By

Pooja Walke

"Lipases (try acryl glycerol acylhydrolase ) are

the enzymes which catalyze the hydrolysis and

the synthesis of ester formed from glycerol and

long chain fatty acid.

During hydrolysis lipases pick acryl group from

glycosides forming lipase acyl complex which

then transfers its acyl group to OH group water.

Enzyme production can be enhanced by

improving the medium. But the use of bacillus

subtilis in solid state fermentation is improved

maximum yield in the production of lipase.

Isolation of bacillus subtilis from soil

collected from the Mahalaxmi oil industry,

Nanded.

Lipase production by using four different oil

samples by submerged fermentation process.

Comparative study of lipase production on

different oil samples i.e groundnut oil,

coconut oil, soybean oil,gingelly oil etc.

1] Sample collection and enrichment

2] Biochemical test

3] Enzyme extraction

4] SDS-PAGE

SDS-PAGE of proteins was carried out in the

presence of sodium dodecyl sulphate – an

anionic detergent that readily binds and

dissociates oligomeric proteins in the

presence of reducing agent, 2-mercapto

ethanol into their subunits.

The protein SDS complex carries negative

charges, hence move towards the anode and

the separation is based on the size of the

protein.

REAGENTS Acrylamide 30% [W/V] Ammonium per sulfate (APS) 10% [2/V] Sodium dodecyl sulphate (SDS) 10% [W/V] Separating gel buffer 1.5M Tris Hcl (pH

8.8) Stacking gel buffer 1M Tris Hcl (pH6.8)

Sample Buffer Staining solution Destaining Solution Storage Solution Running Buffer

COLUMN CHROMATOGRAPHY

SDS PAGE

Lipase activity calculated by titrating against 0.1 N NaOH,

phenolphthalein as indicator was 0.0095µg/ml.

BAND OF SAMPLES

Bovine serum albumin (66.2 kids)

Effect of temperature on enzyme production:

Thus the maximum activity was reported at temperature 40˚C.

0

0.001

0.002

0.003

0.004

0.005

0.006

0.007

0.008

0.009

0.01

5 10 20 25 30 35 40 45

LIPASE ACTIVITY

Effect of pH on enzyme production:

Thus the maximum activity was reported at

pH 8.5.

0

0.001

0.002

0.003

0.004

0.005

0.006

0.007

0.008

0.009

0.01

5 6 6.5 7 7.5 8 8.5 9

LIPASE ACTIVITY

Biochemical Tests Results

Starch Hydrolysis +

Catalase Test +

Oxidase Test -

Lipases are extremely versatile enzyme, showing many interesting properties of industrial applications.

The production of lipase is significantly influenced by carbon sources (substrate), temperature, pH

The maximum production of lipase was obtained by substrate soya bean oil having pH 8.5 and temperature 40˚C.

Fermentation in shake flask improved the lipase yield with an activity 0.0095 µg/ml/min in 48 h using soybean oil as substrate by Bacillus subtillis with moisture content of 70%.

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