Sushama M. Gaikwad

Insect trehalase: Physiological significance and potential applications

Trehalose, a non-reducing disaccharide, is widespread throughout the biological world. It is the major blood sugar in insects playing a crucial role as an instant source of energy and in dealing with abiotic stresses. The hydrolysis of trehalose is under the enzymatic control of trehalase. The enzyme trehalase is gaining interest in insect physiology as it regulates energy metabolism and glucose generation via trehalose catabolism. The two forms of insect trehalase namely, Tre-1 and Tre-2, are important in energy supply, growth, metamorphosis, stress recovery, chitin synthesis and insect flight. Insect trehalase has not been reviewed in depth and the information available is quite scattered. The present mini review discusses our recent understanding of the regulation, mechanism and biochemical characterization of insect trehalase with respect to its physiological role in vital life functions. We also highlight the molecular and biochemical properties of insect trehalase that makes it amenable to competitive inhibition by most glycosidase inhibitors. Due to its crucial role in carbon metabolism in insects, application of inhibitors against trehalose can form a promising area towards formulating strategies for insect pest control.

Trehalose as an indicator of desiccation stress in Drosophila melanogaster larvae: A potential marker of anhydrobiosis

In the current scenario of global climate change, desiccation is considered as one of the major environmental stressors for the biota exposed to altered levels of ambient temperature and humidity. Drosophila melanogaster, a cosmopolitan terrestrial insect has been chosen as a humidity-sensitive bioindicator model for the present study since its habitat undergoes frequent stochastic and/or seasonally aggravated dehydration regimes. We report here for the first time the occurrence of anhydrobiosis in D. melanogaster larvae by subjecting them to desiccation stress under laboratory conditions. Larvae desiccated for ten hours at <5% relative humidity could enter anhydrobiosis and could revive upon rehydration followed by resumption of active metabolism. As revealed by FTIR and HPLC analyzes, our findings strongly indicated the synthesis and accumulation of trehalose in the desiccating larvae. Biochemical measurements pointed out the desiccation-responsive trehalose metabolic pathway that was found to be coordinated in concert with the enzymes trehalose 6-phosphate synthase and trehalase. Further, an inhibitor-based experimental approach using deoxynojirimycin, a specific trehalase inhibitor, demonstrated the pivotal role of trehalose in larval anhydrobiosis of D. melanogaster. We therefore propose trehalose as a potential marker for the assessment of anhydrobiosis in Drosophila. The present findings thus add to the growing list of novel biochemical markers in specific bioindicator organisms for fulfilling the urgent need of environmental biomonitoring of climate change.

α-Galactoside binding lectin from Artocarpus hirsuta: characterization of the sugar specificity and binding site

The hemagglutinin from the seeds of Artocarpus hirsuta was purified to homogeneity by ion-exchange chromatography on DEAE-cellulose and CM-sephadex. The native protein of molecular mass 60,000 (gel filtration) is made up of two pairs of unidentical subunits, alpha and beta with molecular masses of 15,800 and 14,130. The lectin is basic in nature (pI 8.5) and a glycoprotein with neutral sugar content of 6.25%. Rabbit as well as human erythrocytes (A, B and O) are agglutinated by the lectin. The lectin activity is neither affected by Ca2+, Mg2+ or Mn2+ nor by EDTA. Methyl alpha-D-galactopyranoside, pNP-alpha-D-galactopyranoside and pNP-alpha-D-N-acetylgalactosamine are the best inhibitors of the lectin. 4-Methylumbelliferyl-alpha-galactopyranoside fluorescence was quenched on binding to A. hirsuta lectin. The sugar has two binding sites per tetramer of the lectin with a Ka of 3.5x105 M-1 at 25 degrees C. Chemical modification studies indicate that the net positive charge associated with epsilon-NH2 of lysine residues and the phenyl ring of tyrosine are essential for the sugar binding activity of A. hirsuta lectin.

Studies on recombinant single chain Jacalin lectin reveal reduced affinity for saccharides despite normal folding like native Jacalin

Sugar binding studies, inactivation, unfolding, and refolding of native Jacalin (nJacalin) from Artocarpus integrifolia and recombinant single‐chain Jacalin (rJacalin) expressed in Escherichia coli were studied by intrinsic fluorescence and thermal and chemical denaturation approaches. Interestingly, rJacalin does not undergo any proteolytic processing in an E. coli environment. It has 100fold less affinity for methyl‐α‐galactose (Ka: 2.48 × 102) in comparison to nJacalin (Ka: 1.58 × 104), and it also binds Thomsen‐Friedenreich (TF) disaccharide (Galβ1–3GalNAc) with less affinity. Overall sugar binding characteristics of rJacalin are qualitatively similar to that of nJacalin (Gal

Evidence for the essential histidine residue at the active site of glucose/xylose isomerase from Streptomyces*

Modification of glucose/xylose isomerase from Streptomyces sp. NCIM 2730 by diethylpyrocarbonate (DEPC) or its photo-oxidation in presence of rose bengal or methylene blue caused rapid loss in its activity. The inactivation of the enzyme was accompanied by an increase in the absorbance at 240 nm and was reversed by hydroxylamine. Glucose and xylose but not Mg++ and Co++ afforded significant protection to the enzyme from inactivation by DEPC. Inactivation followed pseudo-first-order kinetics and modification of a single histidine residue per mole of enzyme was indicated.

Induction and catabolite repression of cellulase inPenicillium funiculosum

The regulation of endoglucanase synthesis inPenicillium funiculosum is investigated using a method based on the viscosity lowering effect on carboxy methyl cellulose (CMC) by endoglucanase. Cellobiose (1 mg/L) causes induction, whereas glucose (5 g/L) does not repress the enzyme formation. Lactose (5 g/L) has no effect on the synthesis of cellulase. Avicel and cellulose powder (CP) are the best inducers of cellulase and xylanase activity. Both endoglucanase and xylanase activity were induced by CMC, whereas xylan induced only xylanase activity. The effect of protease on induction of cellulase activity is discussed.

D-glucose/xylose isomerase from Streptomyces : differential roles of magnesium and cobalt ions

Abstract The presence of Mg 2+ and Co 2+ is essential for the maximum activity of glucose/xylose isomerase (GXI) from Streptomyces sp. NCIM 2730. Mg 2+ is superior to Co 2+ as an activator but fails to protect the enzyme against inactivation by denaturants. The inactivation of GXI by urea, guanidinium chloride, heat, and acid pH is protected by Co 2+ . Thermal dissociation of the active dimer into inactive monomer was partly prevented by Co 2+ , suggesting that Co 2+ has an essential role in holding the ordered conformation, especially the quaternary structure of the enzyme.

Immobilization of glucose isomerase on Indion 48-R

Abstract The glucose isomerase from Streptomyces sp. NCIM 2730 was produced using wheat bran or bagasse hydrolyzate as an inducer and immobilized on an inexpensive anion exchange resin Indion 48-R. The adsorption capacity and efficiency of the resin were found to be 25 U g−1 and 60%, respectively. The properties of soluble and immobilized glucose isomerase were compared. The stability of the enzyme at higher temperatures and acidic pH is increased due to immobilization. Concentrated glucose syrup (25%) was efficiently converted to fructose by the immobilized enzyme. After the seventh reuse, the conversion capacity of immobilized glucose isomerase was reduced to 61%, with a 35% loss in enzyme activity.

Crystallization and preliminary X-ray characterization of a lectin from Cicer arietinum (chickpea)

The lectin isolated from mature seeds of Cicer arietinum (CAL) agglutinates pronase-treated rabbit and human erythrocytes and its haemagglutination activity is inhibited by fetuin and desialated fetuin but not by simple monosaccharides or oligosaccharides. The purified lectin is a dimer of molecular weight 43,000 Da composed of two identical subunits (MW 21,500), as confirmed by SDS-PAGE. The lectin has been crystallized using the hanging-drop vapour-diffusion method at 295 K over a well solution containing 0.2 M sodium acetate, 0.1 M sodium phosphate buffer pH 6.5 and 14%(w/v) polyethylene glycol 8000. The triangular prism-shaped crystals belong to space group R3 and have unit-cell parameters a = b = 81.2, c = 69.4 A. The diffraction data are 93.8% complete to 2.3 A Bragg spacing with an Rmerge of 0.103.

Polyproline fold—In imparting kinetic stability to an alkaline serine endopeptidase

Polyproline II (PPII) fold, an unusual structural element was detected in the serine protease from Nocardiopsis sp. NCIM 5124 (NprotI) based on far UV circular dichroism spectrum, structural transitions of the enzyme in presence of GdnHCl and a distinct isodichroic point in chemical and thermal denaturation. The functional activity and conformational transitions of the enzyme were studied under various denaturing conditions. Enzymatic activity of NprotI was stable in the vicinity of GdnHCl upto 6.0M concentration, organic solvents viz. methanol, ethanol, propanol (all 90% v/v), acetonitrile (75% v/v) and proteases such as trypsin, chymotrypsin and proteinase K (NprotI:protease 10:1). NprotI seems to be a kinetically stable protease with a high energy barrier between folded and unfolded states. Also, an enhancement in the activity of the enzyme was observed in 1M GdnHCl upto 8h, in organic solvents (75% v/v) for 72h and in presence of proteolytic enzymes. The polyproline fold remained unaltered or became more prominent under the above mentioned conditions. However, it diminished gradually during thermal denaturation above 60°C. Thermal transition studies by differential scanning calorimetry (DSC) showed scan rate dependence as well as irreversibility of denaturation, the properties characteristic of kinetically stable proteins. This is the first report of PPII helix being the global conformation of a non structural protein, an alkaline serine protease, from a microbial source, imparting kinetic stability to the protein.