Kajal Chakraborty

An extra-cellular alkaline metallolipase from Bacillus licheniformisMTCC 6824: Purification and biochemical characterization

An extra-cellular lipase produced by Bacillus licheniformis MTCC 6824 was purified to homogeneity by ammonium sulphate fractionation, ethanol/ether precipitation, dialysis, followed by anion-exchange chromatography on Amberlite IRA 410 (Cl(-) form) and gel exclusion chromatography on Sephadex G 100 using Tris-HCl buffer (pH 8.0). The crude lipase extract had an activity of 41.7LU/ml of culture medium when the bacterium was cultured for 48h at 37°C and pH 8.0 with nutrient broth supplemented with sardine oil as carbon source. The enzyme was purified 208-fold with 8.36% recovery and a specific activity of 520LU/mg after gel exclusion chromatography. The pure enzyme is a monomeric protein and has an apparent molecular mass of 74.8kDa. The lipase had a Vmax and Km of 0.64mM/mg/min and 29mM, respectively, with 4-nitro phenylpalmitate as a substrate, as calculated from the Lineweaver-Burk plot. The lipase exhibited optimum activity at 45°C and pH 8.0, respectively. The enzyme had half-lives (T1/2) of 82min at 45°C, and 48min at 55°C. The catalytic activity was enhanced by Ca(2+) (18%) and Mg(2+) (12%) at 30mM. The lipase was inhibited by Co(2+), Cu(2+), Zn(2+), Fe(2) even at low concentration (10mM). EDTA, at 70mM concentration, significantly inhibited the activity of lipase. Phenyl methyl sulfonyl fluoride (PMSF, 70mM) completely inactivated the original lipase. A combination of Ca(2+) and sorbitol induced a synergistic effect on the activity of lipase with a significantly high residual activity (100%), even after 45min, as compared to 91.5% when incubated with Ca(2+) alone. The lipase was found to be hydrolytically resistant toward triacylglycerols with more double bonds.

Guaiane sesquiterpenes from seaweed Ulva fasciata Delile and their antibacterial properties

Two new guaiane sesquiterpene derivatives, guai-2-en-10alpha-ol (1) and guai-2-en-10alpha-methanol (2), were chromatographically purified as major constituents of the CHCl3/CH3OH (1:1, v/v) soluble fraction of Ulva fasciata. Acetylation of 2 furnished guai-2-en-10alpha-methyl methanoate (3) with acetyl group at C11 position. The structures of the compounds were elucidated using one and two-dimensional NMR and mass spectrometric analysis. Compounds 2 and 3 exhibited significant inhibition to the growth of Vibrio parahaemolyticus with minimum inhibitory concentrations of 25 and 35 microg/mL, respectively. The electronegative C10 acetyl group with high polarisability (7.02x10(-24) cm3) in 3 appeared to withdraw electron cloud from substituted cycloheptyl ring and (R)-3-methylcyclohept-1-ene moiety, thus acting as the nucleophilic center of the molecule resulting in high bioactivity.

Amino Acid Compositions of 27 Food Fishes and Their Importance in Clinical Nutrition

Proteins and amino acids are important biomolecules which regulate key metabolic pathways and serve as precursors for synthesis of biologically important substances; moreover, amino acids are building blocks of proteins. Fish is an important dietary source of quality animal proteins and amino acids and play important role in human nutrition. In the present investigation, crude protein content and amino acid compositions of important food fishes from different habitats have been studied. Crude protein content was determined by Kjeldahl method and amino acid composition was analyzed by high performance liquid chromatography and information on 27 food fishes was generated. The analysis showed that the cold water species are rich in lysine and aspartic acid, marine fishes in leucine, small indigenous fishes in histidine, and the carps and catfishes in glutamic acid and glycine. The enriched nutrition knowledge base would enhance the utility of fish as a source of quality animal proteins and amino acids and aid in their inclusion in dietary counseling and patient guidance for specific nutritional needs.

Purification and biochemical characterization of an extracellular lipase from Pseudomonas fluorescens MTCC 2421.

An extracellular lipase produced by Pseudomonas fluorescens MTCC 2421 was purified 184.37-fold with a specific activity of 424.04 LU/mg after anion exchange and gel exclusion chromatography. The enzyme is a homomeric protein with an apparent molecular mass of 65.3 kDa. The lipase exhibited hydrolytic resistance toward triglycerides with longer fatty acyl chain length containing unsaturation as evident from the lower V(max) (0.23 mM/mg/min) of the lipase toward glycerol trioleate (C(18:1n9)) compared with the fatty acid triglycerides having short to medium carbon chain lengths (C(18:0-12:0), V(max) 0.32-0.51 mM/mg/min). This indicates a preferential specificity of the lipase toward cleaving shorter carbon chain length fatty acid triglycerides. The lipase exhibited optimum activity at 40 degrees C and pH 8.0, respectively. A combination of Ca(2+) and sorbitol induced a synergistic effect on the thermostability of lipase with a significantly high residual activity (100%) after 30 min at 40 degrees C, as compared to 90.6% after incubation with Ca(2+) alone. The lipase activity was inhibited by Cu(2+) and Fe(2+) (42 and 48%, respectively) at 10 mM. The enzyme lost 31% of its initial activity by 0.001 mM EDTA and 42% by 0.1 mM EDTA. Significant reduction in lipase activity was apparent by 2-mercaptoethanol and phenylmethanesulfonyl fluoride at diluted concentration (0.001 mM), thereby indicating an important role of sulfhydryl groups in the catalytic mechanism.

Enrichment of eicosapentaenoic acid from sardine oil with Delta5-olefinic bond specific lipase from Bacillus licheniformis MTCC 6824.

Lipase derived from Bacillus licheniformis MTCC 6824 was purified to homogeneity by anion exchange chromatography on Amberlite IRA 410 (Cl-) and gel filtration using Sephadex G-100 as judged by denaturing polyacrylamide gel electrophoresis. The purified lipase was used for hydrolysis of triacylglycerol in sardine oil to enrich Delta5-polyunsaturated fatty acids (Delta5-PUFAs) namely, arachidonic acid (5,8,11,14-eicosatetraenoic acid, ARA, 20:4n-6) and eicosapentaenoic acid (5,8,11,14,17-eicosapentaenoic acid, EPA, 20:5n-3). The individual fatty acids were determined as fatty acid methyl esters (FAMEs) by gas-liquid chromatography and gas chromatography-mass spectroscopy as FAMEs and N-acyl pyrrolidides. The enzyme exhibited hydrolytic resistance toward ester bonds of Delta5-PUFAs as compared to those of other fatty acids and was proved to be effective for increasing the concentration of EPA and ARA from sardine oil. Utilizing this fatty acid specificity, EPA and ARA from sardine oil were enriched by lipase-mediated hydrolysis followed by urea fractionation at 4 degrees C. The purified lipase produced the highest degree of hydrolysis for SFAs and MUFAs (81.5 and 72.3%, respectively, from their initial content in sardine oil) after 9 h. The profile of conversion by lipase catalysis showed a steady increase up to 6 h and thereafter plateaued down. Lipase-catalyzed hydrolysis of sardine oil followed by urea adduction with methanol provided free fatty acids containing 55.4% EPA and 5.8% ARA, respectively, after complexation of saturated and less unsaturated fatty acids. The combination of enzymatic hydrolysis and urea complexation proved to be a promising method to obtain highly concentrated EPA and ARA from sardine oil.

Aquaculture related invasion of the exotic Artemia franciscana and displacement of the autochthonous Artemia populations from the hypersaline habitats of India

Autochthonous parthenogentic Artemia populations have been reported from Indian hypersaline habitats since 1950s. Exotic Artemia franciscana was imported and introduced into India as live food for aquaculture since the early eighties. To assess the present status of the Artemia populations and the possibility of invasion by the introduced A. franciscana in Indian Salinas, an extensive study was conducted using conventional and molecular approaches. Morphological and biometric observations, crossbreeding experiments and molecular and phylogenetic analysis using Internally Transcribed Spacer-1 sequence revealed the extensive presence of alien, sexual A. franciscana populations in various hypersaline areas. Individual culture experiments and crossbreeding studies further confirmed the absence of autochthonous parthenogentic Artemia populations. Lack of regional endemism in populations of distant origins was evident, indicating that the invaded populations have naturalized and are in the process of evolution. This forms the first report of invasion by A.franciscana in hypersaline habitats of Indian subcontinent and further studies are required to assess the biological implications of this invasion.

Two rare antioxidative prenylated terpenoids from loop-root Asiatic mangrove Rhizophora mucronata (Family Rhizophoraceae) and their activity against pro-inflammatory cyclooxygenases and lipoxidase.

Abstract Two new biogenic prenylated terpenoids were isolated from the methanol extract of Rhizophora mucronata. The extended C20 sesquiterpenoid with prenylated guaiane framework was characterised as (4E, 8Z)-3, 3a, 6, 7-tetrahydro-3, 9-dimethyl-5-(6-methylheptan-2-yl) cycloocta[b]furan-2-(9aH)-one (1). (35E)-1,2,3,5,6,6-icosahydro-4,4,8b,10,14,17,20,20-octamethylpicen-3-yl-34,35-dimethyloct-31-enoate (2) represents the first example of naturally occurring C40 prenylated oleanane-type triterpenoid, whereas one 4,5-dimethyloct-5-enoate side chain remains attached at C-3 position of the oleanane framework formed by the E-ring closure of C30 saccharide moiety. The structures of the compounds were elucidated using NMR and mass spectrometric analysis. Compound 1 was found to have significantly greater antioxidant activities (IC50 ~ 0.75 mg/mL) compared to 2 (IC50 > 0.80 mg/mL). No significant differences in anti-cyclooxygenase-2 of these compounds were discernable (IC50 0.8 – 0.9 mg/mL), whilst compound 1 showed greater anti-5-lipoxidase activities (IC50 ~ 0.8 mg/mL) those that of 2 (IC50 0.96 mg/mL). Bioactivities of the prenylated terpenoids were inversely proportional to lipophilic and bulk descriptors.

DHA and EPA Content and Fatty Acid Profile of 39 Food Fishes from India

Docosahexaenoic acid (DHA) is the principal constituent of a variety of cells especially the brain neurons and retinal cells and plays important role in fetal brain development, development of motor skills, and visual acuity in infants, lipid metabolism, and cognitive support and along with eicosapentaenoic acid (EPA) it plays important role in preventing atherosclerosis, dementia, rheumatoid arthritis, Alzheimers disease, and so forth. Being an essential nutrient, it is to be obtained through diet and therefore searching for affordable sources of these ω-3 polyunsaturated fatty acids (PUFA) is important for consumer guidance and dietary counseling. Fish is an important source of PUFA and has unique advantage that there are many food fish species available and consumers have a wide choice owing to availability and affordability. The Indian subcontinent harbors a rich fish biodiversity which markedly varies in their nutrient composition. Here we report the DHA and EPA content and fatty acid profile of 39 important food fishes (including finfishes, shellfishes, and edible molluscs from both marine water and freshwater) from India. The study showed that fishes Tenualosa ilisha, Sardinella longiceps, Nemipterus japonicus, and Anabas testudineus are rich sources of DHA and EPA. Promotion of these species as DHA rich species would enhance their utility in public health nutrition.

O-heterocyclic derivatives with antibacterial properties from marine bacterium Bacillus subtilis associated with seaweed, Sargassum myriocystum

The brown seaweed, Sargassum myriocystum associated with heterotrophic bacterium, Bacillus subtilis MTCC 10407 (JF834075) exhibited broad-spectra of potent antibacterial activities against pathogenic bacteria Aeromonas hydrophila, Vibrio vulnificus, and Vibrio parahaemolyticus. B. subtilis MTCC 10407 was found to be positive for polyketide synthetase (pks) gene, and therefore, was considered to characterize secondary metabolites bearing polyketide backbone. Using bioassay-guided fractionation, two new antibacterial O-heterocyclic compounds belonging to pyranyl benzoate analogs of polyketide origin, with activity against pathogenic bacteria, have been isolated from the ethyl acetate extract of B. subtilis MTCC 10407. In the present study, the secondary metabolites of B. subtilis MTCC 10407 with potent antibacterial action against bacterial pathogens was recognized to represent the platform of pks-1 gene-encoded products. Two homologous compounds 3 (3-(methoxycarbonyl)-4-(5-(2-ethylbutyl)-5,6-dihydro-3-methyl-2H-pyran-2-yl)-butyl benzoate) and 4 [2-(8-butyl-3-ethyl-3,4,4a,5,6,8a-hexahydro-2H-chromen-6-yl)-ethyl benzoate] also have been isolated from the ethyl acetate extract of host seaweed S. myriocystum. The two compounds isolated from ethyl acetate extract of S. myriocystum with lesser antibacterial properties shared similar structures with the compounds purified from B. subtilis that suggested the ecological and metabolic relationship between these compounds in seaweed-bacterial relationship. Tetrahydropyran-2-one moiety of the tetrahydropyrano-[3,2b]-pyran-2(3H)-one system of 1 might be cleaved by the metabolic pool of seaweeds to afford methyl 3-(dihydro-3-methyl-2H-pyranyl)-propanoate moiety of 3, which was found to have no significant antibacterial activity. It is therefore imperative that the presence of dihydro-methyl-2H-pyran-2-yl propanoate system is essentially required to impart the greater activity. The direct involvement of polarisability (Pl) with the target bioactivity in 2 implied that inductive (field/polar) rather than the steric effect (parachor) appears to be the key factor influencing the induction of antibacterial activity. The present work may have a footprint on the use of novel O-heterocyclic polyketide products from seaweed-associated bacterium for biotechnological, food, and pharmaceutical applications mainly as novel antimicrobial secondary metabolites.

Micronutrient Composition of 35 Food Fishes from India and Their Significance in Human Nutrition

The micronutrients (vitamins and minerals) are required in small amounts but are essential for health, development, and growth. Micronutrient deficiencies, which affect over two billion people around the globe, are the leading cause of many ailments including mental retardation, preventable blindness, and death during childbirth. Fish is an important dietary source of micronutrients and plays important role in human nutrition. In the present investigation, micronutrient composition of 35 food fishes (includes both finfishes and shellfishes) was investigated from varying aquatic habitats. Macrominerals (Na, K, Ca, Mg) and trace elements (Fe, Cu, Zn, Mn, Se) were determined by either atomic absorption spectroscopy (AAS) or inductively coupled plasma mass spectrometry (ICP-MS)/atomic emission spectrometry (ICP-AES). Phosphorus content was determined either spectrophotometrically or by ICP-AES. Fat-soluble vitamins (A, D, E, K) were analyzed by high-performance liquid chromatography (HPLC). The analysis showed that, in general, the marine fishes were rich in sodium and potassium; small indigenous fishes (SIFs) in calcium, iron, and manganese; coldwater fishes in selenium; and the brackishwater fishes in phosphorous. The marine fishes Sardinella longiceps and Epinephelus spp. and the SIFs were rich in all fat-soluble vitamins. All these recommendations were made according to the potential contribution (daily value %) of the species to the recommended daily allowance (RDA). Information on the micronutrients generated would enhance the utility of fish in both community and clinical nutrition.