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.

1. By
Pooja Walke

2.  "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.

3.  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.

4. 1] Sample collection and enrichment
2] Biochemical test
3] Enzyme extraction
4] SDS-PAGE

5.  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.

6. 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

7. COLUMN CHROMATOGRAPHY
SDS PAGE

8.  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)

9.  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

10.  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

11. Biochemical Tests Results
Starch Hydrolysis +
Catalase Test +
Oxidase Test -

12.  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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