Showing papers by "Sathyanarayana N. Gummadi published in 2012"

Production and downstream processing of (1→3)-β-D-glucan from mutant strain of Agrobacterium sp . ATCC 31750

Abstract: We isolated a mutant that produced higher levels of curdlan than the wild strain Agrobacterium sp. ATCC 31750 by chemical mutagenesis using N-methyl-N-nitro-nitrosoguanidine. The mutant strain produced 66 g/L of curdlan in 120 h with a yield of (0.88) while, the wild strain produced 41 g/L in 120 h with a yield of (0.62) in a stirred bioreactor. The mutant could not produce curdlan when the pH was shifted from 7.0 to 5.5 after nitrogen depletion as followed for wild strain. In contrast, pH optimum for cell growth and curdlan production for mutant was found to be 7.0. We optimized the downstream processing of curdlan by varying different volumes of NaOH and HCl for extraction and precipitation of curdlan. The molecular weight of the purified curdlan from the wild and mutant strain was 6.6 × 105 Da and 5.8 × 105 Da respectively. The monosaccharide analyses confirm that curdlan from both wild and mutant strain contains only glucose units. From the NMR and FTIR data, it has been confirmed that curdlan was exclusively composed of β (1 → 3)-D-glucan residues.

Physiology, biochemistry and possible applications of microbial caffeine degradation

Abstract: Caffeine, a purine alkaloid is a constituent of widely consumed beverages. The scientific evidence which has proved the harm of this alkaloid has paved the way for innumerable research in the area of caffeine degradation. In addition to this, the fact that the by-products of the coffee and tea industry pollute the environment has called for the need of decaffeinating coffee and tea industry’s by-products. Though physical and chemical methods for decaffeination are available, the lack of specificity for removal of caffeine in these techniques and their non-eco-friendly nature has opened the area of microbial and enzymatic degradation of caffeine. Another important application of microbial caffeine degradation apart from its advantages like specificity, eco-friendliness and cost-effectiveness is the fact that this process will enable the production of industrially and medically useful components of the caffeine degradation pathway like theobromine and theophylline. This is a comprehensive review which mainly focuses on caffeine degradation, large-scale degradation of the same and its applications in the industrial world.

Recovery of functionally active recombinant human phospholipid scramblase 1 from inclusion bodies using N-lauroyl sarcosine

Abstract: Human phospholipid scramblase (hPLSCR1) is a transmembrane protein involved in rapid bidirectional scrambling of phospholipids across the plasma membrane in response to elevated intracellular calcium (Ca2+) levels. Overexpression of recombinant hPLSCR1 in Escherichia coli BL21 (DE3) leads to its deposition in inclusion bodies (IBs). N-lauroyl sarcosine was used to solubilize IBs and to recover functionally active hPLSCR1 from them. Protein was purified to homogeneity by nickel-nitrilotriacetic acid (Ni2+–NTA) affinity chromatography and was >98% pure. Functional activity of the purified protein was validated by in vitro reconstitution studies, ~18% of 7-nitrobenz-2-oxa-1, 3-diazol-4-yl-phosphatidylcholine (NBD-PC) phospholipids was translocated across the lipid bilayer in the presence of Ca2+ ions. Far ultraviolet circular dichroism (UV-CD) studies reveal that the secondary structure of protein is predominantly an α-helix, and under nondenaturing conditions, the protein exists as a monomer. Here we describe a method to purify recombinant membrane protein with higher yield than previously described methods involving renaturation techniques.

Biochemical and functional characterization of human phospholipid scramblase 4 (hPLSCR4).

Abstract: Human phospholipid scramblase 4 (hPLSCR4), an isoform of the scramblase family, is a type II single-pass transmembrane protein whose function remains unknown. To understand its role, recombinant hPLSCR4 was obtained by cloning the ORF into a pET28 a(+) vector and overexpressed in Escherichia coli. Functional assay showed that Ca2+, Mg2+, and Zn2+ activate hPLSCR4 and mediate scrambling activity independent of the phospholipid head group. Far-UV-CD and fluorescence spectroscopy revealed that Ca2+ and Mg2+ binding induces conformation change in hPLSCR4, exposing hydrophobic patches of the protein, and Ca2+ has more affinity than Mg2+ and Zn2+. Stains-all studies further confirm that hPLSCR4 is a Ca2+-binding protein. Point mutation (Asp290→Ala) in hPLSCR4 decreased the Ca2+-binding affinity as well as Tb3+ luminescence, suggesting residues of the predicted Ca2+-binding motif are involved in Ca2+ binding. Functional reconstitution with (Asp290→Ala) mutant led to ~50% and ~40% decrease in scramblase activity in the presence of Ca2+ and Mg2+, respectively.

A Statistical Approach to Optimize Xylitol Production by Debaryomyces nepalensis NCYC 3413 in Vitro

Abstract: Debaryomyces nepalensis NCYC 3413, halotolerant yeast isolated from rotten apple, was capable of utilizing components of hemicellulose hydrolysate such as glucose, galactose, mannose, xylose and arabinose. The organism utilizes xylose as a sole carbon source and produces xylitol. The Plackett-Burman design was applied to determine the specific medium components affecting xylitol production and found that xylose, K2HPO4, and ZnSO4 were critical in augmenting xylitol production. These significant parameters were further optimized using response surface methodology. The optimum concentrations of xylose, K2HPO4, and ZnSO4 were found to be 100 g/l, 10.6 g/l and 8.9 mg/l respectively. Under these optimal conditions the xylitol production increased from 27 g/l to 36 g/l with a yield of 0.44 g/g (57% increase in total yield). In addition, formation of the by product (glycerol) was decreased under optimal conditions.

Biotechnological Approach to Caffeine Degradation: Current Trends and Perspectives

Abstract: Caffeine (1,3,7-trimethylxanthine) is a naturally occurring, ­commercially important alkaloid. It is an active psychostimulant which increases alertness and sustains concentration by overcoming fatigue. This makes caffeine one of the most widely consumed dietary chemicals, with the global consumption ranging from 80 to 400 mg caffeine per person per day. During coffee processing, various byproducts like pulp water, husk etc. containing appreciably high content of caffeine are generated and are discarded as wastes. Pulp, husk and other waste products from coffee and tea industries increase toxicity of surrounding landmass due to their caffeine content resulting in soil infertility. Effluents are often discharged into water bodies, contaminating drinking and surface water with caffeine and affecting the saprophytic organisms involved in essential biotransformation in the environment. Decaffeination therefore becomes an important step in coffee processing. In this aspect, microbial cells and enzymes, which are biological and non toxic, have been found to be more beneficial than conventional techniques using chemicals and energy. Several microbial strains and enzyme systems such as N-demethylases and caffeine oxidases have been discovered over the last two decades which serve are potential candidates for development of biodecaffeination techniques. The rarity of strains and the unstable nature of caffeine degrading enzymes are some of the challenges with scope for research and development in the area of biodecaffeination.

Inhibition of biogenic membrane flippase activity in reconstituted ER proteoliposomes in the presence of low cholesterol levels.

Abstract: Biogenic membranes or self-synthesizing membranes are the site of synthesis of new lipids such as the endoplasmic reticulum (ER) in eukaryotes. Newly synthesized phospholipids (PLs) at the cytosolic leaflet of ER need to be translocated to the lumen side for membrane biogenesis and this is facilitated by a special class of lipid translocators called biogenic membrane flippase. Even though ER is the major site of cholesterol synthesis, it contains very low amounts of cholesterol, since newly synthesized cholesterol in ER is rapidly transported to other organelles and is highly enriched in plasma membrane. Thus, only low levels of cholesterol are present at the biosynthetic compartment (ER), which results in loose packing of ER lipids. We hypothesize that the prevalence of cholesterol in biogenic membranes might affect the rapid flip-flop. To validate our hypothesis, detergent solubilized ER membranes from both bovine liver and spinach leaves were reconstituted into proteoliposomes with varying mol% of cholesterol. Our results show that (i) with increase in the cholesterol/PL ratio, the half-life time of PL translocation increased, suggesting that cholesterol affects the kinetics of flipping, (ii) flipping activity was completely inhibited in proteoliposomes reconstituted with 1 mol% cholesterol, and (iii) FRAP and DSC experiments revealed that 1 mol% cholesterol did not alter the bilayer properties significantly and that flippase activity inhibition is probably mediated by interaction of cholesterol with the protein.