Isolation and Characterization of Two New Isolates of Sprirulina Platensis from the Rice Fields of Visakhapatnam

Spirulina belongs to the family Oscillatoriaceae which is characterized by spiral shaped chains of cells. Spirulina is one of the rich sources of proteins and is used as a protein supplement by humans. It is also used as a supplement is poultry industry and aquaculture units. It is also cultivated for its bluish green pigment, called Phycocyanin and of all the microbial sources Spirulina is the major source of phycocyanin. Purification and stability of the phycocyanin is often the limiting step in its use in the pharmaceutical applications. In the present study, two new cultures of Spirulina Platensis were isolated from the rice fields of Visakhapatnam and characterized. The two cultures were named SP2 and SP3. Phycocyanin was extracted from these two cultures using three different methods viz., Liquid Nitrogen method, freezing and thawing method and Lysozyme methods. Among these three methods, Liquid Nitrogen method was found to yield highest quantity of Phycocyanin. Absorption spectral scanning of phycocyanin was done using spectrophotometer at 500, 540, 580, 620, 660, 700 ?A. The readings at these absorbances were 0.278, 0.59, 1.538, 2.157, 0.573, and 0.075 respectively. The highest absorbance values were obtained at 620 ?A. The extracted phycocyanin was purified in a step wise manner using Ammonium sulphate precipitation, dialysis, Sephadex G25 and G100 column chromatography. The purity of the phycocyanin was checked by measuring the ratio of A620/A280 values. Maximum purity of 4.98 was obtained after the sephadex G100 step. With an aim of cloning the Phycocyanin gene from these new cultures, primers were designed using the sequence of phycocyanin gene from Synechococcus sp. in the NCBI nucleotide database. A partial sequence of apo ? phycocyanin was amplified and cloned consisting of 271 bp of ? subunit and 363 bp of ? subunit. The ? subunit and ? subunit were separated by 103 bp intergenic sequence.