Title of Invention

A PROCESS FOR THE PREPARATION OF FLUORESCENT PIGMENTS FROM BLUE-GREEN ALGAE.

Abstract

This invention relates to a process for the preparation of fluorescent pigments from blue-green algae. The process is for the extraction and isolation of C-phycocyanin. C-phycoerythrin and phycoerythrocyanin an ideal candidates for the use as fluorescent labels in immunoassay studies from blue-green algae Oscillatoria agardhii and Tolypothrix rechingeri. In the process extraction and purification of thermolabile, water soluble and fluorescence causing pigment protein fractions from Oscillatioria agardhii and Tolypothrix rechinger is carried out. In the process very low concentration of enzyme is used and the process is cost effective,economical and minimizes the loss ofNH2SO4

Full Text

Field of the Invention This invention relates to a process for the preparation of fluorescent pigments from blue-green algae (Osdllatoria agardhii and Tolypothrix rechingerf). This invention particularly relates to a process for the extraction and isolation of C-phycocyanin, C-phycoerythrin and phycoerythrocyanin an ideal candidates for the use as fluorescent labels in immunoassay studies from blue-green algae Osdllatoria agardhii_jmd Tolypothrix rechingeri Background of the Invention The phycobiliproteins are high molecular weight globular proteins found in three groups of algae, the red (Rhodophyta), blue-green (Cyanophyta) and Cryptomonad (Cryptomonadophyta). These phycobiliproteins are localized in the stroma of the photosynthetic tissue (Girand, 1966, Fuhs, 1964). Their function in the process of photosynthesis appears to be that of accessory light absorbers, trapped light energy being handed on to the primary photochemical agent, chlorophyll (Duysens, 1952). These phycobiliproteins have very low fluorescence yield in vivo, but increases enormously on extraction when transfer to chlorophyll is prevented. The actual light absorbing species are linear tetrapyrole derivatives; the phycobilins. These are attached to the apoprotein covalently, phycourobilin by a thioester link (Oh Eochna 1965 a). Phycobilisomes are made up entirely of protein. In cyanobacterial phycobilisomes, phycobiliproteins make up some 80% of the mass. The balance is made up of a linker poly-peptides, some of which also carry bilin prosthetic groups (Tandeau de Marsac and Cohen-Bazire 1977, Yamanaka et al., 1978; Lundell etal, 1981 a). The spectroscopic properties of these phycobiliproteins exhibit several unique qualitative and quantitative features making them ideal for use as fluorescent labels in immunoassay (Oi etal; 1982). The use of phycobiliproteins as covalently linked fluorescent tags are possible and practical and could result in many advantages when compared to conventional labels such as fluorescein. Extinction coefficients of phycobiliproteins is thirty times better than the conventional synthetic labels and their enormous extinction coefficient and high quantum efficiency can increase the sensitivity of fluorescent assays (Hermila, 1985). Abo-shaely A.M. etal., (1992) purified phycocyanin from marine blue-green algae Spirulina platensis. The culture medium was that of Zarrouk (1966) containing 0.25% NaNO3 and 8% NaCl. The light intensity used was 1000 lux at 35° C temperature and culturing was done for a period of 20 days. Zhang Cheng W etal (1996) isolated phycocyanin and allophycocyanin from Spirulina platensis with phosphate buffer pH 7 and (NH^SC^ was used to precipitate the biliprotein which was separated on Sephadex G-100 and by electrophoresis. Dainippon Ink and Chemicals Inc., (Jpn Kokai Tokyo Koho 8077890, 1980) used dried Spirulina for phycocyanin extraction. The dried alga was treated with protease enzyme (9001-92-7) in phosphate buffer pH 7.4 at 20° C for 5 h, and the extract was purified and concentrated by ultrafiltration and treated with pronase E at 40° C for 30 min. and the solution was concentrated in vacuum and'lyophilized. Wake, Hitoshi etal, (Jpn Kokai Jp 0746993, 1995) used ATCC culture of unicellular marine blue-green alga Synechococcus 27194, for phycocyanin production. The alga was grown on BG11 medium containing NaCl under illumination and at 25° C. Thermophilic blue green unicellular alga Synechococcus sp was used by kumano Masanobu, (Jpn Kokai Jp 6206691 1987). The alga was grown in a medium containing NaNC3, KH2PO4, Mg SO4, EDTA, Na2CO3, transition metal salts and distilled water. The algal strain was cultured at 50° C under 2000-5000 lux light intensity for 10 days. The isolation method for phycocyanin extraction involved sonication of the cells in phosphate buffer pH6, precipitation of the phycocyanin proteins with saturated and ultra filtration of the protein using dialysis membrane. Glazer and Suen Fang (1973) have used sodium acetate buffer, pH 5.5 to extract phycoerythrin and phycocyanin from Synechococus sp 630. The algal cells were broken by means of French pressure cells at 17000 psi at 4° C. The pigment proteins were precipitated with (NH^SC^ and dialysed at 4° C. Further purification of the proteins were done on DEAE cellulose DE-52 yielding phycoerythrin and phycocyanin. Akihiko Hattori and Yoshihiko Fujita (1959) isolated phycocyanin, allophycocyanin and phycoerythrin from Tolypothrix tenuis grown under incandescent light. Sonic disrupter was used to break the algal cells and centrifuged at 20,000 g to 1,00,000 g for 15 min. to 1 hr, the proteins were precipitated using (NFL^SC^ at 0.5 M saturation. The precipitate was dialyzed and absorbed on Ca3(PO4)2 gel column and eluted with 0.05 M and 0.1 M KH2P04 . This process was repeated 2-3 time:; and the protein purified by column chromatography and dialyzed. Most of the algae used during earlier investigations were marine , thermophilic forms and procured from ATCC. The extraction procedures including growth conditions required lower temperatures ranging from 4° C to 20° C. Rupturing of the algal cells were done mostly by sonication and by high pressure technique (French pressure cells), and when enzymes were used they were in higher concentration and the process repeated more than once. The algae reported in this invention are tropical fresh water forms, available in plenty in moist places and does not need extra culture conditions and can be grown under room temperature. The applicants have used low concentrations of enzyme to break the cells. The advantage of the present process is that the extraction procedure will be much economical and less time censuring than the reported procedures. Objects of the Invention The main objective of the present invention is to provide simple and cost effective process for the preparation of fluorescent pigments from blue-green algae. Another objective of the invention is to use naturally available species of blue-green algae for the preparation of fluorescent pigments. The source of blue green algae is ponds located at Osmania University campus, Hyderabad and was deposited on 24-4-200 at Indian Institute of Chemical Technology,Hyderabad. Summary of the Invention This invention particularly relates to a process for the extraction and isolation of C-phycocyanin, C-phycoerythrin and phycoerythrocyanin an ideal candidates for the use as fluorescent labels in immunoassay studies from blue-green algae Oscillatoria agardhii and Tolypothrix rechingeri. Detailed description of the Invention Accordingly the present invention provides a process for the preparation of fluorescent pigments from blue-green algae selected from Oscillatonia agardhii and Tolypothrix rechingeri which comprises preparing algal pellet by centrifugation., treating the algal pellet with lysozyme or papain such as herein described or thawing in the presence of phosphate or acetate buffer at pH in the range of 4.5 to 6.0, separating the lysed cells from the mixture by centrifugation to get the supernatant, precipitating the protein pigment by conventional precipitating agent (NH^SCU, purifying the precipitated pigment by known column chromatographic methods and removing the inorganic matter by dialysis from the fluorescent pigment and drying the purified fluorescent pigment by lyophilization. In an embodiment of the present invention the blue-green algae Oscillatoria agardhii and Tolypothrix rechingeri may be a potential source for C-phycocyanin, C-phycoerythin and phycoerythrocyanin production, as these algal species have not been investigated before. In another embodiment the algal cells are centrifuged at 6000-8000 rpm in a centrifuge at 24-30° C for 20-35 minutes to obtain the algal pellet. In still another embodiment all solutions used in extraction processes contained 0.1 mM- 0.1 MEDIA. » In still another embodiment lysozyme (Muramidase; mucopeptide N-acetyl muramoyl hydrolase EC 32.1.17 from chicken egg white Hi-media grade), papain (purified from papaya latex 100 T4 Loba grade) may be used for breaking the algal cell wall. In still another embodiment the buffer used is Na2HPO4-KH2PO4 (phosphate buffer) at pH ranging from 5-6, and of 0.05-0.1 M concentration. Sodium acetate-sodium chloride of pH 4.5-5.5 and cone. 0.05-0.1 M may also be used as buffer. In still another embodiment the lysed cell are separated by known method such as centrifugation at 6000-12,000 rpm and 24 -30 ° C for 20-30 min. In still another embodiment the precipitating agents used is 60% w/v Acetic acid and its chlorinated derivatives may also be used as precipitating agents but are likely to cause deactivation of protein pigments. In still another embodiment the absorbent used in column chromatography such as DEAE cellulose (Diethylaminoethyl cellulose an ion exchanger). Sephadex anion exchanger may also be used as adsorbent for purification of protein pigments. In still another embodiment the eluent used is phosphate buffer (NaiHPCV KH2PO4) of 0.05-0.1 M concentration. Sodium acetate- sodium chloride buffer of same range of concentration may also be used. In still another embodiment the removal of inorganic matter is effected by conventional method such as dialysis using cellulose dialysis sacks 16 mm x 30 cm (250-94, Sigma). In still another embodiment the dialysis is effected in distilled water for 12-24 hours at room temperature (26° C - 30° C). In yet another embodiment the drying is effected by lyophilization. The present invention resulted in the separation of water soluble, thermolabile C-phycocyanin from Oscillatoria agardhii and C-phycoerythrin and phycoerythrocyanin from Tolypothrix rechingeri. The phycobiliproteins are fluorescent emitting at all stages of their preparation and are identified based on their absorption maxima and fluorescene excitation maxima as C-phycocyanin(614,652nm);C-phycoerythrin(565,580nm) and phycoerythrocyanin(568,620). The source, blue-green algae (Oscillatoria agardhii and Tolypothrix rechingeri) are found to grow in moist places, water bodies, sewage canals, waste waters and rice fields rich in organic and inorganic carbon, nitrogen and sulfur compounds. The collection of the micro organism is by way of collecting the mixed population of the organisms along with the water in which they flourish in natural condition. The organism from the natural source is grown in the laboratory by purifying the organism from the mixed population on 2% agar with BG11 medium. The composition of the BG11 medium being NaNO3, 1.5 g; KH2PO4.3H2O, 0.04 g; MgSO4.7H20, 0.075g; CaCl2.2H2O, 0.036 g; citric acid 0.006 g; Ferric ammonium citrate, 0.006 g; EDTA (Ethylene diamine tetra acetic acid, disodium salt), 0.001 g; Na2CO3 0.020 g in one litre of distilled water adding 1 ml. of trace metal mix designated as AS . The trace metal mix A5 is composed of the following salts H3BO3, 2.860 mg/ml; MnCl2.4H2O, 1.810 mg/ml; ZnSO4.7H2O,0.22 mg/ml; Na2MoO4.2H2O, 0.390 mg/ml; CuSO4.5H20, 0.070 mg/ml and Co (NO3)2.6H2O, 0.0494 mg/ml. After purifying and isolating the algae on 2% agar these algae are grown in large quantities in liquid BG11 medium at pH 8.5 under white fluorescent lights with 4000 lux light intensity and the temperature ranging from 26° C to 30° C. The cultures are flushed with air at the rate of 200 ml/min throughout the experiments. The algae, Oscillatoria agardhii takes one month time for harvesting while Tolypothrix rechingeri takes three months, and the yields of algae on wet weight basis are 6gm/L and 8 gm /L respectively. The procedures for the isolation of C-phycocyanin, C-phycoerythrin and phycoerythrocyanin are simple and the results are reproducible. The pigments are thermolabile and produce high fluorescence at 652 nm, 580 nm and 620 nm, respectively. Accordingly the present invention provides a process for the extraction and purification of thermolabile, water soluble and fluorescence causing pigment protein fractions from Oscillatoria agardhii and Tolypothrix rechingeri. The algae after attaining sufficient growth are harvested from the growth medium by centrifuging the cultures in a cold centrifuge at 6000 rpm for 20 min, the supernatant liquid is discarded and the algal pellet is dispersed in distilled water and centrifuged for 20 min. in a cold centrifuge. This washing process is repeated twice discarding the supernatant liquid and collecting the algal pellets. To release the chromoproteins the algal pellets are dispersed in phosphate buffer pH 6 (0.1 m Na2HPO4-kH2 PCXO containing 6 mg/ml of lysozyme (muramidase; mucopeptide N-acetylmuramoyl hydrolase EC32.1.17 protein, Hi-Media) in the ratio of 1:10 (1 gm. wet weight of the algae in 10 ml. of lysozyme phoshate buffer solution) and kept overnight to break the cells. Later the lysed cells are separated from the pigment mixture by centrifuging the contents at 10,000 rpm in a cold centrifuge for 20 min. The supernatant solution containing the chromoproteins are purified by precipitating with 60% w/v (NH4)2SO4 and centrifuing at 12,000 rpm for 20 min. in a cold centrifuge. The supernatant discarded and the chromoprotein precipitate is dissolved in minimum volume (20 ml) of 0.1 M (Na2HPO4-KH2PO4) phosphate buffer pH 6 and re-precipitated with 60% (NH4)2SO4; the precipitate is dissolved in minimum quantity (20 ml) of phosphate buffer pH 6 and 15 ml. of these chromoprotein portions are chromatographed on 30 cm x 1.5 cm column of DEAE - Cellulose equilibrated with same phosphate buffer pH 6 and developed with the same buffer. Separate columns are run for separate blue-green algae. The pigment protein fractions of C-phycocyanin from Oscillatoria agardhii and C-phycoerythrin and phycoerythrocyanin from Tolypothrix rechingeri are collected and precipitated with 60% w/v (NH4)2SO4 centrifuged at 12,000 rpm for 20 mts. and dissolved in minimum volume (10 ml.) of 0.1 M phosphate buffer pH 6 and dialysed overnight against 100 vol. of distilled water in cellulose dialysis sacks 16 mm x 30 cm (250 - 94, Sigma). The dialysed pure C-phycocyanin from Oscillatoria agardhii has an absorption maxima of 614 nm +and fluorescence excitation maxima of 652 nm and C-phycoerythrin and phycoerythrocyanin from Tolypothrix rechingeri have absorption maxima of 565mm and 568 nm and fluorescence excitation maxima of 580 nm and 620 nm. The dialysed pure C-phycocyanin from Oscillatoria agardhii and C-phycoerythrin and phycoerythrocyanin from Tolypothrix rechingeri are made to powder form by lyophilization. The following examples are given by way of illustration and therefore should not be construed to limit the scope of the present invention. Example 1 The blue-green algae Oscillatoria agardhii was cultured in BG11 medium pH 8.5 in 650 ml. Glass bottles with continuous aeration at the rate of 200 ml/min. throughout the experiment. The cultures were illuminated with white fluorescent lights of 4000 lux intensity and maintained at a temperature ranging from 26° C to 30° C. Sufficient quantities of biomass generated 6 gm/L for Oscillatoria agardhii in one month time. The algae were centrifuged at 6000 rpm for 20 min. in a cold centrifuge, the supernatant liquid discarded and the algal pellets dispersed in distilled water and re-centrifuged. This process was repeated twice. The washed algal pellet weighing around 6 gm wet weight, for Oscillatoria agardhii were dispersed in phosphate buffer pH 6 containing 6 mg/ml of lysozyme in the ratio 1:10 (for every gram wet weight of the algae 10 ml. of lysozyme phosphate buffer used) and kept overnight to break the cells. The lysed cells were separated from the pigment mixture by centrifuging the contents at 10,000rpm in a cold centrifuge for 25 min. The supernatant solution containing the chromo-proteins purified by precipitating with 60% w/v (NH4)2SO4and centrifuging at 10,000 rpm for 25 min. in a cold centrifuge. The supernatant discarded and the chromoprotein precipitate dissolved in minimum volume (20 ml) of 0.1 M phosphate buffer pH 5 and re-precipitated with 60% w/v (NH4)2SO4 ; the precipitate was dissolved in minimum quantity (20 ml) of 0.1 M phosphate buffer pH 5 and 15 ml. of this chromoprotein portions chromatographed on 30 cm x 1.5 cm column of DEAE-cellulose equilibrated with same phosphate buffer pH 5 and developed with the same buffer. The purified pigment protein fractions of C-phycocyanin from Oscillatoria agardhii was collected and precipitated with 60% w/v (NH4)2SO4 and centrifuged at 10,000 rpm for 25 min. and dissolved in minimum quantity (10ml) of 0.1 M phosphate buffer pH 5 and dialysed overnight against 100 vol. of distilled water in cellulose dialysis 100 vol. of distilled water in cellulose dialysis sacks 16 mm x 30 cm (250 gm.Sigma). The dialysed pure C-phycocyanin from Oscillatoria agardhii has an absorption maxima of 614 nm and fluorescence excitation maxima of 652 nm. The dialysed pure C-phycocyanin from Oscillatoria agardhii was made to powder form by lyophilization. The yields after lyophilization were 0.3% of C-phycocyanin from Oscillatoria agardhii. Example 2 The blue-green algae Tolypothrix rechingeri was cultured in BG11 medium pH 8.5 in 650 ml. Glass bottles with continuous aeration at the rate of 200 ml/min. throughout the experiment. The cultures were illuminated with white fluorescent lights of 4000 lux intensity and maintained at a temperature ranging from 26° C to 30° C. Sufficient quantities of biomass 8 g/L for Tolypothrix rechingeri in three months time. The algae were centrifuged at 6000 rpm for 20 min. in a cold centrifuge, the supernatant liquid discarded and the algal pellets dispersed in distilled water and re-centrifuged. This process was repeated twice. The washed algal pellet weighing around » 8 gms. for Tolypothrix rechingeri were dispersed in phosphate buffer pH 6 containing 6 mg/ml of lysozyme in the ratio 1:10 (for every gram wet weight of the algae 10 ml. of lysozyme phosphate buffer used) and kept overnight to break the cells. The lysed cells were separated from the pigment mixture by centrifuging the contents at 12,000 ipm in a cold centrifuge for 20 min. The supernatant solution containing the chromo-proteins purified by precipitating with 60% w/v (NH^a SO4, and centrifuging at 12,000 rpm for 20min. in a cold centrifuge. The supernatant discarded and the chromoprotein precipitate dissolved in minimum volume (20 ml) of 0.05 M phosphate buffer pH 6 and re-precipitated with 60% w/v (NH4)2SO4 ; the precipitate was dissolved in minimum quantity (20 ml) of 0.05 M phosphate buffer pH 6 and 15 ml. of this chromoprotein portions chromatographed on 30 cm x 1.5 cm column of DEAE-cellulose equilibrated with same phosphate buffer pH 6 and developed with the same buffer. The purified pigment protein fractions of C-phycoerythrin and phycoerythrocyanin from Tolypothrix rechingeri were collected and precipitated with 60% w/v (NH4)2SO4 and centrifuged at 12,000 rpm for 20 min. and dissolved in minimum quantity (10ml) of 0.05 M phosphate buffer pH 6 and dialysed overnight against 100 vol. of distilled water in cellulose dialysis 100 vol. of distilled water in cellulose dialysis sacks 16 mm x 30 cm (250 gm Sigma). The dialysed pure C-phycoerythrin and C-phycoerythrocyanin from Tolypothrix rechingeri have absorption maxima of 565 nm and 568 nm and fluorescence excitation maxima of 580 nm and 620 nm respectively. The dialysed pure C-phycoerythrin and phycoerythrocyanin from Tolypothrix rechingeri were made to powder form by lyophilization. The yields after lyophilization were 0.65% of C-phycoerythrin and 0.10% of phycoerythrocyanin from Tolypothrix rechingeri. Example 3 The blue-green algae Tolypothrix rechingeri was cultured in BG11 medium pH 8.5 in 650 ml. glass bottles with continuous aeration at the rate of 200 ml/min. throughout the experiment. The cultures were illuminated with white fluorescent lights of 4000 lux intensity and maintained at a temperature ranging from 26° C to 30° C Tolypothrix rechingeri generated around 7.5 gm/L of biomass on wet weight basis in one months time. The algae were centrifuged at 6000 rpm for 20 min. in a cold centrifuge, the supernatant liquid discarded and the algal pellets dispersed in distilled water and re-centrifuged, this process repeated twice. The washed algal pellets were taken into 20 ml. of acetate buffer and the algae were frozen overnight. The thawed cells were centrifuged at 10,000 rpm for 20 min. to remove particulate matter and the supernatant collected. The algal pellets washed with 20 ml. of acetate buffer each time and centrifuged, this process repeated twice and the coloured chromoprotein mixture in the supernatant pooled and precipitated with 60% (NHj^SC^ and centrifuged at 12,000 rpm for 20 min. in cold centrifuge. The coloured protein precipitate dissolved in 20 ml. of acetate buffer and 15 ml. of this protein portion chromatographed on 30 mm x 1.5 cm column of DEAE-cellulose equilibrated and developed with same phosphate buffer pH 6. The purified pigment protein fraction from Tolypothrix rechingeri was collected and precipitated with 60% w/v (NH4)2SO4 and centrifuged at 12,000 rpm for 20 min. the precipitate thus obtained was dissolved in minimum (10 ml.) quantity of 0.1 M acetate buffer pH 5.0 and dialysed overnight against 100vol. of distilled water in cellulose dialysis sacks 16 mm x 30 cm (250-94, Sigma). The dialysed pure C-phycoerythrin and Phycoerythrocyanin from Tolypothrix rechingeri have absorption maxima of 565 nm and 568 nm and fluorescence excitation maxima of 580 nm and 620 nm respectively. The dialysed pure C-phycoerythrin from Tolypothrix rechingeri was made to powder form by lyophilization. The yields after lyphilization were 0.35% phycoerythrin and 0.02% phycoerythrocyanin from Tolypothrix rechingeri on dry weight basis. Example 4 The blue-green algae Tolypothrix rechingeri were cultured in BG11 medium pH 8.5 in 650 ml. glass bottles with continuous aeration at the rate of 200 ml/min. throughout the experiment. The cultures were illuminated with white fluorescent lights of 4000 lux intensity and maintained at a temperature ranging from 26° C to 30° C Tolypothrix rechingeri generated around 7.6 gm/L of biomass on wet weight basis in three months time. The algae were centrifuged at 6000 rpm for 20 min. in a cold centrifuge, the supernatant liquid discarded and the algal pellets dispersed in distilled water and re-centrifuged, this process repeated twice. The washed algal pellets were taken into 20 ml. of phosphate buffer and the algae were frozen overnight. The thawed cells were centrifuged at 10,000 rpm for 20 min. to remove paniculate matter and the supernatant collected. The algal pellets washed with 20 ml. of phosphate buffer each time and centrifuged, this process repeated twice and the coloured chromoprotein mixture in the supernatant pooled and precipitated with 60% (NH4)2SO4 and centrifuged at 12,000 rpm for 20 min. in cold centrifuge. The coloured protein precipitate dissolved in 20 ml. of phosphate buffer and 15 ml. of this protein portion chromatographed on 30 mm x 1.5 cm column of Sepahdex anian exchanger equilibrated and developed with same phosphate buffer pH 6. The purified pigment protein fraction C-phycoerythrin and phycoerythrocyanin from Tolypotherix rechingeri were collected and precipitated with 60% w/v (NH4)2SO4 and centrifuged at 12,000 rpm for 20 min. the precipitate thus obtained was dissolved in minimum (10 ml.) quantity of 0.1 M phosphate buffer pH 6 and dialysed overnight against 100vol. of distilled water in cellulose dialysis sacks 16 mm x 30 cm (250-94, Sigma). The dialysed pure C-phycoerythrin and phycoerythrocyanin from Tolypothrix rechingeri have absorption maxima of 565 nm and 568 nm and fluorescence excitation maxima of 580 nm and 620 nm respectively. The dialysed pure C-phycoerythrin and phycoerythrocyanin from Tolypothrix rechingeri were made to powder form by lyophilization. The yields after lyphilization were 0.4% of C-phycoerythrin and 0.02% of phycoerythrocyanin from Tolypothrix • rechingeri on dry weight basis. Example 5 The blue-green algae Oscillatoria agardhii was cultured in BG11 medium pH8.5 in 650 ml. Glass bottles with continuous aeration at the rate of 200 ml/min. throughout the experiment. The cultures were illuminated with white fluorescent lights of 4000 lux intensity and maintained at a temperature ranging from 26° to 30 ° C. The biomass generated for Oscillatoria agardhii for one month was 6.5 gm/L. The algae were harvested by centrifuging the algal cultures at 6000 rpm for 20min. in a cold centrifuge, discarding the supernatant the algal pellets were dispensed in distilled water and re-centrifuged, the process of washing repeated twice. The washed algal pellets were dispersed in phosphate buffer pH 6 containing 60 mg/ml of papain (purified from papaya latex 100 T4 Loba grade) in the ratio of 1:10 (for every gram wet weight of algae 10 ml. of phosphate buffer pH 6 taken) and kept overnight to rupture the cells. The cell debris is separated from the pigment mixture by centrifuging the contents at 10,000 rpm in a cold centrifuge for 20 min. the supernatant solution containing the proteins purified by precipitating with 60% w/v (NH4)2SO4 and centrifuging at 12,000 rpm for 20 min. in a cold centrifuge. The supernatant discarded and the coloured protein precipitate dissolved in minimum volume (20ml) of 0.1 M phosphate buffer pH 6 and re-precipitated with 60% w/v (NH4)2SO4; the precipitate was dissolved in 20 ml. of phosphate buffer pH 6 and 15 ml. of this portion chromatographed on 30 cm x 1.5 cm column of DEAE - Cellulose equlibrated with same phosphate buffer pH 6 and developed with the same buffer. The purified pigment protein fraction so obtained were precipitated with 60% w/v (NH4)2SO4 and centrifuged at 12,000 rpm for 20 min. and dissolved in 10 ml. of 0.1 M phosphate buffer pH 6 and dialysed overnight against 100 vol of distilled water in cellulose dialysis sacks 16 mm x 30 cm (250-94 Sigma). The dialysed pure C-phycocyanin from Oscillatoria agardhii has an absorption maxima of 614 nm and fluorescence excitation maxima of 652 nm; The dialysed pigment fractions were made to powder form by lyophilization. * The yields after lyophilization were 0.25% of C-phycocyanin from Oscillatoria agardhii.. Example 6 The blue-green algae Tolypothrix rechingeri were cultured in BG11 medium pH8.5 in 650 ml. Glass bottles with continuous aeration at the rate of 200 ml/min. throughout the experiment. The cultures were illuminated with white fluorescent lights of 4000 lux intensity and maintained at a temperature ranging from 26° to 30 ° C. The biomass generated for Tolypothrix rechingeri 7.8 gm/L in three months. The algae were harvested by centrifuging the algal cultures at 6000 rpm for 20min. in a cold centrifuge, discarding the supernatant the algal pellets were dispensed in distilled water and re-centrifuged, the process of washing repeated twice. The washed algal pellets were dispersed in acetate buffer pH 5 containing 60 mg/ml of papain (purified from papaya latex 100 T4 Loba grade) in the ratio of 1:10 (for every gram wet weight of algae 10 ml. of phosphate buffer pH 5 taken) and kept overnight to rupture the cells. The cell debris is separated from the pigment mixture by centrifuging the contents at 10,000 rpm in a cold centrifuge for 20 min. the supernatant solution containing the proteins purified by precipitating with 60% w/v (NH4)2SO4 and centrifuging at 12,000 rpm for 20 min. in a cold centrifuge. The supernatant discarded and the coloured protein precipitate dissolved in minimum volume (20ml) of 0.1 M acetate buffer pH 5 and re-precipitated with 60% w/v (NH4)2SO4, the precipitate was dissolved in 20 ml. of acetate buffer pH 5 and 15 ml. of this portion chromatographed on 30 cm x 1.5 cm column of DEAE - Cellulose equlibrated with same acetate buffer pH 5 and developed with the same buffer. The purified pigment protein fraction so obtained were precipitated with 60% w/v (NH4)2SO4 and centrifuged at 12,000 rpm for 20 min. and dissolved in 10 ml. of 0.1 M acetate buffer pH 5 and dialysed overnight against 100 vol of distilled water in cellulose dialysis sacks 16 mm x 30 cm (250-94 Sigma). The dialysed pure C- * phycoerythrin and phycoerythrocyanin from Tolypothrix rechingeri have absorption maxima of 565 nm and 568 nm and fluorescence excitation maxima of 580 nm and 620 nm respectively. The dialysed pigment fractions were made to powder form by lyophilization. The yields after lyophilization were 0.5% of C-phycoerythrin and 0.07% of phycoerythrocyanin from Tolypothrix rechingeri on dry weight basis. The present invention has following advantages: The blue-green algae investigated were local tropical species, available easily and abound in most places, fresh water ponds and rice fields and were stable at room temperature conditions. Rupturing of the blue-green algal cells in previous works were done using high pressure French Pressure cell at 17,000 psi at 4° C, sonic disrupters and enzymes. At times the enzyme treatments were repeated too, while we have used very low concentration (6 mg.) of the enzyme. To sum up our approach is more cost effective and economical and industrial level work could minimise the loss of (NH4)2SO4 by reusing the same after drying and crystallizing the same chemical. We Claim: 1. A process for the preparation of fluorescent pigments from blue-green algae selected from Oscillatonia agardhii and Tolypothrix rechingeri which comprises preparing algal pellet by centrifugation., treating the algal pellet with lysozyme or papain such as herein described or thawing in the presence of phosphate or acetate buffer at pH in the range of 4.5 to 6.0, separating the lysed cells from the mixture by centrifugation to get the supernatant, precipitating the protein pigment by conventional precipitating agent (NH^SO^ purifying the precipitated pigment by known column chromatographic methods and removing the inorganic matter by dialysis from the fluorescent pigment and drying the purified fluorescent pigment by lyophilization. 2. A process as claimed in claim 1 wherein the algal pellet prepared by known method by centrifuging at 6000-12000 rpm at 24 - 30° C for 20 - 30 min. 3. A process as claimed in claims 1 - 2 wherein the lysozyme used for breaking the algal cell wall is muramidase, mucopeptide n-acetylmuramoyl hydrolase; lyophillid powder from chicken egg white, Hi-Media; papain purified from papaya latex. 4. A process as claimed in claims 1 -3 wherein the buffer used is Na2HPO4-KH2PO4 or CH3COONg-NaC at pH in the range of 5 - 6 and 0.05 - 0. IM concentration containing 0.1 m M-0.1 MEDIA. 5. A process as claimed in claims 1-4 wherein the lysed cells are separated by known methods such as centrifugation at 6000-12000 rpm at 24° - 30° C for 20 - 30 min. 6. A process as claimed in claims 1-5 wherein the precipitating agent used in 60% w/v (NH4)SO4 containing 0.1 M - 0.1 M EDTA. 7. A process as claimed in claims 1 - 6 wherein the absorbent used in column chromatography in DEAE cellulose is Diethylaminoethyl cellulose, Ion exchanger or Sephadex anion exchanger. 8. A process as claimed in claims 1-7 wherein the eluent used is phosphate buffer (Na2HPO4KH2PO4) or acetate buffer of 0.05 - 0.1 M concentration containing 0.1 m M EDTA-0.1 MEDIA. 9. A process as claimed in claims 1 - 8 wherein the removal of inorganic matter is effected by conventional methods such as dialysis using cellulose dialysis sacks 16 mm x30 cm (250-94, Sigma). 10. A process as claimed in claims 1-9 wherein the dialysis is affected in distilled water for 12- 24 hours at room temperature of 26° - 30°C. 11. A process as claimed in 1 - 10 wherein the drying is effected by lyophilzation. 12. A process for the preparation of fluorescent pigments from blue-green algae as herein described with reference to the examples.

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331-del-2001-description (complete).pdf

331-del-2001-form-1.pdf

331-DEL-2001-Form-18.pdf

331-del-2001-form-2.pdf

331-DEL-2001-Form-3.pdf

331-del-2001-petition-138.pdf