The increase in the atmospheric concentrations of one of the vital green house gasses, carbon dioxide, due to anthropogenic interventions has led to several undesirable consequences such as global warming and related changes. In the global effort to combat the predicted disaster, several CO 2 capture and storage technologies are being deliberated. One of the most promising biological carbon dioxide sequestration technologies is the enzyme catalyzed carbon dioxide sequestration into bicarbonates which was endeavored in this study with a purified C. freundii SW3 b-carbonic anhydrase (CA). An extensive screening process for biological sequestration using CA has been defined. Six bacteria with high CA activity were screened out of 102 colonies based on plate assay and presence of CA in these bacteria was further emphasized by activity staining and Western blot. The identity of selected bacteria was confirmed by 16S rDNA analysis. CA was purified to homogeneity from C. freundii SW3 by subsequent gel filtration and ion exchange chromatography which resulted in a 24 kDa polypeptide and this is in accordance with the Western blot results. The effect of host on metal ions, cations and anions which influence activity of the enzyme in sequestration studies suggests that mercury and HCO 3 -ion almost completely inhibit the enzyme whereas sulfate ion and zinc enhances carbonic anhydrase activity. Calcium carbonate deposition was observed in calcium chloride solution saturated with carbon dioxide catalyzed by purified enzyme and whereas a sharp decrease in calcium carbonate formation has been noted in purified enzyme samples inhibited by EDTA and acetazolamide.

Carbonic anhydrase enzyme, one of the fastest known enzymes, remains largely unexplored in prokaryotes when compared to its mammalian counterparts despite its ubiquity. In this study, the enzyme has been purified from Bacillus subtilis SA3 using sequential Sephadex G-75 chromatography, DEAE cellulose chromatography, and sepharose-4B-L-tyrosinesulphanilamide affinity chromatography and characterized to provide additional insights into its properties. The apparent molecular mass of carbonic anhydrase obtained by SDS-PAGE was found to be approximately 37 kDa. Isoelectric focusing of the purified enzyme revealed an isoelectric point (pI) of around 6.1 when compared with marker. The presence of metal ions such as Zn, and anion SO 4 − increased enzyme activity while strong inhibition was observed in the presence of Hg 2+ , Cl − , HCO 3 − , and metal chelator EDTA. The optimum pH and temperature for the enzyme were found to be 8.3 and 37°C, respectively. Enzyme kinetics with p-nitrophenyl acetate as substrate at pH 8.3 and 37°C determined the V max and K m values of the enzyme to be 714.28 µmol/mg protein/min and 9.09 mM, respectively. The K i value for acetazolamide was 0.22 mM, compared to 0.099 mM for sulphanilamide. The results from N-terminal amino acid sequencing imply the purified protein is a putative beta-carbonic anhydrase with close similarities to CAs from plants, microorganisms. © KSBB

Carbon concentrating mechanisms play a vital role in photosynthesis in microalgae and cyanobacteria especially in the proper functioning of Rubisco and assimilation of carbon via the Calvin cycle. This study evaluates the role of carbon dioxide on carbon concentrating mechanism (CCM) in a cynaobacteria, Spirulina platensis and a microalga, Chlorella sp. 786. The study organisms were grown in both atmospheric (control sample, 0.035%) and high (exposed sample, 10%) CO 2 concentrations. Second dimension (2D) electrophoresis revealed a huge difference in the protein profiles of both organisms suggesting the induction of CCM related proteins in the sample maintained at atmospheric CO 2 concentration and the repression of CCM related proteins in the sample maintained at 10% CO 2 . Liquid chromatography-mass spectroscopy analysis revealed the presence of two important C i transporter proteins in the control sample of S. platensis, namely ferredoxin-NADP + reductase and ATP binding cassette (ABC) transport system protein. These proteins were only expressed in the control sample and were downregulated or not expressed at all in the exposed sample. Consequently, this study conclusively proves that CCMs are only inducted at low CO 2 concentrations and are not functional at high CO 2 concentration.Key Words: carbon concentrating mechanism (CCM); Chlorella sp.; cyanobacteria; proteomics; Rubisco; Spirulina platensis INTRODUCTIONNatural photosynthesis in green plants achieves carbon dioxide (CO 2 ) fixation on a global scale. The incorporation of CO 2 into the biosphere by the photosynthetic action of plants and microorganisms has been estimated to amount to about 10 11 tons of CO 2 per year Somanchi 1999, Prentice 2001). However, the efficiency of solar energy conversion in plant production under optimal growth conditions is only 5-6%. The global average efficiency has been estimated as 0.15% (Price et al. 2008). Photosynthesis is much more efficient in single celled organisms such as microalgae and cyanobacteria than in terrestrial C 3 and C 4 plants ( Kaplan and Reinhold 1999). This high efficiency is primarily due to two factors: the action of carbonic anhydrase (CA), both extracellular and intracellular, and the CO 2 concentrating mechanisms (CCM) ( Van et al. 2001, Spalding et al. 2002, Vance and Spalding 2005. CO 2 concentration plays a vital role in the induction or repression of CCM in microalgae and cyanobacteria. It has been proven that CCM is induced in low CO 2 concentrations, however, there is little informa- 296exposed sample was maintained at a CO 2 concentration of 10% (10,000 ppm). Both the cultures were grown for a period of 15-20 days in the reactor (Ramanan et al. 2010).The algal cells were centrifuged at 5,000 ×g for 5 min and to 0.5 g of algal pellet, 10 mL of algal culture medium with 2% Triton X-100 was added. The algal pellet was resuspended and pelleted. Supernatant which had greenish-yellow tint was poured off. The pellet was once again rinsed with culture medium and centrifuged again. The super...

Background & objectives: Coronary artery disease (CAD), a leading cause of mortality and morbidity worldwide has multifactorial origin. Epicardial adipose tissue (EAT) has complex mechanical and thermogenic functions and paracrine actions via various cytokines released by it, which can have both pro- and anti-inflammatory actions on myocardium and adjacent coronaries. The alteration of EAT gene expression in CAD is speculated, but poorly understood. This study was undertaken to find out the difference in gene expression of epicardial fat in CAD and non-CAD patients. Methods: Twenty seven patients undergoing coronary artery bypass graft (CABG) and 16 controls (non-CAD patients undergoing valvular heart surgeries) were included in the study and their EAT samples were obtained. Gene expressions of uncoupling protein-1, monocyte chemoattractant protein-1 (MCP-1), adiponectin, adenosine A1 receptor (ADORA-1), vascular cell adhesion molecule-1 ( VCAM-1 ) and tumour necrosis factor-alpha ( TNF-α ) were studied by real-time reverse transcription-polymerase chain reaction. Glucose, insulin, lipid profile, high-sensitivity C-reactive protein, homocysteine, vitamin D, TNF-α and leptin levels were estimated in fasting blood samples and analyzed. Results: Leptin levels were significantly higher in CABG group as compared to controls ( P <0.05), whereas other metabolic parameters were not significantly different between the two groups. MCP-1 , VCAM-1 and TNF-α were upregulated in the CABG group as compared to controls. Further, multivariate analysis showed significantly reduced adjusted odds ratio for MCP-1 [0.27; 95% confidence interval: 0.08-0.91] in the CABG group as compared to controls ( P <0.05). Interpretation & conclusions: Our findings showed an alteration in EAT gene expression in CAD patients with significant upregulation of MCP-1 . Further studies with a large sample need to be done to confirm these findings.

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