A novel inhibition based biosensor using urease nanoconjugate entrapped biocomposite membrane for potentiometric glyphosate detection.

A potentiometric biosensor based on agarose-guar gum (A-G) entrapped bio-nanoconjugate of urease with gold nanoparticles (AUNps), has been reported for the first time for glyphosate detection. The biosensor is based on inhibition of urease activity by glyphosate, which was measured by direct potentiometry using ammonium ion selective electrode covered with A-G-urease nanoconjugate membrane. TEM and FTIR analysis revealed nanoconjugate formation and its immobilization in A-G matrix respectively. The composite biopolymer employed for immobilization yields thin, transparent, flexible membrane having superior mechanical strength and stability. It retains the maximum activity (92%) of urease with negligible leaching. The conjugation of urease with AUNps allows improvement in response characteristics for potentiometric measurement. The biosensor shows a linear response in the glyphosate concentration range from 0.5ppm-50ppm, with limit of detection at 0.5ppm, which covers maximum residual limit set by WHO for drinking water. The inhibition of catalytic activity of urease nanoconjugate by gyphosate was confirmed by FTIR analysis. The response of fabricated biosensor is selective towards glyphosate as against various other pesticides. The biosensor exhibits good performance in terms of reproducibility and prolonged storage stability of 180days. Thus, the present biosensor provides an alternative method for simple, selective and cost effective detection of glyphosate based on urease inhibition.

The contribution of two disulfide bonds in the trypsin binding domain of horsegram (Dolichos biflorus) Bowman-Birk inhibitor to thermal stability and functionality.

The major Bowman-Birk inhibitor (BBIs) of horsegram (Dolichos biflorus) HGI-III, contains seven interweaving disulfides and is extremely stable to high temperatures. The contributions of two disulfide bonds in the trypsin domain to thermal stability and functionality were evaluated using disulfide deletion variants of wild type protein. Thermal denaturation kinetics, differential scanning calorimetry and urea denaturation studies indicate that the absence of either of the two disulfides destabilizes the protein significantly. C20-C66 contributes substantially to both thermal stability and controls trypsin and chymotrypsin inhibitor activity. These two disulfides act in synergy as deletion of both disulfides leads to a complete loss of thermal stability. The data indicate that the two subdomains are not entirely independent of each other. Long range interactions, between the domains are facilitated by C20-C66. The deletion of the disulfide bonds also increased proteolytic susceptibility in a manner similar to the decreased thermal stability. From this study of rHGI a prototype of legume BBIs in can be concluded that among the array of seven evolutionarily conserved disulfide bonds, the disulfide C20-C66 that connects a residue in the trypsin domain with a residue at the border of the same domain plays a dominant role in maintaining functional and structural stability.

Characterization of α-mannosidase from Dolichos lablab seeds: partial amino acid sequencing and N-glycan analysis.

α-Mannosidase is a key enzyme in processing and degradation of N-glycans in plants and animals. In the present study α-mannosidase from crude extracts of Dolichos lablab (Indian beans) has been purified by ammonium sulfate precipitation, anion exchange, galactose Sepharose, phenyl Sepharose, gel permeation and Con A Sepharose chromatography. The purified protein migrated as a single band corresponding to 116 kDa on SDS-PAGE under reducing conditions. The pH and temperature optima of α-mannosidase activity determined by use of p-nitrophenyl-α-D-mannopyranoside as substrate were found to be 5.0 and 60-65°C, respectively. The KM was 1.48 mM and swainsonine was a potent inhibitor of the enzyme with IC(50) value 50-80 nM. Additionally, the de novo amino acid sequencing showed active site regions highly conserved among other plant acidic α-mannosidases and yielded sequence coverage of approximately 32.5%. N-glycopeptide analysis revealed the presence of paucimannosidic type structure in a conserved N-glycosylation site as well as at least one oligo mannosidic glycan at an undetermined site after ZIC-HILIC enrichment of proteolytic glycopeptides. The partial biochemical and molecular characterization of this enzyme reveals that it is a class II α-mannosidase from the glycosyl hydrolase family 38.

The unique enzymatic function of field bean (Dolichos lablab) D-galactose specific lectin: a polyphenol oxidase.

The polyphenol oxidase (PPO) of field bean (Dolichos lablab) is a tetramer made up of two subunits of mass 29,000 and 31,000 Da. The amino acid sequence of the tryptic peptides showed approximately 90% sequence identity to the D: -galactose specific legume lectins. The haemagglutinating activity of a pure and homogenous preparation of PPO measured using human erythrocytes was 1261 HAU mg(-1) protein and was inhibited by D: -galactose. Purification by galactose-sepharose chromatography also indicated that the PPO and haemagglutinating activities were associated with a single protein. Crude extracts of other legumes did not exhibit PPO activity, yet cross reacted with anti-PPO antibodies. This dual function protein with PPO and haemagglutinating activity is unique to field bean. The two activities are independent of each other occurring at different loci on the protein. These observations further evidence and strengthen the assumption that galactose specific legume lectins have enzymatic function. Both PPO and lectins are proteins that play a vital role in the defense mechanism of plants. The complementarity of these two simultaneous and independent powerful defense mechanisms exhibited by a single protein renders it a candidate gene for the development of inbuilt plant protection.

Dual-function protein in plant defence: seed lectin from Dolichos biflorus (horse gram) exhibits lipoxygenase activity.

Plant-pathogen interactions play a vital role in developing resistance to pests. Dolichos biflorus (horse gram), a leguminous pulse crop of the subtropics, exhibits amazing defence against attack by pests/pathogens. Investigations to locate the possible source of the indomitable pest resistance of D. biflorus, which is the richest source of LOX (lipoxygenase) activity, have led to a molecule that exhibits LOX-like functions. The LOX-like activity associated with the molecule, identified by its structure and stability to be a tetrameric lectin, was found to be unusual. The evidence for the lectin protein with LOX activity has come from (i) MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS, (ii) N-terminal sequencing, (iii) partial sequencing of the tryptic fragments of the protein, (iv) amino acid composition, and (v) the presence of an Mn2+ ion. A hydrophobic binding site of the tetrameric lectin, along with the presence of an Mn2+ ion, accounts for the observed LOX like activity. This is the first ever report of a protein exhibiting both haemagglutination and LOX-like activity. The two activities are associated with separate loci on the same protein. LOX activity associated with this molecule adds a new dimension to our understanding of lectin functions. This observation has wide implications for the understanding of plant defence mechanisms against pests and the cellular complexity in plant-pathogen interactions that may lead to the design of transgenics with potential to impart pest resistance to other crops.

The conformational state of polyphenol oxidase from field bean (Dolichos lablab) upon SDS and acid-pH activation.

Field bean (Dolichos lablab) contains a single isoform of PPO (polyphenol oxidase)--a type III copper protein that catalyses the o-hydroxylation of monophenols and oxidation of o-diphenols using molecular oxygen--and is a homotetramer with a molecular mass of 120 kDa. The enzyme is activated manyfold either in the presence of the anionic detergent SDS below its critical micellar concentration or on exposure to acid-pH. The enhancement of kcat upon activation is accompanied by a marked shift in the pH optimum for the oxidation of t-butyl catechol from 4.5 to 6.0, an increased sensitivity to tropolone, altered susceptibility to proteolytic degradation and decreased thermostability. The Stokes radius of the native enzyme is found to increase from 49.1+/-2 to 75.9+/-0.6 A (1 A=0.1 nm). The activation by SDS and acid-pH results in a localized conformational change that is anchored around the catalytic site of PPO that alters the microenvironment of an essential glutamic residue. Chemical modification of field bean and sweet potato PPO with 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide followed by kinetic analysis leads to the conclusion that both the enzymes possess a core carboxylate essential to activity. This enhanced catalytic efficiency of PPO, considered as an inducible defence oxidative enzyme, is vital to the physiological defence strategy adapted by plants to insect herbivory and pathogen attack.