Swagata Pal

Purification and characterization of a thermostable intra-cellular β-glucosidase with transglycosylation properties from filamentous fungus Termitomyces clypeatus

An intra-cellular beta-glucosidase was purified to homogeneity by gel filtration, ion exchange chromatography and HPGPLC from mycelial extract of Termitomyces clypeatus in the presence of the glycosylation inhibitor 2-deoxy-d-glucose. CD spectroscopy demonstrated that the purified enzyme exhibited alpha-helical conformation. MALDI-TOF identified the enzymes molecular weight as 6688Daltons, but SDS-PAGE and immunoblotting indicated that the enzyme formed aggregates. The enzyme also showed unique properties of co-aggregation with sucrase in the fungus. The enzyme showed around 80% stability up to 60 degrees C and residual activity was 80-100% between pH ranges 5-8. The enzyme had higher specific activity against p-nitrophenyl-d-glucopyranoside than cellobiose and HPLC showed that the enzyme possesses transglycosylation activity and synthesizes cello-oligosaccharides by addition of glucose. The enzyme will be useful in synthetic biology to produce complex bioactive glycosides and to avoid chemical hazards. This is the first report of a beta-glucosidase enzyme with such a low monomeric unit size.

Enhanced activity and stability of cellobiase (β-glucosidase: EC 3.2.1.21) produced in the presence of 2-deoxy-D-glucose from the fungus Termitomyces clypeatus.

Generally less glycosylation or deglycosylation has a detrimental effect on enzyme activity and stability. Increased production and secretion of cellobiase was earlier obtained in the presence of the glycosylation inhibitor 2-deoxy-d-glucose in filamentous fungus Termitomyces clypeatus [Mukherjee, S.; Chowdhury, S.; Ghorai, S.; Pal, S.; Khowala, S. Biotechnol. Lett.2006, 28, 1773-1778]. In this study the enzyme was purified from the culture medium by ultrafiltration and gel-permeation, ion-exchange and high-performance liquid chromatography, and its catalytic activity was six times higher compared to the control enzyme. K(m) and V(max) of the purified enzyme were measured as 0.187 mM and 0.018 U mg(-1), respectively, using pNPG as the substrate. The enzyme had temperature and pH optima at 45 degrees C and pH 5.4, respectively, and retained full activity in a pH range of 5-8 and temperatures of 30-60 degrees C. Interestingly less glycosylated cellobiase was resistant towards proteolytic as well as endoglycosidase-H digestion and showed higher stability than native enzyme due to increased aggregation of the protein. The enzyme also showed higher specific activity in the presence of cellobiose and pNPG and less susceptibility towards salts and different chemical agents. The beta-glucosidase can be considered as a potentially useful enzyme in various food-processing, pharmaceutical and fermentation industries.

Synergistic protective effect of folic acid and vitamin B12 against nicotine-induced oxidative stress and apoptosis in pancreatic islets of the rat

Abstract Context: Nicotine is an abundant and most significant component of cigarette smoke. Epidemiological evidence strongly suggests an association between cigarette smoking and pancreatic injury, although effects of smoking on endocrine pancreas are still controversial. Objective: We examined the impact and underlying mechanisms of action of folic acid and vitamin B12 on nicotine-induced damage in pancreatic islets of rats. Materials and methods: Male Wistar rats were treated with nicotine (3 mg/kg body weight/d, intraperitonealy) with or without folic acid (36 µg/kg body weight/d, orally) and vitamin B12 (0.63 µg/kg body weight/d, orally) for 21 d. Fasting blood glucose, oral glucose tolerance test, HBA1c, insulin, oxidative stress parameters, proinflammatory cytokines, and CRP level were measured. Histological evaluation, TUNEL assay, and immunohistochemical staining of NF-κB and caspase-3 were also performed. Results: Folic acid and vitamin B12 blunted the nicotine-induced impairment in fasting blood glucose (51–56% recovery), HbA1c (64–76% recovery), oral glucose tolerance, insulin level (23–40% recovery), and islet cell counts (26–74% recovery) in rats. Moreover, folic acid in combination with vitamin B12 also attenuated the nicotine-induced changes in markers of oxidative stress (17–88% recovery), TNF-α (40–99% recovery), and IL-6 level (47–65% recovery), CRP level (59–73% recovery), expression of NF-κB and caspase-3, and apoptosis in pancreatic islet cells. Discussion and conclusion: The present study shows that folic acid and vitamin B12 supplementation can reduce nicotine-induced impairment in glucose homeostasis and apoptosis and damage of pancreatic islet cells by modulating oxidative stress, levels of proinflammatory cytokines, and expression of NF-κB.

An Interactive Software for the Analysis of Thermal Characteristics of Capacitor-Run Single-Phase Induction Motors

This paper reports on the development of a software package, in FORTRAN 90, for determination of thermal characteristics of a capacitor-run single-phase induction motor. The software can be used for (i) calculating the steady-state temperature of the body and any internal part of the motor structure, (ii) heating and cooling curves at no load, and (iii) transient thermal characteristics for surface and stator slot temperatures at different loading. For normal motor operation, the steady-state temperature rise can be used as a direct measure of loss rather than the small difference between input and output power. In the software, the design details of the motor have been used as the input data, and a mixed-parameter thermal model has been developed based on dimensional information and constant thermal coeffi cients. The thermal model developed by the authors has been tested with a 3.5-HP, 400-V, 50-Hz capacitor run totally enclosed fan cooled (TEFC) single-phase motor. Its performance has been found to be ...This paper reports on the development of a software package, in FORTRAN 90, for determination of thermal characteristics of a capacitor-run single-phase induction motor. The software can be used for (i) calculating the steady-state temperature of the body and any internal part of the motor structure, (ii) heating and cooling curves at no load, and (iii) transient thermal characteristics for surface and stator slot temperatures at different loading. For normal motor operation, the steady-state temperature rise can be used as a direct measure of loss rather than the small difference between input and output power. In the software, the design details of the motor have been used as the input data, and a mixed-parameter thermal model has been developed based on dimensional information and constant thermal coeffi cients. The thermal model developed by the authors has been tested with a 3.5-HP, 400-V, 50-Hz capacitor run totally enclosed fan cooled (TEFC) single-phase motor. Its performance has been found to be in conformity with experimental temperature data for the body and the conductors inside the slot of the motor, obtained by varying the load for different values of the run capacitance. A computer-aided data acquisition system has also been developed for the on-line temperature monitoring of the stator slot. The conductor temperature also has been estimated by resistance method.

Effect of Alocasia indica Tuber Extract on Reducing Hepatotoxicity and Liver Apoptosis in Alcohol Intoxicated Rats

The possible protective role of ethanolic extract of A. indica tuber (EEAIT) in hepatotoxicity and apoptosis of liver caused by alcohol in rats was investigated. Treatment of rats with alcohol (3 g ethanol per kg body weight per day for 15 days intraperitoneally) produced marked elevation of liver biomarkers such as serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyl transpeptidase (γ-GT), and total bilirubin levels which were reduced by EEAIT in a dose-dependent manner. Furthermore, EEAIT improved antioxidant status (MDA, NO, and GSH) and preserved hepatic cell architecture. Simultaneous supplementation with EEAIT significantly restored hepatic catalase (CAT) and superoxide dismutase (SOD) activity levels towards normal. The studies with biochemical markers were strongly supported by the histopathological evaluation of the liver tissue. EEAIT also attenuated apoptosis and necrosis features of liver cell found in immunohistochemical evaluation. HPLC analysis of the extract showed the presence of three major peaks of which peak 2 (RT: 33.33 min) contains the highest area (%) and UV spectrum analysis identified it as flavonoids. It is therefore suggested that EEAIT can provide a definite protective effect against chronic hepatic injury caused by alcohol in rats, which may mainly be associated with its antioxidative effect.

In situ reversible aggregation of extracellular cellobiase in the filamentous fungus Termitomyces clypeatus

Cellobiase (E.C. 3.2.1.21), is a widely exploited industrial glycosidase with a major role in biofuel industry. Its stability and shelf life are major bottlenecks in achieving a superior formulation for industry. In the filamentous fungus Termitomyces clypeatus, the enzyme is secreted in a co-aggregated form with sucrase; the separation of this co-aggregation results in substantial loss of the enzyme’s activity. The aim of the present study was to examine the mode of aggregation of the secreted cellobiase-sucrase coaggregate and its role in the stabilization of cellobiase. Transmission electron microscopy and dynamic light scattering of purified co-aggregates revealed reversible, concentration driven self-aggregation of the extracellular enzymes to form larger entities. However, the intracellular enzyme aggregates were rigid, non-interacting, and possessed a higher percentage of disulphide bonds. Circular dichroic spectra of the two coaggregates indicated no significant difference in secondary structures. Self-association increased the stability of extracellular aggregates towards heat by 1.5 fold, SDS by 4 ∼ 7 fold, and chaotropic agents, by 1.5 ∼ 2 fold, than the intracellular counterpart. The Km of extracellular aggregate varied between 0.29 and 0.45 mM as a result of spontaneous aggregation and disaggregation, whereas that of intracellular aggregate was 0.22 mM irrespective of its concentration status. In situ detection of cellobiase in native PAGE revealed two activity bands of the extracellular enzyme, which indicated a minimum of two active dissociated aggregate species, as compared to a single band for the intracellular enzyme. These studies are believed to improve the understanding of aggregation of the fungal glycosidases, which remains to be a blackbox, to increase the efficacy of these enzymes.

Possible involvement of iNOS and TNF-α in nutritional intervention against nicotine-induced pancreatic islet cell damage

Nicotine is the more abundant and most significant components of cigarette smoke. Epidemiological evidence strongly suggests an association between cigarette smoking and pancreatic injury. Although effects of smoking on endocrine pancreas are still controversial Here, we examined the impact and underlying mechanisms of action of folic acid and vitamin B12 on nicotine induced damage in pancreatic islets of rats. Male Wistar rats were treated with nicotine (3mg/kg body weight/day, intraperitonealy) with or without folic acid (36μg/kg body weight/day, orally) and vitamin B12 (0.63μg/kg body weight/day, orally) for 21days. Supplementation with folic acid and vitamin B12 suppressed the nicotine induced changes in HbA1c, insulin, TNF-α, IL-6, generation of reactive oxygen species, and attenuated the changes in markers of oxidative stress. Moreover, folic acid and vitamin B12 also counteracted the increased expression of protein and mRNA contents of TNF-α and iNOS produced by nicotine. Further, folic acid and vitamin B12 in combination limits the nicotine induced changes in cell cycle and excessive apoptosis of the pancreatic β-cells and also successfully blunted the nicotine induced alteration in loss of mitochondrial membrane potential. In conclusion, data demonstrate that folic acid and vitamin B12 may be possible nutritional intervention against cellular oxidative stress, which is a critical step in nicotine-mediated islet injury, and improves islet cell functional status by scavenging free radicals and by inhibiting the generation of pro-inflammatory mediators.

Protective efficacy of folic acid and vitamin B12 against nicotine-induced toxicity in pancreatic islets of the rat

Abstract Although cigarette smoking is associated with insulin resistance and an increased risk for type 2 diabetes, few studies have examined the effect of nicotine on the adult endocrine pancreas. In this study, male Wister rats were treated with nicotine (3 mg/kg body weight/day) with or without supplementation of folic acid (36 μg/kg body weight/day) or vitamin B12 (0.63 μg/kg body weight/day) alone or in combination. Fasting blood glucose, insulin and HBA1C level and different oxidative and anti-oxidative stress parameters were measured and pancreatic tissue sections were stained with eosin-haematoxylene. Data were analysed by nonparametric statistics. The results revealed that nicotine induced prediabetes condition with subsequent damage to pancreatic islets in rats. Nicotine also caused oxidative stress in pancreatic tissue as evidenced by increased nitric oxide and malondialdehyde level and decreased superoxide dismutase, catalase and reduced glutathione level. Compared to vitamin B12 supplementation, folic acid blunted the nicotine-induced toxicity in pancreatic islets with higher efficacy. Further, folic acid and vitamin B12 in combination were able to confer significant protection on pancreatic islets against nicotine induced toxicity. These results suggest that supplementation of folic acid and vitamin B12 in combination may be a possible strategy of detoxification against nicotine-induced toxicity in pancreatic islets of the rat.

Increased enzyme secretion by 2-deoxy-d-glucose in presence of succinate by suppression of metabolic enzymes in Termitomyces clypeatus

Regulatory mode of secretion of proteins was detected for the industrial glycosidase, cellobiase, under secreting conditions (in presence of TCA cycle intermediates like succinate etc.) in the filamentous fungus Termitomyces clypeatus. The titers of key metabolic enzymes were investigated under secreting and non-secreting conditions of growth and compared to the corresponding production of intra and extracellular levels of cellobiase. Results were compared in presence of 2-deoxy-D-glucose, a potent glycosylation inhibitor in the secreting media. Inclusion of 2-deoxy-D-glucose in presence of succinate caused about 10 to 100 times decrease in titers of the metabolic enzymes hexokinase, fructose-1,6-bisphosphatase, isocitrate lyase and malate dehydrogenase leading to increased secretion of cellobiase by more than 100 times. The intracellular concentration of cAMP (86-fold decrease in presence of 2-deoxy-D-glucose under secreting conditions) and turnover rate of proteins also dropped significantly. In this suppressed metabolic state, a 10-fold increase in the titer of the secreted cellobiase was noticed. The results indicated elucidation of carbon catabolite repression like phenomenon in the fungus under secreting conditions which was more pronounced by 2-deoxy-D-glucose. The interdependence between secretion and regulation of metabolic enzymes will help in better understanding of the physiology of these highly adapted organisms for increasing their secretion potential of glycosidases like cellobiase with high industrial value.

Performance prediction of single phase induction motors using field and thermal models

The paper describes a software package for modeling a single-phase induction motor. The authors have developed a software package, in which the performance parameters of a capacitor start and capacitor run single-phase induction motor is evaluated. The inductance parameters and the core loss are evaluated mostly by using finite element (FE) field calculation technique and partly by some standard empirical formulae. The secondary resistance is estimated by using lumped parameter circuits which take into account the deep bar effects. The performance characteristics of the machine are then estimated by solving the equivalent circuit. The steady state and transient thermal characteristics of the motor is also estimated from a lumped parameter thermal model. Experiments were performed on a 3.5 H.P. capacitor run single-phase induction motor and the simulated results were verified with experimental data. It was found that the software gave a satisfactory estimation of the single-phase induction motor examined. The software uses all the motor dimensions as input data for the field and thermal models, so it is useful at the design stage for performance prediction of the designed machine.