al.. Cyclodextrin glucosyltransferase (CGTase) producing alkalophilic bacteria were isolated from the water samples collected from Marneri pond of Tirunelveli, Tamil Nadu by serial dilution and plating method. Totally 22 bacteria were isolated from the collected water samples and screened for CGTase activity by Horikoshis medium II agar plate method. Among 22 bacterial isolates, 15 isolates showed CGTase activity and better zone producing strain was selected for further studies. The potential strain was identified as Bacillus circulans by 16S rDNA gene sequencing experiment. The best enzyme activity was observed at pH 10.5, 32°C, supplemented with cassava as carbon source an d beef extract as nitrogen source.
This organism was cultured under solid-state fermentation for 72 h using wheat bran as the substrate. After 72 h, crude enzyme was extracted from the culture medium. The fibrinolytic enzyme was purified from the crude sample by various steps: ammonium sulfate precipitation, dialysis, ion exchange chromatography, and casein-agarose affinity chromatography. All purification steps were performed at 4°C until otherwise stated. The crude enzyme was precipitated at 70% saturation of ammonium sulfate, and the protein was collected by centrifugation (10,000×g for 10 min).
The tube was placed back in incubation for 96 more hours to observe any more positives. 2.10 Catalase Test A trypticase soy agar plate was used and after incubation, four drops of 3% Hydrogen Peroxide was added to the plate to flow over the bacterial growth. A presence of bubbling was observed. 2.11 Starch Hydrolysis A starch agar plate was inoculated with a streak of the unknown bacteria and then incubated. On the second day of incubation, the plate was removed from the incubator and placed over a hot plate heating Iodine solids.
The supernatant was then removed without disturbing the pellet. This process was repeated twice more until a total of 2.5 mL of cell suspension was removed from each culture and all the supernatant was removed from each micro centrifuge tube. The original protocol called for a total of 1.2 mL of cell suspension to be taken from each culture, but the laboratory staff determined that a greater volume was required in order to get a sufficient DNA concentration to be used for the DNA digestion. To each tube was added 200 μL Cell Suspension Solution, followed by vortexing until the pellets were completely re-suspended. Next 200 μL Cell Lysis Solution was added to each tube and then mixed
Smaller and smaller organelles may be sendimented by successive centrifugation at increasing speed. 1ml of the homogenate (H) was pipetted into a clean microfuge tube labelled P1. The tube labelled ‘P1’ was centrifuged at 1,000g for 10 minutes to sediment the first pellet (P1). The supernatant was carefully removed with an automatic pipette and placed in a micro centrifuge tube labelled ‘P2’. The P2 tube was then placed in the refrigerated centrifuge at a speed of 15,000g for 30-60 minutes at 4°C.
Remove excess water from the surface of the gram stained glass slide and observed under 1000X oil immersion microscope. Gram-staining have performed for Staphylococcus aureus (control); Enterococcus faecalis (control); Nostril microflora in NA, MSA, and PYCa. Gram-staining provides results of the bacteria morphology, type of gram-stain. Catalase test was also done prior to gram-staining. MicrobactTM Biochemical Identification Kit was used for identifying gram-negative aerobic and facultatively anaerobic bacteria.
The media used in this experiment was Trypticase nitrate broth. The reagents used (A and B) were sulfanilic acid and alpha-naphthylamine (respectively). Using aseptic technique, the bacterium (16A and 16B) were inoculated into labeled broth test tubes. The tubes were incubated for 48 hours at 37 degrees Celsius. When the incubation was complete 5 drops of reagent A and 5 drops of reagent B were added to each of the broths.
Although the astaxanthin content n Haematococcus accounts to more than 2% on dry weight basis, the tough cell wail of cyst cells hinders solvent extraction and intestinal absorption of the pigment from the intact algal biomass. Methods of cell wall disruption which have been applied include mechanical breakage, chemical hydrolysis and lytic enzymes ( Okabue, RN and -ewis MJ 1983 Biotechnol.Lett 5: 731-736., Grung M, D'Souza, FML, Borowitzka M, and Jaaen Jensen S 1992, J appi Phycol 4: 165-171) either denature astaxanthin or are cumbersome and difficult to apply on a large scale. Therefore the present invention provides an alternate improved process for the extraction of carotenoids from encysted Haematococcus cells which facilitated extraction of carotenoids with out homogenisation of cells or use of lytic enzymes. Procedures currently reported for the extraction of astaxanthin from microorganisms are the
The extraction, purification, characterisation and its biological activity of each prawn/shrimp lectin are discussed here. Introduction Lectins have been known from the beginning of 19th century. Agglutinin/lectins are proteins or glycoproteins which have the ability to bind to specific carbohydrates expressed on different cell surfaces and cause agglutination. This functional
The sterile medium was inoculated with known volume of inoculum and incubated at different temperatures (25, 30 and 350C) for different periods (48,72 and 96h) of fermentation. After fermentation the mouldy substrate was mixed with distilled water (1:4 w/v), agitated at through cheese cloth followed by centrifugation at 20,000 rpm for 20 minutes. The clear supernatant was used as crude enzyme. Assay of glucoamylase The assay of glucoamylase was carried out according to the method of Shazia Malik, Tehreema Iftikhar and Ikram Ul Haq13. One unit (U) of glucoamylase is defined as the amount that liberates 1 µmol of reducing sugar as glucose/ml/min under the assay condition.