Papers by Sukesh Chander Sharma
Role of Hal5p protein kinase under ethanol stress in Saccharomyces cerevisiae
The Applied Biology & Chemistry Journal
Yeast Saccharomyces cerevisiae is one of the simplest eukaryotic model organisms to study various... more Yeast Saccharomyces cerevisiae is one of the simplest eukaryotic model organisms to study various parameters of ethanol tolerance and has attracted a huge consideration for the cost-conscious production of bioethanol by the fermentation of soluble sugars. Ethanol tolerance is crucial for the efficient production of bioethanol from S. cerevisiae. In this study, we examined the role of HAL5 protein kinase in ethanol tolerance in S. cerevisiae by comparing the cell growth, survival, and stress responses of wild-type, hal4,5 mutant, and Hal5p overexpressed strains under 10 % (V/V) ethanol stress. Our findings suggest that overexpression of HAL5 protein kinase enhances ethanol tolerance in S. cerevisiae by improving cellular responses to ethanol-induced stress, including trehalose accumulation, reactive oxygen species (ROS) management, glutathione homeostasis, lipid peroxidation, and protein carbonyl contents.
Membrane Lipids from Cellular Barriers to Health and Homeostasis
CRC Press eBooks, Mar 21, 2023
Frontiers in Microbiology
Applied Biochemistry and Biotechnology, 1996
The effect of salt stress on lipid composition and its relationship with ethanol tolerance in Sac... more The effect of salt stress on lipid composition and its relationship with ethanol tolerance in Saccharomyces cerevisiae was studied. Amounts of phospholipids as well as that of sterols decreased, whereas that of protein and glycolipids increased with increasing salt concentration. Relative proportion of amino phospholipids (phosphatidylethanolamine and phosphatidylserine) decreased, whereas that of phosphatidylcholine showed a reverse trend. Cells grown under increasing salt concentration were more resistant to ethanol-induced leakage of UVabsorbing substances, an index of ethanol endurance. Results showed an overlap between osmotolerance and ethanol tolerance in this strain.
International Journal of Molecular Sciences, 2022
Xylan is one of the major structural components of the plant cell wall. Xylan present in the huma... more Xylan is one of the major structural components of the plant cell wall. Xylan present in the human diet reaches the large intestine undigested and becomes a substrate to species of the gut microbiota. Here, we characterised the capacity of Limosilactobacillus reuteri and Blautia producta strains to utilise xylan derivatives. We showed that L. reuteri ATCC 53608 and B. producta ATCC 27340 produced β-D-xylosidases, enabling growth on xylooligosaccharide (XOS). The recombinant enzymes were highly active on artificial (p-nitrophenyl β-D-xylopyranoside) and natural (xylobiose, xylotriose, and xylotetraose) substrates, and showed transxylosylation activity and tolerance to xylose inhibition. The enzymes belong to glycoside hydrolase family 120 with Asp as nucleophile and Glu as proton donor, as shown by homology modelling and confirmed by site-directed mutagenesis. In silico analysis revealed that these enzymes were part of a gene cluster in L. reuteri but not in Blautia strains, and quan...
Trehalase (α, α-trehalose-1-C-glucohydrolase, EC 3.2.1.28) is a glycoside hydrolase enzyme that c... more Trehalase (α, α-trehalose-1-C-glucohydrolase, EC 3.2.1.28) is a glycoside hydrolase enzyme that catalyzes the conversion of trehalose to glucose. Trehalose hydrolysis by trehalase enzyme is an important physiological process for various organisms, such as fungal spore germination, insect flight, and the resumption of growth in resting cells. So by studying biochemical properties of trehalase we can control the spore germination of harmful fungus, Apergillus niger. Till now trehalase has been reported from many other organisms including plants and animals, Trehalase has been purified and characterized from various organisms such as Saccharomyces cerevisiae, Lentinula edodes, Acidobacterium capsulatum, and Medicago sativa, as well as root nodules of Phaseolus vulgaris, soybean and from the from the seeds of Triticum aestivum. In this study, trehalase was purified from Aspergillus niger grown on cellulosic medium. And this purified trehalase was tested for various requirements it may n...
Biochemistry and Biophysics Reports, 2019
Dextransuccrase (E.C 2.4.1.5) is a key enzyme in S. mutans for the metabolism of sucrose which he... more Dextransuccrase (E.C 2.4.1.5) is a key enzyme in S. mutans for the metabolism of sucrose which helps in the adherence and accumulation of bacteria on tooth surface leading to the formation of dental caries. Dextransuccrase resembles in its catalytic properties with the brush boarder sucrase and exhibits pH dependent inhibitory and stimulatory effects in response to Na +. In this communication we studied the effect of monovalent cations on the activity of dextransuccrase from S. mutans. The percentage inhibition of dextransuccrase was 65% at 0.5 mM NaCl which enhanced to 90% at 20 mM sodium concentration. However there was no effect on dextransucrase activity in presence of other monovalent cations (Rb + , Cs + , and K +) tested. Enzyme activity was enhanced 20-24% in acidic pH but was strongly inhibited (59-89%) around neutral and alkaline pH by 0.5-2.0 mM sodium chloride. Upon dialysis, 86% of enzyme activity was restored to control values. There was no effect of 2 mM NaCl on glucosyltransferase activity of the enzyme. Kinetic studies revealed that enzyme showed biphasic effects in response to Na + ions. At acidic pH the enzyme exhibited mixed type of activation affecting both Vmax and Km, while in alkaline pH, the enzyme showed V-type effect reducing Vmax by 74% without affecting Km. The effects of sodium ions on dextransuccrase activity were specific, thus it can be useful to block its catalytic activity, and reducing the cariogenic potential of S. mutans. 2. Materials and methods 2.1. Reagents and buffers All the chemicals used in the study were of analytical grade. Agar, Brain Heart Infusion Broth (BHI), exogenous dextrose, glucose, sucrose, peptone and maleic acid, sodium chloride, yeast, and ethanol were obtained from HiMedia Pvt. Ltd. (Mumbai, India). All other chemical used were obtained from E. Merck Pvt. Ltd. (Mumbai, India) or Sisco Research Lab Pvt. Ltd. (Mumbai, India). Universal buffer of 20 mM of various pH containing lithium hydroxide were prepared as described by Alvarado (1979) [6]. 2.2. Bacterial strain and growth media A lyophilized culture of Streptococcus mutans (MTCC 890) was
Molecular and Cellular Biology, 2007
K+ transport in living cells must be tightly controlled because it affects basic physiological pa... more K+ transport in living cells must be tightly controlled because it affects basic physiological parameters such as turgor, membrane potential, ionic strength, and pH. In yeast, the major high-affinity K+ transporter, Trk1, is inhibited by high intracellular K+ levels and positively regulated by two redundant “halotolerance” protein kinases, Sat4/Hal4 and Hal5. Here we show that these kinases are not required for Trk1 activity; rather, they stabilize the transporter at the plasma membrane under low K+ conditions, preventing its endocytosis and vacuolar degradation. High concentrations (0.2 M) of K+, but not Na+ or sorbitol, transported by undefined low-affinity systems, maintain Trk1 at the plasma membrane in the hal4 hal5 mutant. Other nutrient transporters, such as Can1 (arginine permease), Fur4 (uracil permease), and Hxt1 (low-affinity glucose permease), are also destabilized in the hal4 hal5 mutant under low K+ conditions and, in the case of Can1, are stabilized by high K+ concent...
Interactions of dextransucrase purified from Streptococcus mutans 890 with plant polyphenols
Biochemistry and Biophysics Reports, 2021
Plant polyphenols have been extensively studied for their chemopreventive properties for human he... more Plant polyphenols have been extensively studied for their chemopreventive properties for human health. Dextransucrase plays an essential role in synthesizing exopolysaccharides from its exclusive substrate sucrose in Streptococcus mutans. In the present study, the effect of polyphenols gallic acid and tannic acid was investigated on the dextransucrase activity. The enzyme was purified by ethanol precipitation followed by column chromatography by Sephadex G-200 gel chromatography, followed by PEG-400 treatment. The purified enzyme exhibited 52 fold enrichment with 17.5% yield and specific activity of 3.54 Units/mg protein. On SDS-PAGE enzyme protein gave a single band with a molecular weight of 160 kDa. Dextransucrase activity was inhibited 80–90% by 0.04 mM tannic acid (TA) or 0.4 mM gallic acid (GA) suggesting that tannic acid has 10- fold more inhibitory potential than gallic acid on the activity of dextransucrase. CD/ORD studies revealed modifications in the tertiary structure of...
Antibodies against dextransucrase from Streptococcus mutans display anti-biofilm and growth suppressing activities
Dextransucrase produced by Streptococcus mutans play an essential role in the formation of dental... more Dextransucrase produced by Streptococcus mutans play an essential role in the formation of dental caries by synthesizing exopolysaccharides from sucrose, an important metabolite of the organism. In this study we report the location of dextransucrase in Streptococcus mutans cells and describe that antibodies raised against dextransucrase inhibited biofilm formation and reduced the adherence and hydrophobic properties of Streptococcus mutans. Western blot analysis and immunoelectron microscopy revealed that dextransucrase is located abundantly in the membrane fraction in S. mutans cells. Scanning electron microscopy and fluorescence microscopy revealed reduced cell density, impaired bioflim (plaque) formation in presence of dextransucrase antibodies. Genes associated with bioflim formation in S. mutans such as GtfB, GtfC, BrpA, relA, Smu630, vicK were down regulated (50–97%) in presence of the enzyme antibody. Presence of enzyme antibodies reduced adherence of S. mutans cells to glass...
Involvement of Streptococcus mutans in the pathogenesis of dental caries among human populations ... more Involvement of Streptococcus mutans in the pathogenesis of dental caries among human populations is well established. Here, we studied the effect of gallic acid, a naturally occurring polyphenol on certain cariogenic activities of S. mutans. Gallic acid inhibited the glycosyltransferase activity, a key enzyme of sucrose metabolism by 27-36% in S. mutans. Minimal inhibitory concentration (MIC) of gallic acid (136 μg/mL) inhibited the growth of S. mutans by 50%. About 0.4 mM of the polyphenol reduced biofilm formation by 40%, hydrophobicity 60% and acid production 36% by the organism under in vitro growth conditions. Fluorescence microscopy revealed that in absence of gallic acid, the cells were present as clumps, however in the presence of gallic acid (68 μg/mL), they were well segregated due to the inhibition of biofilm formation. The present findings suggest that gallic acid has cariostatic activity against S. mutans, which may have potential application in prevention of dental car...
Inhibition Analysis Of Tris-Like Compounds On Trehalase Activity In Aspergillus Niger
Aspergillus niger, a black spored fungus produces enzyme trehalase, which is responsible for conv... more Aspergillus niger, a black spored fungus produces enzyme trehalase, which is responsible for conversion of trehalose to glucose, it is responsible for spore germination. So by studying this enzyme's inhibition properties, one may learn to control germination of this fungus. This fungus is responsible for disease called black mould on certain fruits and vegetables such as grapes, apricots, onions, and peanuts, and is a common contaminant of food. So, in this study structure-inhibition analysis of trehalase was performed using tris-like compounds, and it was seen that Tris inhibits trehalase activity by 73%. And effect of different buffer systems was studied.
Sterols are essential components of the plasma membrane in eukaryotic cells. Nystatin-resistant e... more Sterols are essential components of the plasma membrane in eukaryotic cells. Nystatin-resistant erg mutants were used in the present study to investigate the in vitro effects of altered sterol structure on membrane lipid composition, fluidity, and asymmetry of phospholipids. Quantitative analyses of the wild type and mutants erg2, erg3 and erg6 revealed that mutants have lower sterol (free)-tophospholipid molar ratios than the wild type. Phosphatidylcholine content was decreased in erg2 and erg3 mutants; however, it was increased in erg6 strains as compared to normals. Phosphatidylserine content was increased in the erg6 mutant only. Fluorescence anisotropy decreased with temperature in both probes, and was lower for mutants than for the wild type, suggesting an increased freedom in rotational movement due to decreased membrane order. Investigation of changes in the aminophospholipid transbilayer distribution using two chemical probes, trinitrobenzene sulfonic acid and fluorescamine...
Applied Microbiology and Biotechnology
Streptococcus mutans is a common principal causative agent of dental caries. In this communicatio... more Streptococcus mutans is a common principal causative agent of dental caries. In this communication, we describe that the antibodies raised against purified dextransucrase effectively inhibited the growth of S. mutans. The purified enzyme showed 58-fold enrichment, 17.5% yield and a specific activity of 3.96 units/mg protein. Purified IgG fraction of the antibody showed significant affinity with the antigenic protein. Immunotritation of the enzyme with dextransucrase antibody showed a gradual increase in inhibition of dextransucrase activity. The growth of S. mutans was also inhibited by 85% in the presence of 28 μg of IgG fraction of the antibody. Antibodies also impaired glucosyltransferase activity (72.8%) and biofilm formation by 92.6% in S. mutans. Western blot analysis revealed no cross reactivity with the various tissues of mice, rat, rabbit and humans. Dot blot analysis showed little reactivity with Lactobacillus acidophilus and Staphylococcus aureus and there was no reactivity with other bacterial strains like Enterococcus faecalis, Escherichia coli and Salmonella typhimurium. These findings suggest that antibody raised against dextransucrase exhibit inhibitory effects on the growth of S. mutans and biofilm formation with no reactivity with various mammalian tissues, thus it could be an effective anticariogenic agent.
The importance of antioxidants and plasma membrane ion transporters which play role in cellular response against salt induced oxidative stress
Ecological Genetics and Genomics
Abstract Salt-induced oxidative stress accounts for redox imbalance and cell death in the food sp... more Abstract Salt-induced oxidative stress accounts for redox imbalance and cell death in the food spoilage yeasts. However, the physiological mechanisms underlying this imbalance and succeeding effective tolerance are still poorly understood. To elucidate this cellular protective mechanism governing in moderately halotolerant yeasts, Zygosaccharomyces bisporus was analyzed under sub-lethal conditions of sodium salt. For most stress intervals; high activity of enzymic antioxidants suggested their broad panorama in rescuing salt prompted apoptosis, attenuating intracellular degenerative reactive species with simultaneous reversal of characteristic apoptotic changes as nuclear fragmentation and chromatin condensation. In addition, the synergistic role of cell membrane cation transporters; plasma membrane ATPase and Na+/H+ like antiporter have been investigated by quantifying the external pH of the medium under stress, with the aid of specific ion transporter inhibitors like N,N'-Dicyclohexylcarbodiimide, sodium azide, and amiloride respectively. Taken together, the major outcomes from this study provide the evidence of salt imposed cell death by triggering unbalanced redox status via several key mitochondrial reactive species, allied with yeast defensive pleiotropic manipulations like antioxidants, expulsion of toxic Na+ by Nha transporters and re-establishing the optimal growth pH in alliance with PM-ATPase as a stress response resulting redox homeostasis restoration and osmoregulation. This study critically claims a proteostatic crosstalk mechanism between mitochondria and the cytosol, thus plays an important role in cell survival and advance halo-tolerance. Thereby, this inter-relationship can be further exploited for biotechnological and industrial applications in spoilage yeasts.
Effect Of Valproic Acid On Growth And Different Stress Indicators With Hal5 Gene In Saccharomyces Cerevisiae Strains
Biotech Today : An International Journal of Biological Sciences
FEBS letters, Jul 9, 2017
We have identified in yeast a connection between two master regulators of cell growth: a biochemi... more We have identified in yeast a connection between two master regulators of cell growth: a biochemical connection involving the TORC1 protein kinase (which activates protein synthesis, nutrient uptake and anabolism) and a biophysical connection involving the plasma membrane proton-pumping H(+) -ATPase Pma1 (which drives nutrient and K(+) uptake and regulates pH homeostasis). Raising the temperature to non-permissive values in a TOR thermosensitive mutant decreases Pma1 activity. Rapamycin, a TORC1 inhibitor, inhibits Pma1 dependent on its receptor Fpr1 and on the protein phosphatase Sit4, a TORC1 effector. Mutation of either Sit4 or Tco89, a non-essential subunit of TORC1, decreases proton efflux, K(+) uptake, intracellular pH, cell growth, and tolerance to weak organic acids. Tco89 does not affect Pma1 activity but activates K(+) transport. This article is protected by copyright. All rights reserved.
Changes in the composition and peroxidation of yeast membrane lipids during ethanol stress
Acta Microbiologica Et Immunologica Hungarica, Feb 1, 1994
Lipid peroxidation in Saccharomyces cerevisiae cells increased with ethanol treatment of the cell... more Lipid peroxidation in Saccharomyces cerevisiae cells increased with ethanol treatment of the cells. Such cells have decreased amount of total lipid, phospholipid and free sterols. Sterol:phospholipid ratio decreased slightly in the cells treated with 4% ethanol. However, this ratio significantly increased with further rise in ethanol concentration to 12%. Relative content of glycolipids (glycolipid to phospholipid ratio) increased in ethanol-treated yeast cells. Diphosphatidylglycerol content increased significantly and phosphatidylcholine to phosphatidylethanolamine ratio decreased in ethanol-treated cells. The amount of alpha-tocopherol decreased during ethanol stress. Catalase failed to counter the effect of ethanol. The results from the present study indicated that ethanol might be interfering with the antioxidant defence mechanisms of the yeast cells.
Indian Journal of Biochemistry Biophysics, Feb 1, 2013
Streptococcus mutans is responsible for causing dental caries in humans and utilizes sucrose for ... more Streptococcus mutans is responsible for causing dental caries in humans and utilizes sucrose for its growth. The dextransucrase (EC 2.4.1.5) is responsible for sucrose metabolism, which exhibits both hydrolytic and glucosyltransferase activities. In this study, we examined the effects of the plant phenols, namely gallic, tannic and syringic acids and aqueous extracts of certain traditionally used chewing sticks (Acacia arabica, Azadirachta indica, Pongamia pinnata and Salvadora persica) for prevention of dental caries on hydrolytic activity of dextransucrsae in S. mutans. Gallic acid (4-5 mM) produced 80-90% inhibition of the enzyme, while tannic acid (0.2 mM) and syringic acid (5 mM) inhibited the enzyme activity 80% and 48%, respectively in vitro. The aqueous extracts of chewing sticks produced 35-40% inhibition of dextransucrase activity at 5 mg phenol concentration. Kinetic analysis revealed mixed-type of enzyme inhibition by polyphenols, where both K m and V max were altered. The value of K i for tannic, gallic and syringic acids were 0.35, 1.6 and 1.94 mM, respectively. The enzyme inhibition by polyphenols was optimum at pH 7-7.5, while by plant extract was maximum at pH 5-6. These results suggest that plant polyphenols may find potential applications in the prevention and control of dental caries by inhibiting dextransucrase activity in S. mutans.
Uptake of inorganic phosphate is a limiting factor for Saccharomyces cerevisiae during growth at low temperatures
FEMS yeast research, Jan 26, 2015
The fermenting ability of Saccharomyces at low temperature is crucial for the development of alco... more The fermenting ability of Saccharomyces at low temperature is crucial for the development of alcoholic beverages, but the key factors for cold tolerance of yeast are not well known. In this report we present results of a screening for genes able to confer cold tolerance by overexpression in a laboratory yeast strain auxotrophic for tryptophan. We identified genes of tryptophan permeases (TAT1 and TAT2), suggesting that the first limiting factor for growth at low temperature of tryptophan-auxotrophic yeast is tryptophan uptake. This fact is of little relevance to industrial strains which are prototrophic for tryptophan. Then we screened for genes able to confer growth at low temperatures in tryptophan-rich medium and found several genes related to phosphate uptake (PHO84, PHO87, PHO90 and GTR1). This suggests that without tryptophan limitation, uptake of inorganic phosphate becomes the limiting factor. We have found that overexpression of the previously uncharacterized ORF YCR015c/CT...
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Papers by Sukesh Chander Sharma