Balasubramaniem Ashokkumar

Optimization of media for β-fructofuranosidase production by Aspergillus niger in submerged and solid state fermentation

Abstract The kinetics of β-fructofuranosidase (Ffase) production by Aspergillus niger in submerged (SmF) and solid-state fermentation (SSF) systems was investigated. The maximum productivity of Ffase (81.8 U/litre/h) was obtained in SSF for 72 h while it was 18.3 U/litre/h in SmF for 120 h. The productivity of extra cellular Ffase produced in SSF was five-fold higher than in SmF. Optimization of fermentation medium for Ffase production was carried out using De Meos fractional factorial design with seven components such as (NH 4 ) 2 SO 4 , KH 2 PO 4 , FeSO 4 , MgSO 4  · 7H 2 O, sucrose, urea and yeast extract. The media designed for SmF after two steps of optimization supported the growth of A. niger and higher productivity of Ffase (58.3 U/litre/h) than with the medium before optimization. The optimized medium of SmF when used in SSF, did not improve the Ffase productivity and therefore the medium for SSF was optimized independent of SmF. After two optimization steps, the media was defined for SSF which supported the growth and high level of Ffase productivity (149.1 U/litre/h) in SSF compared to the medium before optimization (81.8 U/litre/h) and optimized medium for SmF (58.3 U/litre/h). The results suggested that the optimized media for SmF and SSF for the production of Ffase have to be different.

Characterization and fatty acid profiling in two fresh water microalgae for biodiesel production: Lipid enhancement methods and media optimization using response surface methodology

Two fresh water microalgae, Coelastrella sp. M-60 and Micractinium sp. M-13 were investigated in this study for their potential of biodiesel production. For increasing biomass and lipid production, these microalgae were subjected to nutrient starvation (nitrogen, phosphorous, iron), salinity stress and nutrient supplementation with sugarcane industry effluent, citric acid, glucose and vitamin B12. The lipid productivity obtained from the isolates Coelastrella sp. M-60 (13.9 ± 0.4 mg/L/day) and Micractinium sp. M-13 (11.1 ± 0.2 mg/L/day) was maximum in salinity stress. The media supplemented with all the four nutrients yielded higher lipid productivity than the control. The response surface methodology (RSM) was employed to evaluate the effect of sugarcane industry effluent and citric acid on growth and lipid yield. Fatty acid profile of Coelastrella sp. M-60 and Micractinium sp. M-13 were composed of C-14, C-16:0, C-18:0, C-18:1 and C-18:2 and their fuel properties were also in accordance with international standards.

Mechanism and regulation of folate uptake by pancreatic acinar cells: effect of chronic alcohol consumption

Folate plays an essential role in one-carbon metabolism, and a relationship exists between methyl group metabolism and pancreatic exocrine function. Little, however, is known about the mechanism(s) and regulation of folate uptake by pancreatic acinar cells and the effect of chronic alcohol use on the process. We addressed these issues using the rat-derived pancreatic acinar cell line AR42J and freshly isolated primary rat pancreatic acinar cells as models. We found [(3)H]folic acid uptake to be 1) temperature and pH dependent with a higher uptake at acidic than at neutral/alkaline pH; 2) saturable as a function of substrate concentration at both buffer pH 7.4 and 6.0; 3) inhibited by folate structural analogs and by anion transport inhibitors at both buffer pH 7.4 and 6.0; 4) trans-stimulated by unlabeled folate; 5) adaptively regulated by the prevailing extracellular folate level, and 6) inhibited by modulators of the cAMP/PKA-mediated pathway. Both the reduced folate carrier (RFC) and the proton-coupled folate transporter (PCFT) were found to be expressed in AR42J and in primary pancreatic acinar cells, as well as in native human pancreas with expression of RFC being higher than PCFT. Chronic alcohol feeding of rats (4 wk; 36% of calories from ethanol) led to a significant decrease in folate uptake by freshly isolated primary pancreatic acinar cells compared with cells from pair-fed controls; this effect was associated with a parallel decrease in the level of expression of RFC and PCFT. These studies reveal that folate uptake by pancreatic acinar cells is via a regulated carrier-mediated process which may involve RFC and PCFT. In addition, chronic alcohol feeding leads to a marked inhibition in folate uptake by pancreatic acinar cells, an effect that is associated with reduction in level of expression of RFC and PCFT.

Biodegradation and utilization of dimethylformamide by biofilm forming Paracoccus sp. strains MKU1 and MKU2

Two bacterial strains capable of degrading N,N-dimethylformamide (DMF) were isolated from the effluent and sludge samples of textile and tyre industries. The 16S rRNA gene analysis revealed that bacterial strains belonged to the genera Paracoccus and named as Paracoccus sp. MKU1 and Paracoccus sp. MKU2. The DMF degradation experiments conducted at a DMF concentration of 1% v/v and HPLC analysis revealed that MKU1 and MKU2 degraded 55% and 46% of DMF after 120 h of growth. Biofilm quantification by microtiter plate assay revealed that both the bacterial isolates can form efficient biofilm on during DMF utilization. The presence of secondary carbon sources influenced the DMF degradation and biofilm formation where highest biofilm formation was observed in the presence of acetate and enhanced the DMF degradation to a maximum of 86.59% with MKU1 whereas glucose and acetate enhanced DMF degradation by MKU2 to a maximum of 82.7% and 80% respectively.

Identification of dynein light chain road block-1 as a novel interaction partner with the human reduced folate carrier

The reduced folate carrier (RFC) is a major folate transport system in mammalian cells. RFC is highly expressed in the intestine and believed to play a role in folate absorption. Studies from our laboratory and others have characterized different aspects of the intestinal folate absorption process, but little is known about possible existence of accessory protein(s) that interacts with RFC and influences its physiology and/or cell biology. We investigated this issue by employing a bacterial two-hybrid system to screen a BacterioMatch II human intestinal cDNA library using the large intracellular loop between transmembrane domains 6 and 7 of the human RFC (hRFC) as bait. Our screening has resulted in the identification of dynein light chain road block-1 (DYNLRB1) as an interacting partner with hRFC. Existence of a direct protein-protein interaction between hRFC and DYNLRB1 was confirmed by in vitro pull-down assay and in vivo mammalian two-hybrid luciferase assay and coimmunoprecipitation analysis. Furthermore, confocal imaging of live human intestinal epithelial HuTu-80 cells demonstrated colocalization of DYNLRB1 with hRFC. Coexpression of DYNLRB1 with hRFC led to a significant (P < 0.05) increase in folate uptake. On the other hand, inhibiting the endogenous DYNLRB1 with gene-specific small interfering RNA or pharmacologically with a specific inhibitor (vanadate) led to a significant (P < 0.05) decrease in folate uptake. This study demonstrates for the first time the identification of DYNLRB1 as an interacting protein partner with hRFC. Furthermore, DYNLRB1 appears to influence the function and cell biology of hRFC.

Enhancement of lipid production and fatty acid profiling in Chlamydomonas reinhardtii, CC1010 for biodiesel production

Lipid from microalgae is one of the putative oil resources to facilitate the biodiesel production during this era of energy dissipation and environmental pollution. In this study, the key parameters such as biomass productivity, lipid productivity and lipid content were evaluated at the early stationary phase of Chlamydomonas reinhardtii, CC1010 cultivated in nutrient starved (nitrogen, phosphorous), glucose (0.05%, 0.1%, 0.15% and 0.2%) and vitamin B12 supplementation (0.001%, 0.002% and 0.003%) in Tris-Acetate-Phosphate (TAP) medium. The lipid content in nitrogen starved media was 61% which is 2.34 folds higher than nutrient sufficient TAP medium. Glucose supplementation has lead to proportional increase in biomass productivity with the increasing concentration of glucose whereas vitamin B12 supplementations had not shown any influence in lipid and biomass production. Further, fatty acid methyl ester (FAME) profiling of C. reinhardtii, CC 1010 has revealed more than 80% of total SFA (saturated fatty acid) and MUFA (mono unsaturated fatty acid) content. Quality checking parameters of biodiesel like cetane number, saponification value, iodine number and degree of unsaturation were analyzed and the biodiesel fuel properties were found to be appropriate as per the international standards, EN 14214 and ASTM D6751. Conclusively, among all the treatments, nitrogen starvation with 0.1% glucose supplementation had yielded high lipid content in C. reinhardtii, CC 1010.

Enteropathogenic Escherichia coli inhibits intestinal vitamin B1 (thiamin) uptake: studies with human-derived intestinal epithelial Caco-2 cells

Infection with the gram-negative enteropathogenic Escherichia coli (EPEC), a food-borne pathogen, represents a significant risk to human health. Whereas diarrhea is a major consequence of this infection, malnutrition also occurs especially in severe and prolonged cases, which may aggravate the health status of the infected hosts. Here we examined the effect of EPEC infection on the intestinal uptake of the water-soluble vitamin B1 (thiamin) using an established human intestinal epithelial Caco-2 cell model. The results showed that infecting Caco-2 cells with wild-type EPEC (but not with nonpathogenic E. coli, killed EPEC, or filtered supernatant) leads to a significant (P < 0.01) inhibition in thiamin uptake. Kinetic parameters of both the nanomolar (mediated by THTR-2) and the micromolar (mediated by THTR-1) saturable thiamin uptake processes were affected by EPEC infection. Cell surface expression of hTHTR-1 and -2 proteins, (determined by the biotinylation method) showed a significantly (P < 0.01) lower expression in EPEC-treated cells compared with controls. EPEC infection also affected the steady-state mRNA levels as well as promoter activity of the SLC19A2 and SLC19A3 genes. Infecting Caco-2 cells with EPEC mutants that harbor mutations in the escN gene (which encodes a putative ATPase for the EPEC type III secretion system, TTSS) or the espA, espB, or espD genes (which encode structural components of the TTSS) did not affect thiamin uptake. On the other hand, mutations in espF and espH genes (which encode effector proteins) exhibited partial inhibition in thiamin uptake. These results demonstrate for the first time that EPEC infection of human intestinal epithelial cells leads to inhibition in thiamin uptake via effects on physiological and molecular parameters of hTHTR-1 and -2. Furthermore, the inhibition appears to be dependent on a functional TTSS of EPEC.

Green renewable energy production from Myxosarcina sp.: media optimization and assessment of biodiesel fuel properties

Renewable energy resources, like biomass from plants and algae, have gained more interest for biodiesel production as an energy source to reduce the consumption of fossil fuels and elevated global warming. In this study, Myxosarcina sp., a unicellular cyanobacterium, was evaluated for higher biomass and lipid production via the supplementation of sugar industrial waste (SIW) and sodium chloride (NaCl), and medium optimization with response surface methodology (RSM) for biodiesel production. The outcome of the findings was that greater biomass and lipid productivities of 28.5 ± 2.4 (1.2 fold) and 3.4 ± 0.2 (1.3 fold) mg L−1 per day were observed in BG-11 medium supplemented with SIW when compared with the control (24 ± 1 and 2.6 ± 0.4 mg L−1 per day). However, a higher lipid content 20.6 ± 1.8% (1.5 fold) was achieved with RSM optimized medium, including NaCl at 0.8 M, SIW at 2 mL L−1, sodium nitrate (NaNO3) at 2.5 g L−1 and magnesium sulphate (MgSO4) at 0.075 g L−1, than in the control (13.6 ± 1.4%). Fatty acid characterization by GC-MS analysis revealed that Myxosarcina sp. yielded 39 ± 5.3% of saturated fatty acids (SFA) and 61 ± 5.3% of monounsaturated fatty acid (MUFA) and its biodiesel fuel properties, evaluated using empirical equations, showed that almost all the properties calculated were in compliance with the national and international biodiesel standards EN 14214 (Europe), ASTM D6751-02 (US) and IS 15607 (India). Thus, Myxosarcina sp. can be utilized as the environmentally friendly biodiesel feedstock for high quality biodiesel production in the current scenario to meet escalating energy demand.

Hemocompatible glutaminase free L-asparaginase from marine Bacillus tequilensis PV9W with anticancer potential modulating p53 expression

Bacillus tequilensis PV9W, a marine bacterial isolate obtained from Gulf of Mannar, Rameswaram, India, produced glutaminase free L-asparaginase which was purified to homogeneity with a significant increase (13 fold) in specific activity. The apparent Km (0.045 ± 0.013 mM) and Vmax (7.465 ± 0.372 μmol ml−1 min−1) values of this purified L-asparaginase was identified and it was found that the maximum activity of the L-asparaginase was at a pH 8.5 and a temperature of 35 °C. The enzyme is a mixed α/β protein and the influence of different effectors were documented. The purified L-asparaginase had effective acrylamide degradation activity (6 IU per ml) and cytotoxic activity against HeLa cell lines with an IC50 of 0.036 ± 0.009 IU per ml. Furthermore, the purified enzyme showed p53 dependent G2 arrest in HeLa cells analyzed by FACS and was hemocompatible. Thus, this study highlights the marine isolate PV9W as a potential source for glutaminase free L-asparaginase with industrial as well as pharmaceutical applications. This study also paves a way for a possible therapeutic drug with the least amount of side effects.

Industrial effluent as a substrate for glutaminase free L-asparaginase production from Pseudomonas plecoglossicida strain RS1; media optimization, enzyme purification and its characterization

Glutaminase free L-asparaginase is a vital enzyme because of its anticancer potential. A potent bacterium isolated from a marine environment which produces glutaminase free L-asparaginase using M-9 medium with L-asparagine, was identified as Pseudomonas plecoglossicida RS1 by 16S rRNA gene sequencing. Statistical modeling was employed to optimize the medium using sugar cane industry effluent as the sole substrate for L-asparaginase production. The enzyme activity of L-asparaginase was higher with M-9 medium containing 0.8% effluent (3.25 ± 0.12 IU mL−1) compared to M-9 medium containing 0.3% L-asparagine (0.73 ± 0.08 IU mL−1). The apparent Km and Vmax of the purified L-asparaginase was 2.25 ± 0.61 mM and 8.9 ± 0.81 IU mL−1 min−1 respectively and the optimal activity of L-asparaginase was at pH 8.5 and 55 °C. This study highlights the use of industrial effluent as an alternate to L-asparagine for the production of L-asparaginase and how to improve the cost effectiveness of this enzyme.