Structural Characterization of an Exopolysaccharide Isolated from Enterococcus faecalis, and Study on its Antioxidant Activity, and Cytotoxicity Against HeLa Cells.

An exopolysaccharide (EPS-I) having the molecular weight ~ 2.6 × 105 Da, was isolated from a Zinc resistant strain of Enterococcus faecalis from costal area. The exopolysaccharide consists of D-mannose, D-glucose, and L-fucose in molar ratio of 9:4:1. The monosaccharide units in the EPS-1 were determined through chemical (total acid hydrolysis and methylation analysis) and spectroscopic (FTIR and 1H NMR experiment) analysis. The mannose-rich EPS-1 showed total antioxidant activity (1 mg mL-1 of EPS-I as functional as approximately to 500 ± 5.2 µM of ascorbic acid) and Fe2+ metal ion chelation activity (EC50 = 405.6 µg mL-1) and hydroxyl radical scavenging activity (EC50 = 219.5 µg mL-1). The in vitro cytotoxicity experiment of EPS-I against cervical carcinoma cell line, HeLa cells showed strong cytotoxic effect (LC50 = 267.3 µg mL-1) and at that concentration, it found almost nontoxic against normal healthy cells (HEK-293).

Thermodynamics and kinetic properties of halostable endoglucanase from Aspergillus fumigatus ABK9.

An endoglucanase from Aspergillus fumigatus ABK9 was purified from the culture extract of solid-state fermentation and its some characteristics were evaluated. The molecular weight of the purified enzyme (56.3 kDa) was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, zymogram analysis and confirmed by MALDI-TOF mass spectrometry. The enzyme was active optimally at 50 °C, pH 5.0 and stable over a broad range of pH (4.0-7.0) and NaCl concentration of 0-3.0 M. The pKa1 and pKa2 of the ionizable groups of the active sites were 2.94 and 6.53, respectively. The apparent Km , Vmax , and Kcat values for carboxymethyl cellulose were 6.7 mg ml(-1), 775.4 µmol min(-1) , and 42.84 × 10(4) s(-1), respectively. Thermostability of the enzyme was evidenced by the high activation energy (91.45 kJ mol(-1)), large enthalpy for activation of denaturation (88.77 kJ mol(-1)), longer half-life (T1/2) (433 min at 50 °C), higher melting temperature (Tm ) (73.5 °C), and Q10 (1.3) values. All the characteristics favors its suitability as halotolerant and thermostable enzyme during bioprocessing of lignocellulosic materials.

Analysis of alteration of gut microbial population under the exposure of graded hyperbaric pressures: application of metagenomic approach.

Gastroenterological disorders are very common at hyperbaric conditions. The present study was conducted to find out the impact of gut flora on the gastrointestinal disorders created at such environmental circumstances. For this, male albino rat were exposed to graded hyperbaric pressures (915 and 1277 mmHg) and large intestinal content was examined for microbial composition using culture based and PCR-DGGE tools. After 30 day exposure, total aerobes (38.54 and 375.57 folds, 1.35 and 1.58 gdi) and E. coli (126.05 and 873.23 folds, 1.31 and 1.44 gdi) were increased whereas total anaerobes (7.01 x 10(4) and 8.84 x 10(3) folds, -1.56 and -1.39 gdi), Enterobacter spp. (-2.45 and -1.00 gdi) and Clostridium perfringens (12.88 and 54.16 folds, -1.38 and -1.75 gdi) were decreased significantly in respect to control after exposure of simulated hyperbaric pressures like at 915 and 1277 mmHg, respectively. Metagenomics study revealed an overall reduction in total microbial profile was noted than control at higher level hyperbaric pressure, i.e., 1277 mmHg air pressure for highest duration of exposure. Though, some new bands also appeared which indicated the expansion of dormant or new microbiota, Variation in the numbers of these newly dominated bacteria was correlated to dose and duration of hyperbaric treatment. The histological results clearly indicated that hyperbaric environment induced severe inflammation in the mucosal and submucosal layer of large intestine. Thus, the result suggest that hyperbaric pressure is an important exogenous factor that strongly modulated the intestinal morphology and microbial ecology, and induced several gastrointestinal ailments during hyperbarism.

Exploitation of fermented shrimp-shells hydrolysate as functional food: assessment of antioxidant, hypocholesterolemic and prebiotic activities.

In the present study the bioactivities of chitooligosaccharides of fermented shrimp-shell hydrolysate (SSH) in respect to hypocholesterolemic, antioxidant and prebiotic activity were tested in male albino rat. Rats were treated with four different diets, viz., (i) cholesterol-rich (5%) basal diet (ChB), (ii) ChB+10% chitin, (iii) ChB+10% SSH and (iv) control group (without cholesterol). After 4 weeks of treatment, body mass index, liver weight, serum total cholesterol and LDL-cholesterol in groups (ii) and (iii) were decreased significantly than group (i). SSH supplementation significantly resists oxidative stress by reducing the thiobarbituric acid reactive substances and by increasing catalase, superoxide dismutase and free radical scavenging activity. The colonization of Lactobacillus and Bifidobacterium population in small and large intestine were more in group (iii) than other groups. Reduction of Clostridium perfringens population and non-significant changes of E. coli was also noted in SSH supplement group. Histological study revealed that the villus height and villus:crypt of the small intestine were increased significantly in SSH supplemented group (iii) without any diarrheal symptoms. The results demonstrated that the shrimp-shells hydrolysate has hypocholesterolemic effect, can resist lipid peroxidation and can influence the growth of health beneficial microbes, hence can be used as functional food for hypercholesterolemic patients.

Study on thermodynamics and adsorption kinetics of purified endoglucanase (CMCase) from Penicillium notatum NCIM NO-923 produced under mixed solid-state fermentation of waste cabbage and bagasse.

In the current study, one thermostable endoglucanase was purified from Penicillium notatum NCIM NO-923 through mixed solid state fermentation of waste cabbage and bagasse. The molecular weight of the purified enzyme was 55kDa as determined by SDS polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme had low activation energy (Ea) of 36.39KJ mol(-1) for carboxymethyl cellulose hydrolysis and the enthalpy and entropy for irreversible inactivation was 87 kJ mol (-1) and 59.3 J mol (-1) K(-1) respectively. The enzyme was quite thermostable with a Tm value of 62.2°C. The pKa1 and pKa2 of ionizable groups of the active sites were 2.5 and 5.3 respectively. Apparent Km, Vmax and Kcat of the enzyme were found to be 5.2 mg mL(-1), 80 U/gds and 322.4 sec(-1) respectively. The enzyme showed about 1.4 fold increased activity in presence of 10mM MgSO4. Adsorption of endoglucanase on Avicel at wide pH range was studied at different temperatures. Langmuir type adsorption isotherm at 10°C showed maximum adsorption strength of enzyme at pH 3.0, which was in a range of optimum pH of the enzyme.

Rapid screening of tannase producing microbes by using natural tannin.

Use of natural tannin in the screening of tannase producing microbes is really promising. The present work describes about the possibility and integrity of the newly formulated method over the previously reported methods. Tannin isolated from Terminalia belerica Roxb. (Bahera) was used to differentiate between tanninolytic and nontanninolytic microbes. The method is simple, sensitive and superior for the rapid screening and isolation of tannase-producing microbes.

Structure and biological properties of exopolysaccharide isolated from Citrobacter freundii.

This study aimed to investigate the molecular characterization, antioxidant activity in vitro, cytotoxicity study of an exopolysaccharide isolated from Citrobacter freundii. Firstly, the culture conditions were standardized by the Design of experiments (DoE) based approach, and the final yield of thecrude exopolysaccharide was optimized at 2568 ± 169 mg L-1. One large fraction of exopolysaccharide was obtained from the culture filtrate by size exclusion chromatography and molecular characteristics were studied. A new mannose rich exopolysaccharide (Fraction-I) with average molecular weight ~ 1.34 × 105 Da was isolated. The sugar analysis showed the presence of mannose and glucose in a molar ratio of nearly 7:2 respectively. The structure of the repeating unit in the exopolysaccharide was determined through chemical and 1D/2D- NMR experiments as: Finally, the antioxidant activity, and the cytotoxicity of the exopolysaccharide were investigated and the relationship with molecular properties was discussed as well.

Tissue specific expression and in-silico characterization of a putative cysteine synthase gene from Lathyrus sativus L.

Grass pea (Lathyrus sativus L.) is a worldwide popular pulse crop especially for its protein rich seeds with least production cost. However, the use of the crop became controversial due to the presence of non-protein amino acid, ß-N-oxalyl-L-α, ß-diaminopropionic acid (ß-ODAP) in its seed and leaf, which is known as the principle neurotoxin to cause neurolathyrism (a motor neurodegenerative disease of humans and animals) during prolonged consumption as regular diet. Till date, the knowledge on ß-ODAP biosynthesis in Lathyrus sp. is limited only to a small part of the complex bio-chemical steps involved including a few known sulfur-containing enzymes (viz. cysteine synthase, ODAP synthase etc.). In Lathyrus sativus, biosynthesis of ß-ODAP varies differentially in a tissue-specific manner as well as in response to several environmental stresses viz. zinc deficiency, iron over-exposure, moisture stress etc. In the present study, a novel cysteine synthase gene (LsCSase) from Lathyrus sativus L was identified and characterized through bioinformatics approaches. The bioinformatic analysis revealed that LsCSase showed maximum similarity with the O-acetyl serine (thiol) lyase of Medicago truncatula with respect to several significant sequence-specific conserved motifs (cysK, CBS like, ADH_zinc_N, PALP), sub-cellular localization (chloroplast or cytoplasm) etc., similar to other members of cysteine synthase protein family. Moreover, the tissue-specific regulation of the LsCSase as well as its transcriptional activation under certain previously reported stressed conditions (low Zn+2-high Fe+2, PEG induced osmotic stress) were also documented through quantitative real-time PCR analyses, suggesting a possible link between the LsCSase gene activation and ß-ODAP biosynthesis to manage external stresses in grass pea. This preliminary study offers a probable way towards the development of less toxic consumer-safe grass pea by down-regulation or deactivation of such gene/s (cysteine synthase) through genetic manipulations.

Production of bioethanol as useful biofuel through the bioconversion of water hyacinth (Eichhornia crassipes).

Water hyacinth (Eichhornia crassipes) represents a promising candidate for fuel ethanol production in tropical countries because of their high availability and high biomass yield. Bioconversion of such biomass to bioethanol could be wisely managed through proper technological approach. In this work, pretreatment of water hyacinth (10 %, w/v) with dilute sulfuric acid (2 %, v/v) at high temperature and pressure was integrated in the simulation and economic assessment of the process for further enzymatic saccharification was studied. The maximum sugar yield (425.6 mg/g) through enzymatic saccharification was greatly influenced by the solid content (5 %), cellulase load (30 FPU), incubation time (24 h), temperature (50 °C), and pH (5.5) of the saccharifying medium. Central composite design optimized an ethanol production of 13.6 mg/ml though a mixed fermentation by Saccharomyces cerevisiae (MTCC 173) and Zymomonas mobilis (MTCC 2428). Thus the experiment imparts an economic value to water hyacinths that are cleared from choking waterways.

Production of chitin and bioactive materials from Black tiger shrimp (Penaeus monodon) shell waste by the treatment of bacterial protease cocktail.

The main objective of this study was to obtain chitin in pure form from a new crustacean waste material for industrial applications. Black tiger shrimp shell wastes are a rich source of protein and valuable bioactive carbohydrate polymers such as chitin. After removal of carotenoid, Black tiger shrimp shell wastes (BTSHWs) were treated with chemicals and protease enzyme to extract chitin. Box-Behnken response surface methodology was applied to optimize the deproteinization process to obtain chitin. At optimal pH (8.82), temperature (50.05 °C), agitation speed (100.98 rpm), enzyme substrate ratio of 1:8 (wv-1) and 72 h of incubation with Paenibacillus woosongensis TKB2 crude protease cocktail, 80 % deproteinization was found along with 77.28 % recovery of chitin. The valuable oligopeptides were determined by MALDI-TOF analysis and analysis of adequate amount of free amino acids in protein hydrolysate from BTSHW, indicating a high nutritional value used for food, feed or as a nitrogen source in growth medium for microorganisms. The chitin obtained was compared with the commercial chitin using scanning electron microscopy, Fourier transform infrared spectrometer, X-ray diffraction and 13C CP/MAS-NMR. Chitin obtained from crude protease treatment showed comparable physicochemical and structural properties to those of the commercial chitin. The carotenoid obtained after treatment can be used for medicinal purpose.

Chitinases biosynthesis by immobilized Aeromonas hydrophila SBK1 by prawn shells valorization and application of enzyme cocktail for fungal protoplast preparation.

Production and optimization of ß-N-acetyl glucosaminidase and chitinase by Ca-alginate immobilized Aeromonas hydrophila SBK1 was carried out using prawn shell as cost-effective substrate. Beads prepared with 5.0% Na-alginate (containing 2.0% colloidal chitin) and 1.0 M CaCl2 showed considerable beads integrity and supported maximum production of chitinolytic enzymes. Bead diameter, 3 mm; temperature, 35°C; pH 7.0; agitation, 90 rpm were found ideal for the maximum production of the enzymes. The fermentation and thermodynamic indices revealed the feasibility of immobilized cells over free cells for enzymes production. Reasonable amount of chitosaccharides (degree of polymerization; 1-6) accumulated in the production media which have paramount antioxidant activity. Scale up experiment was successfully carried out in 5 L fermentor. In immobilized state, the chitosaccharides yield and antioxidant activity increased about 44.76% and 22.22%, whereas specific productivity of ß-N-acetyl glucosaminidase and chitinase increased by 22.86% and 33.37% over free state. The cell entrapped beads can be reused upto ten cycles without marked loss of its biocatalytic efficiency. High level of protoplast of Aspergillus niger was generated by treating mycelia with 10 U/ml of crude chitinase after 4 h at pH 7.0 and in the temperature 35-40°C, and 67% of the protoplasts were found to be regenerated.

Appraisal of antioxidant, anti-hemolytic and DNA shielding potentialities of chitosaccharides produced innovatively from shrimp shell by sequential treatment with immobilized enzymes.

Chitosaccharides (CS) of varied size were prepared from shrimp shell through sequential catalysis, using crude protease and chitinase enzymes immobilized on agar beads. In the optimized state, immobilization yield and activity yield for protease were 84% and 62%, and for chitinase were 75% and 57%, respectively. Immobilized protease and chitinase treatment improved CS yields (101 µg/ml) and retained 63% and 52% of activities after 10 reuses, respectively. Stronger radical-scavenging activity (RSA) of CS against ABTS, DPPH and hydroxyl radical was noted with EC50 values 19.1, 26.4 and 29.6 µg/ml, respectively. Peroxyl and superoxide RSAs of 96.8% and 88.6% were noticed at 70 µg/ml of CS. Singlet oxygen quenching, reducing power and ferrous ion-chelating activities of CS were also pronounced. CS reasonably reduced oxidative damage of DNA, protein and RBC by inhibiting H2O2 and AAPH radicals. Reversible CS-DNA condensation leads to DNA stabilization without changing its conformation and advocates its employment in gene therapy.

Dynamics of predominant microbiota in the human gastrointestinal tract and change in luminal enzymes and immunoglobulin profile during high-altitude adaptation.

High-altitude (HA) visitors like pilgrims, trackers, scientists and military personnel face a group of nonspecific gastrointestinal (GI) symptoms during acclimatization to hypobaric hypoxia. In order to investigate the alteration of indigenous gastrointestinal microbiota in the development of such GI symptoms, an experiment was conducted for the enumeration of dominant cultivable faecal microbiota of 15 soldiers at base level (Delhi) and during their 15-day acclimatization at 3,505 m HA (Leh). At HA, faecal microbiota analysis revealed that total aerobes decreased significantly with increase of total and facultative anaerobes. The strict anaerobes like Bifidobacterium sp., Bacteroidetes sp. and Lactobacillus sp. exhibited positive growth direction index (GDI) like other predominant obligate anaerobes Clostridium perfringens and Peptostreptococcus sp. Different enzymes like amylase, proteinase and polyphenol hydrolase produced by different bacterial populations showed positive GDI, whereas phosphatase producers exhibited negative GDI. The levels of microbe-originated enzymes like amylase, proteinase, alkaline phosphatase and ß-glucuronidase were also elevated during HA acclimatization. In addition, in vitro gas production ability was enhanced with increase of faecal immunoglobulins IgA and IgG. We demonstrated that hypoxic environment at HA had the potential to alter the gut microbial composition and its activities that may cause GI dysfunctions.

Study of the cultivable microflora of the large intestine of the rat under varied environmental hyperbaric pressures.

BACKGROUND/PURPOSE(S): We conducted an in vivo experiment to investigate the effect of hyperbarometric air pressure on the quantity and composition of the cultivable microflora of the large intestine. METHODS: Using selective culture-based methods, we enumerated from the large intestine total aerobes and total anaerobes, and indicator bacteria such as Escherichia coli, other Enterobacteriaceae, Bifidobacterium spp., Lactobacillus spp. and Clostridium perfringens, and studied their quantitative variation. RESULTS: Total aerobes and facultative anaerobes (E. coli and other Enterobacteriaceae) were increased with an increase in air pressure, whereas the increase caused a drastic reduction in the numbers of total anaerobes and Clostridium perfringens. Bifidobacterium spp. and Lactobacillus spp. were affected slightly by the altered air pressures. Variation in the numbers of these groups of bacteria was correlated to dose and duration of hyperbaric treatment. CONCLUSION: We conclude from our results that air pressure is an important exogenous factor that strongly modulates bacterial colonization of the large intestine and the composition of the intestinal microflora, and that the occurrence of gastrointestinal disorders during hyperbarism is a result of alteration in the indigenous microflora.

Regulation of xylanase biosynthesis in Bacillus cereus BSA1.

Microbial xylanases have a promising biotechnological potential to be used in industries. In this study, regulation of xylanase production was examined in Bacillus cereus BSA1. Xylanase production was induced by xylan. The enzyme production further increased in the presence of xylose and arabinose in very low concentration with addition of xylan (0.5% up to 6.02 U/ml). Addition of glucose (about 0.1%) to the media supplemented with xylan repressed xylanase production. Even higher concentration (>0.1%) of xylose and arabinose repressed xylanase biosynthesis. Glucose-mediated repression was partially relived by addition of cyclic adenosine monophosphate. Chemical like 2-4-dinitrophenol, which can inhibit adenosine triphosphate synthesis in cell, repressed xylanase synthesis and it suggested xylanase synthesis to be an energy dependent process.

Production and composition of extracellular polysaccharide synthesized by a Rhizobium isolate of Vigna mungo (L.) Hepper.

An extracellular polysaccharide (EPS) was produced by a Rhizobium sp. isolated from the root nodules of Vigna mungo (L.) Hepper. Maximum EPS production (346 mg l(-1)) was when the yeast extract basal medium was supplemented with mannitol (1%), biotin (1.5 mg l(-1)) and asparagine (0.3%). Ribose (53%) and mannose (47%) were the principle monomers of the EPS. Chemical, chromatographic and spectroscopic analysis showed that this polymer, which has Man(4)Rib(1) as an oligomeric subunit, has an apparent molecular mass of 750 kDa.

Rapid screening of tannase producing microbes by using natural tannin

Use of natural tannin in the screening of tannase producing microbes is really promising. The present work describes about the possibility and integrity of the newly formulated method over the previously reported methods. Tannin isolated from Terminalia belerica Roxb. (Bahera) was used to differentiate between tanninolytic and nontanninolytic microbes. The method is simple, sensitive and superior for the rapid screening and isolation of tannase-producing microbes.

Study on Thermodynamics and Adsorption kinetics of Purified endoglucanase (CMCase) from Penicillium notatum NCIM NO-923 produced under mixed solid-state fermentation of waste cabbage and Bagasse

In the current study, one thermostable endoglucanase was purified from Penicillium notatum NCIM NO-923 through mixed solid state fermentation of waste cabbage and bagasse. The molecular weight of the purified enzyme was 55kDa as determined by SDS polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme had low activation energy (Ea) of 36.39KJ mol-1 for carboxymethyl cellulose hydrolysis and the enthalpy and entropy for irreversible inactivation was 87 kJ mol −1 and 59.3 J mol −1 K−1 respectively. The enzyme was quite thermostable with a Tm value of 62.2˚C. The pKa1 and pKa2 of ionizable groups of the active sites were 2.5 and 5.3 respectively. Apparent Km, Vmax and Kcat of the enzyme were found to be 5.2 mg mL-1, 80 U/gds and 322.4 sec-1 respectively. The enzyme showed about 1.4 fold increased activity in presence of 10mM MgSO4. Adsorption of endoglucanase on Avicel at wide pH range was studied at different temperatures. Langmuir type adsorption isotherm at 10˚C showed maximum adsorption strength of enzyme at pH 3.0, which was in a range of optimum pH of the enzyme.