L9BT862,Y8BT818,Y8BT819

8/2/2019 L9BT862,Y8BT818,Y8BT819

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BY

Shanavaz kamal (L9BT862)

K . Swathi (Y8BT818)

K . Surya Tejaswi (Y8BT819)

Under The Guidance OfDr.K.Sobha

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INTRODUCTION

Lipase is an enzyme that catalyzes the formation orhydrolysis (cleavage) of lipids (fats).

Lipases are a subclass of the esterases.

Lipases perform essential roles in the digestion, transport

and processing of dietary lipids .

Genes encoding lipases are even present incertain viruses.

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Initially lipase digestion occur in the lumen of smallintestine.

Lipase is produced primarily in the pancreas but alsoin mouth and stomach.

The pancreatic enzyme are available in the tabletsand capsule forms.

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APPLICATIONS

DAIRY INDUSTRY

For hydrolysis of milk fat.

Enhancement of cheese like products, cheese ripening,lipolysis of butterfat and cream.

Lipases also play a crucial role in the preparation ofenzyme modified cheeses (EMC).

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DETERGENTS

Used in laundry detergents and automatic dish washingmachine detergents.

Reduce the environmental load of detergent products.

Chemicals in detergents are reduced and arebiodegradable.

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PHARMACEUTICAL INDUSTRY

Lipases are currently being used for the preparation of opticallyactive intermediates on a kilogram scale.

Lipases were successfully applied in the regio-selectivemodification of castanospermine.

Promising drug in the treatment of AIDS.

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MEDICAL APPLICATIONS

Treatment for obesity is an example in which fatty

acid absorption is reduced.

Through the measurement of serum concentration ofpancreatic lipase, pancreatitis can be diagnosed.

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MATERIALS MEDIA PREPARATIONMICRO-ORGANISM

The fungal species Aspergillus japonicus (MTCC-1975)was used.

This organism helps in the production of lipase. It grows at an optimum temperature of 30 degrees C.GROWTH MEDIA AND ITS COMPOSITION

The media is malt extract broth.

composition:a) malt extract - 20.0 g/L

b) water - 1.0 L

c) pH - 7.0

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MEDIA PREPARATION AND PRODUCTION OF LIPASE

The culture media was prepared by weighing 1g ofmalt extract in 50 ml of distilled water and isautoclaved.

The lyophilized powder was put in the culture mediaand incubated for a period of 24 hrs.

After incubation growth can be observed and thisacts as master culture.

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Subcultures were prepared using master culture.

Now 5ml of subculture was taken and inoculated into45ml of solution containing soybean flour (50g/L),wheat mill bran and cheese whey.

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Now the whole setup was placed in a shaker andshaked at 120 RPM at 30 degrees C for 72hrs.

After incubation for 3 days, the media wascentrifuged and the pseudomycelium was removed.

The obtained media was assayed for lipase activity.

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LIPASE ACTIVITY

Lipase activity was determined in an emulsifier free systemusing different oils (Olive oil, Coconut oil, Palm oil, Ricebran oil, Saf flower oil, Groundnut oil, Sunflower oil,Sesame oil, Soybean oil) as substrate.

Reactions were carried out in 100ml conical flask at 40

degrees by immersion in water bath and shaking at 120RPM.

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The reaction mixture consisting of 2ml of 0.1Mpotassium phosphate buffer, Different pH:6,7,8 , 1mlof olive oil and 1ml of culture supernatant wasincubated at 40 degrees for 30 minutes.

The enzyme reaction was terminated by the additionof 5ml 96% ethanol.

Followed by titration with 0.05N potassium hydroxidesolution and phenolphthalein indicator. When reactionmixture turns to pink color titration is stopped andreading is noted.

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Lipase activity: V*Molarity of KOH*1000*1/30

V=Volume of reagent

1000=conversion factor from milli equivalent to microequivalent

One unit of lipase activity was defined as the amountwhich liberated 1 micromole of fatty acid per minuteat 40 degrees C.

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Results and Discussion

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Lipase Activity at different

pHS.No

Oil used pH:6 pH:7 pH:8

1 Rice bran oil 2.5 3.66 3.33

2 Ground nut oil 6.33 9.66 4.5

3 Sunflower oil 4.33 3.83 4.166

4 Mustard oil 6.66 5.55 5.16

5 Cottonseed oil 6.3 4.0 4.8

6 Soybean oil 6.0 4.83 4.5

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S.no Oil used pH:6 pH:7 pH:8

7 sesame oil 7.16 6.66 6.83

8 corn oil + saffloweroil

4.5 5.16 6.16

9 Olive oil 6.3 5.8 8.1

10 Coconut oil 5.5 5.8 4.3

11 Palm oil 2.0 2.6 2.3

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The maximum lipase activity is found with groundnutoil at a pH- 7.

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References Alberghina L, Schmid RD, Verger R, editors. Lipases: structure, mechanism and genetic

engineeringWeinheim:VCH, 2010.

Naharro ,Anguita J, Rodriguez-Aparicio LB, G. Purification, gene cloning, amino acid sequencing analysis and

expression of an extracellular lipase from an Aeromonas hydrophila human isolate. Appl Environ Microbiol2011;59:2411 –7.

Yoshida N, Aoyama S ,Inouya S. Cloning, sequencing and expression of lipase gene from Pseudomonas fragiIFD-12049 in Escherichia coli. FEBS Lett 2006;242:36 –40.

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

Sinisterra JV ,Arroyo M, Sanchez-Montero JM,. Thermal stabilization of immobilized lipase B from

Candidaantarctica on different supports: effect of water activity on enzymatic activity in organic media. EnzymeMicrob Technol 2003;24:3 –12.

Parmar VS ,Azim A, Sharma SK, Olsen CE,. Lipase catalysed synthesis of optically enriched a-haloamides.BioorgMed Chem 2008;9:1345 –8.

Balashev K, Jensen TR, Kjaer K, Bjornholm T. Novel methods for studying lipids and lipases and their mutualinteraction at interfaces: Part I. Atomic force microscopy. Biochimie 2001;83:387 –97.

Malcata FX Balca˜o VM, Paiva AL,. Bioreactors with immobilized lipases: state of the art. Enzyme MicrobTechnol 2003;18:392 –416.

Balca˜o VM, Kennippen A, Malcata FX, Kalo PJ. Lipase catalyzed acidolysis of butter fat with oleic acid:characterization of process and product. Enzyme Microb Technol 1998;33:118 –28.

Antranikian G ,Becker P, Abu-Reesh I, Markossian S,, Markl H. Determination of the kinetic parameters duringcontinuous cultivation of the lipase-producing thermophile Bacillus sp. [HI-9] on olive oil. Appl MicrobiolBiotechnol 2002;42:184 –90.

Beer HD, McCarthy JE, Bornscheuer UT, Schmid RD. Cloning, expression, characterization and role of theleadersequence of a lipase from Rhizopus oryzae. Biochem Biophys Acta 2007;1399:173 –80.

Belarbi EH Molina E Chisti Y. A rocess for hi h ield and scaleable recover of hi h urit eicosa entaenoic

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