Production of Lipase

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"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 bacillussubtilis in solid state fermentation is improved

maximum yield in the production of lipase.


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Isolation ofbacillus subtilis from soil

collected from the Mahalaxmi oil industry,

Nanded.

Lipase production by using four different oilsamples by submerged fermentation process.

Comparative study of lipase production on

different oil samples i.e groundnut oil,

coconut oil, soybean oil,gingelly oil etc.


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1] Sample collection and enrichment

2] Biochemical test

3] Enzyme extraction

4] SDS-PAGE


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SDS-PAGE of proteins was carried out in the

presence of sodium dodecyl sulphate ananionic detergent that readily binds and

dissociates oligomeric proteins in thepresence of reducing agent, 2-mercapto

ethanol into their subunits.

The protein SDS complex carries negative

charges, hence move towards the anode andthe separation is based on the size of the

protein.


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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 BufferStaining solution

Destaining SolutionStorage SolutionRunning Buffer


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COLUMN CHROMATOGRAPHY

SDS PAGE


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Lipase activity calculated by titrating against 0.1 N NaOH,

phenolphthalein as indicator was 0.0095g/ml.

BAND OF SAMPLES

Bovine serum albumin

(66.2 kids)


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Effect of temperature on enzyme production:

Thus the maximum activity was reported at

temperature 40C.

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


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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


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Biochemical Tests Results

Starch Hydrolysis +

Catalase Test +

Oxidase Test -


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Lipases are extremely versatile enzyme, showingmany interesting properties of industrialapplications.

The production of lipase is significantly

influenced by carbon sources (substrate),temperature, pH

The maximum production of lipase was obtainedby substrate soya bean oil having pH 8.5 andtemperature 40C.

Fermentation in shake flask improved the lipaseyield with an activity 0.0095 g/ml/min in 48 husing soybean oil as substrate by Bacillussubtillis with moisture content of 70%.


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Alberghina L, Schmid RD, Verger R, editors. Lipases: structure,mechanism and genetic engineeringWeinheim:VCH, 1991.

Anguita J, Rodriguez-Aparicio LB, Naharro G. Purification, genecloning, amino acid sequencing analysis andexpression of an extracellularlipase from an Aeromonas hydrophila human isolate. Appl EnvironMicrobiol1993;59:2411 7.

Bornscheuer UT, editor. Enzymes in lipid modificationWeinheim: Wiley-

VCH, 2000.Bosley JA, Clayton JC. Blue print for a lipase support use ofhydrophobic controlled pore glasses asmodel systems. Biotechnol Bioeng1994;43:934 8.

Arroyo M, Sinisterra JV. Influence of chiral corvones on selectivity ofpure lipase-B from Candida antarctica.Biotechnol Lett 1995;17:525 30.

Biochimie 2001;83:387 97.Balcao VM, Paiva AL, Malcata FX. Bioreactorswith immobilized lipases: state of the art. Enzyme MicrobTechnol

1996;18:392 416. Becker P, Abu-Reesh I, Markossian S, Antranikian G, Markl H.

Determination of the kinetic parameters duringcontinuous cultivation ofthe lipase-producing thermophile Bacillus sp. [HI-9] on olive oil. ApplMicrobiolBiotechnol 1997;42:184 90.

Berto P, Belingheri L, Dehorter B. Production and purification of a novelextracellular lipase from Alternariabrassicicola. Biotechnol Lett1997;19:533 6.


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Production of Lipase

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