Sridevi Annapurna Singh

Optimisation of downstream processing parameters for the recovery of pectinase from the fermented bran of Aspergillus carbonarius

Abstract Aspergillus carbonarius produces extracellular pectinases when grown on wheat bran in solid-state fermentation (SSF) on a laboratory scale in flasks. The downstream processing parameters for recovery of the crude pectinases from the fermented bran were optimised. Optimum time and temperature of extraction were determined to be 15 min and 30°C. The enzyme could be concentrated at least 4-fold in the extract by sequential extractions. Extraction using water or acetate buffer (pH 4.3) containing sodium sulphate resulted in maximum enzyme recovery. Colour and polysaccharides in the enzyme extract could be minimised by treatment with sodium chloride and activated charcoal. Concentration of the enzyme by ultrafiltration resulted in a 50-fold concentration of activity with 91% recovery.

A simple fractionation protocol for, and a comprehensive study of the molecular properties of, two major endopolygalacturonases from Aspergillus niger

A comprehensive study on purification and characterization of the two endopolygalacturonases from Aspergillus niger, PG II and PG IV, accounting for 70% of the total polygalacturonase activity, is reported. These enzymes were purified to homogeneity using ion‐exchange chromatography and gel filtration. The enzymes had specific activities of 982 and 3750 units/mg, and their molecular masses were 61 and 38 kDa, respectively. The pH optimum of PG II was pH 3.8–4.3 and for PG IV it was between pH 3 and 4.6, and the temperature optima also differed for the enzymes. The enzymes preferred pectic acid as a substrate, cleaving it at random, leading to the release of oligogalacturonides as products. The Km values of the two enzymes were found to be 0.12 and 0.72% respectively. The enzymes were rich in hydrophilic amino acids and relatively low in the sulphur‐containing amino acids. Both enzymes were rich in β‐structure and differed in their tertiary folding. The tryptophan residues were in a hydrophobic environment. The enzymes differed in their thermal stability; the midpoint of thermal inactivation, Tm, of the two enzymes was found to be 43 °C for PG II and 46 °C for PG IV.

Interaction of αS1-casein with curcumin and its biological implications.

alpha(S1)-Casein is one of the major protein components of the casein fraction of milk. Curcumin (diferuloyl methane), the major curcuminoid, constituting about 2-5% of turmeric (Curcuma longa ) is the active ingredient with many physiological, biochemical, and pharmacological properties. On the basis of spectroscopic measurements, it is inferred that curcumin binds to alpha(S1)-casein at pH 7.4 and 27 degrees C with two binding sites, one with high affinity [(2.01 +/- 0.6) x 10(6) M(-1)] and the other with low affinity [(6.3 +/- 0.4) x 10(4) M(-1)]. Binding of curcumin to alpha(S1)-casein is predominantly hydrophobic in nature. The anisotropy of curcumin or conformation of alpha(S1)-casein did not change on interaction. The stability of curcumin in solution at pH 7.2 was enhanced on binding with alpha(S1)-casein. The chaperone-like activity of alpha(S1)-casein gets slightly enhanced on its binding to curcumin. The ability of curcumin to protect erythrocytes against hemolysis was not affected due to curcumin- alpha(S1)-casein interaction. The two binding sites of alpha(S1)-casein for curcumin, along with enhanced solution stability on interaction, may offer an alternative approach in physiological and nutritional applications.

Interaction of sesamol (3,4-methylenedioxyphenol) with tyrosinase and its effect on melanin synthesis

Sesamin, sesamolin (lignans) and sesamol--from sesame seed (Sesamum indicum L.)--are known for their health promoting properties. We examined the inhibition effect of sesamol, a phenolic degradation product of sesamolin, on the key enzyme in melanin synthesis, viz. tyrosinase, in vitro. Sesamol inhibits both diphenolase and monophenolase activities with midpoint concentrations of 1.9 μM and 3.2 μM, respectively. It is a competitive inhibitor of diphenolase activity with a K(i) of 0.57 μM and a non-competitive inhibitor of monophenolase activity with a K(i) of 1.4 μM. Sesamol inhibits melanin synthesis in mouse melanoma B16F10 cells in a concentration dependant manner with 63% decrease in cells exposed to 100 μg/mL sesamol. Apoptosis is induced by sesamol, limiting proliferation. This study of the chemistry and biology of lignans, in relation to the mode of action of bioactive components, may open the door for drug applications targeting enzymes.

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.

Effect of infrared heating on the formation of sesamol and quality of defatted flours from Sesamum indicum L.

Infrared (IR) heating offers several advantages over conventional heating in terms of heat transfer efficiency, compactness of equipment, and quality of the products. Roasting of sesame seeds degrades the lignan sesamolin to sesamol, which increases the oxidative stability of sesame oil synergistically with tocopherols. IR (near infrared, 1.1 to 1.3 microm, 6 kW power) roasting conditions were optimized for the conversion of sesamolin to sesamol. The resultant oil was evaluated for sesamol and tocopherol content as well as oxidative stability. The defatted flours were evaluated for their nutritional content and functionality. IR roasting of sesame seeds at 200 degrees C for 30 min increased the efficiency of conversion of sesamolin to sesamol (51% to 82%) compared to conventional heating. The gamma-tocopherol content decreased by 17% and 25% in oils treated at 200 and 220 degrees C for 30 min, respectively. There were no significant differences in the tocopherol content and oxidative stability of the oil. Methionine and cysteine content of the flours remained unchanged due to roasting. The functional properties of defatted flours obtained from either IR roasted or conventionally roasted sesame seeds remained the same. Practical Applications: Sesame oil is stable to oxidation compared to other vegetable oils. This stability can be attributed to the presence of tocopherols and the formation of sesamol, the thermal degradation product of sesamolin-a lignan present in sesame. Roasting of sesame seeds before oil extraction increases sesamol content which is a more potent antioxidant than the parent molecule. The conversion efficiency of sesamolin to sesamol is increased by 31% by infrared roasting of seeds compared to electric drum roasting. This can be used industrially to obtain roasted oil with greater oxidative stability.

In vivo modulation of LPS induced leukotrienes generation and oxidative stress by sesame lignans

The role of inflammation and oxidative stress is critical during onset of metabolic disorders and this has been sufficiently established in literature. In the present study, we evaluated the effects of sesamol and sesamin, two important bioactive molecules present in sesame oil, on the generation of inflammatory and oxidative stress factors in LPS injected rats. Sesamol and sesamin lowered LPS induced expression of cPLA2 (61 and 56%), 5-LOX (44 and 51%), BLT-1(32 and 35%) and LTC4 synthase (49 and 50%), respectively, in liver homogenate. The diminished serum LTB4 (53 and 64%) and LTC4 (67 and 44%) levels in sesamol and sesamin administered groups, respectively, were found to be concurrent with the observed decrease in the expression of cPLA2 and 5-LOX. The serum levels of TNF-α (29 and 19%), MCP-1 (44 and 57%) and IL-1β (43 and 42%) were found to be reduced in sesamol and sesamin group, respectively, as given in parentheses, compared to LPS group. Sesamol and sesamin offered protection against LPS induced lipid peroxidation in both serum and liver. Sesamol, but not sesamin, significantly restored the loss of catalase and glutathione reductase activity due to LPS (P<.05). However, both sesamol and sesamin reverted SOD activities by 92 and 98%, respectively. Thus, oral supplementation of sesamol and sesamin beneficially modulated the inflammatory and oxidative stress markers, as observed in the present study, in LPS injected rats. Our report further advocates the potential use of sesamol and sesamin as an adjunct therapy wherein, inflammatory and oxidative stress is of major concern.

Inhibition of lipoxygenase by sesamol corroborates its potential anti-inflammatory activity.

Reactive oxygen species, the byproducts of oxygenases reaction, when in excess, promote degenerative diseases like cardiovascular, cancer and arthritis. Sesame lignans- sesamin, sesamolin and the phenolic degradation product of sesamolin, sesamol, are empirically known for their health promoting properties like antioxidant, antimutagenic, antiaging and antiinflammatory activities. In the current study, the effect of sesamol on the inflammatory oxygenase - lipoxygenase (LOX) was investigated. Enzyme kinetics and spectroscopic techniques were used to understand the inhibition mechanism. Sesamol was a potent inhibitor of soy LOX-1. It inhibited soy LOX-1 in a dose dependent manner with IC50 value of 51.84μM and Ki of 4.9μM. Binding studies using circular dichroism and corroborated by surface plasmon resonance, revealed that sesamol does not bind or change the conformation of LOX. Further, sesamol prevented the conversion of inactive LOX (Fe2+) to active LOX (Fe3+) by arresting the oxidation state of iron and prolonging the lag phase by virtue of its ability to scavenge hydroperoxides. Understanding the mechanism of action of such molecules will help in their application and promotion as nutraceuticals.

Porcine pancreatic alpha amylase and its isoforms—Effect of deglycosylation by peptide-N-glycosidase F

Porcine pancreatic alpha-amylase (PPA) and its isoforms (PPA-I and PPA-II) were deglycosylated by peptide-N-glycosidase F (PNGase F) to investigate the role of bound carbohydrate. On deglycosylation, the effect on thermal stability was less pronounced. Deglycosylation resulted in a shift of the mid-point of thermal transition by 1-2 degrees C towards lower temperature. The fluorescence emission maxima of PPA, PPA-I and PPA-II were found to be 340nm indicating the presence of tryptophan residues in a fairly hydrophilic environment. A red shift in emission spectra accompanied by an increase in fluorescence intensity was observed upon deglycosylation.

Effect of arginine:lysine and glycine:methionine intake ratios on dyslipidemia and selected biomarkers implicated in cardiovascular disease: A study with hypercholesterolemic rats

The effect of intake ratios of arginine (Arg): lysine (Lys) and glycine (Gly): methionine (Met) on lipid profile and selected cardiovascular disease markers, was studied, in rats maintained on a hypercholesterolemic diet. The rise in blood cholesterol was countered by 32%, 24%, and 49%, respectively, through increased oral supplementation of Arg, Gly, and Arg+Gly; a corresponding increase in plasma phospholipids at the end of the 8-week study was observed. The elevated plasma cholesterol to phospholipids ratio was countered by 27, 40, and 57%, respectively, through oral supplementation of Arg, Gly, and Arg+Gly. The elevation in hepatic cholesterol was lowered by 18, 29, and 51%, respectively, while phospholipids concentration was concomitantly increased by these amino acids. The elevated cholesterol to phospholipids ratio was, thus, significantly countered in the hypercholesterolemic situation by orally supplemented Arg, Gly, and Arg+Gly. Increased plasma asymmetric dimethylarginine (ADMA) levels, under hypercholesterolemic conditions, were lowered by 12, 15 and 34%, respectively, while plasma symmetric dimethylarginine (SDMA) levels were lowered by 14, 10 and 17%, respectively, with orally supplemented Arg, Gly and Arg+Gly. Only Gly and Arg+Gly decreased plasma homocysteine levels. Total nitric oxide (NO) concentration was considerably increased by Gly supplementation in hypercholesterolemic rats. Thus, altered ratios of Arg:Lys or Gly:Met offered beneficial influence on the lipid profile and plasma levels of ADMA, SDMA and homocysteine in hypercholesterolemic rats. Optimal beneficial effects, among ratios tested, was observed when Arg:Lys and Gly:Met ratios were maintained in ratios of 1:1 and 2:1, respectively.