BY D.SABARIANNAI

BYD.SABARIANNAI

AIM OF THE STUDY

• To screen the cyanobacteria which tolerate high salinity.

• To study what are the Antioxidant enzymes produced in this cyanobacteria.

• To use this cyanobacteria for reclamation of saline soil.

• We selected 10 organism for screening.• Oscillatoria salina 110551.• Oscillatoria boryana 91531.• Oscillatoria willei 130711.• Chrococcus minor 91342.• Synechococcus elongatous 141741.• Spirulina subsalsa 101022.• Phormidium valderianum 20041• Phormidium valderianum 30501.• Phormidium corium 91921• Gleocapsa crepidium 20372.

• Out of 10 organisms Oscillatoria willei Mo711 tolerate high salinity.

• This organism is treated in 3 different conditions 0,25,100 ppt.

• The treatment is done for 24 , 48 hours.• Then it is centrifuged & protein is extracted.• protein is estimated and loaded in native gel

for anti-oxidative enzyme study.

Anti oxidative Enzyme • SOD staining:• Gel is washed in Tris HCl PH 8.• Riboflavin - 4mg, EDTA - 2mg, NBT – 20mg

are added to Tris HCl PH 8.• Incubate gel for 30 min in dark.• Illuminate the gel under light for 10- 15 min at

RT.• Achromatic regions are visualised dark blue

background.

0 25 100 0 25 10024 Hrs 48 Hrs

Total SOD

0 25 100 0 25 100 0 25 100 0 25 10024 Hrs 48 Hrs

24 Hrs 48 Hrs

Types of SOD

Peroxidase3,3‘-diaminobenzidine(DAB) oxidised DAB.

• Gel is washed with sodium acetate buffer PH 4.5.• DAB – 30mg, H202 - 250µl are added to sodium

acetate buffer & incubate gel in this solution until brown bands are visualised.

0 25 100 0 25 100

24 Hrs 48 Hrs

Peroxidase

Catalase• H2O2 H2O+ O2• Gel is washed in potassium phosphate buffer

PH 7.0.• Gel is incubated in potassium phosphate

buffer containing H2O2 →3µl, 2% potassium ferric cyanide, 2% ferric chloride.

• The yellow colour bands are appeared on a dark green background.

• Gel is washed in phosphate buffer pH 7.5. • Gel is incubated in phosphate buffer PH 7.5

containig α- napthyl acetate → 50mg, β -napthyl acetate → 50mg,Fast blue RR → 100mg .

• Incubate the gel in this solution until dark gray colour band appeared.

Esterase

0 25 100 0 25 100

24 Hrs 48 Hrs

• SDS – PAGE still in process.• We are standardizing the SDS-PAGE.

Yet to be done:2D gel

0 25 100 0 25 100

24 Hrs 48 Hrs

SDS-PAGE