by the detergent-solubilized galactosyltransferase. Enzymatic fragmentation analyses of the labeled products showed that a single Gal residue was transferred per acceptor molecule, that the linkage was (1-->6)-alpha, and that there was transfer to alternative Man residues within the acceptor molecules. Analysis of the relative frequencies of transfer to alternative Man residues within acceptor oligosaccharides of different chain length allowed the deduction of the substrate subsite recognition requirement of the galactosyltransferase. The enzyme has a principal recognition sequence of six Man residues, with transfer of Gal to the third Man residue from the nonreducing end of the sequence. These observations are incorporated into a refined model for enzyme interaction in galactomannan biosynthesis.