Bacterial Alkaliphile Lab Report

The 1.0 g of soil sample was transferred to 100 ml sterilized distilled water in 250 ml conical flask and agitated (200 rpm) at 37°C for 15 min in shaker. The suspension was then diluted to 10־7 dilutions. One ml of each diluted sample was spread by spread plate technique into petri plates containing Horikoshii medium and nutrient agar medium and inoculated at 37°C for 24 h12.

Screening of bacterial alkaliphiles:

Individual bacterial colonies were screened for proteolytic activities on Skim milk agar medium. The pH of the medium was adjusted to pH 10 with 1N NaOH before and after sterilization. The inoculated plates were incubated at 37oC for 48 hrs and observed for zones of clearance, indicating proteolytic activities.

Identification of the

Estimation of proteases was carried out with 2.5 ml of casein in a test tube; 1 ml of enzyme source was added and incubated for 10min at room temperature. After incubation 2 ml of TCA was added to stop the reaction and centrifuged the reaction mixture at 5000-8000 rpm for 15 min. Supernatant was separated and 2 ml of Na2CO3 and 1 ml of Folin-Ciacalteau reagent were added in 1 ml of supernatant. The reaction mixture was boiled for 1 min in a boiling water bath and 6 ml of distilled water was added to make a final solution to 10 ml. In control tube, the reaction was terminated the reaction at zero time and the absorbance was read at 650

The activity of the protease was then measured.

Effect of enzyme concentration on alkaline protease activity:

The effect of enzyme concentration on alkaline protease activity was determined by incubating the reaction mixture (pH 10) for 10 minutes at different enzyme concentration ranging from 0.5 ml to 5 ml. The activity of the protease was then measured.

Effect of different metal ions on protease activity:

The effect of different metal ions on alkaline protease activity was determined. The enzyme assay was performed in the reaction mixture as described above in the presence of various metal ions at a final concentration of 1 mM. The activity of the enzyme without any additives was taken as 100 %. The influence of various metal ions such as Potassium chloride¸ Sodium chloride, Ferric chloride, Barium chloride, Cupric chloride and Magnesium chloride (1 mM each) on protease activity was studied by pre-incubating the enzyme with the compounds for 15 min at 37°C. Then, the remaining activity was measured under the enzyme assay