Enzymatic Assay of Lipase

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BACKGROUND

A lipase is an enzyme that catalyzes the hydrolysis of fats.¹ Lipases are a subclass of the esterase. Lipase perform essential role in digestion, transport and processing of dietary lipids.² Most lipases act at a specific position on the glycerol backbone of a lipid substrate.

For example human pancreatic lipase (HPL) which is the main enzyme that breaks down dietary fats in the human digestive system,³ converts triglycerides substrates found in ingested oils to monoglycerides and two fatty acids.

Lipase helps our body to absorb fats. Lipases are generally a group of water soluble enzymes, which exhibit the ability of acting at the interface between aqueous and organic phases. They primarily catalyze the hydrolysis of ester bonds in water insoluble lipid substrates.

So the basic objective is to perform the Enzymatic Assay of Lipase by Titrimetric Assay method.

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REQUIREMENTS

Reagents	200 Mm tris HCl buffer Olive oil substrate solution 95% ethanol 0.9% Thymolphthale Indicator Solution 50 Mm Sodium hydroxide solution Lipase enzyme solution
*Apparatus*	50 ml Erlenmeyer flask 25 ml burette

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PROCEDURE

Preparation of Reagents

The above given reagents are prepared as follows:⁴

For 200 mM tris HCl buffer we have to prepare 100ml in deionized water using Trisma base. The ph is then adjusted to 7.2 & the temperature is adjusted at 37°C with 1M HCl
For Olive oil substrate solution we can generally use olive oil.
For 95% ethanol we need to prepare 50 ml in deionized water using ethyl alcohol.
For 0.9% Thymolphthale Indicator Solution we can directly use Thymolphthale Indicator Solution.
For 50 Mm Sodium hydroxide solutions we need to prepare 100ml in deionized water using Sodium anhydrous hydroxide.
For Lipase enzyme solution: it should be prepared immediately before use. For this we need to prepare a solution containing 500-1000 units/ml of Lipase in cold deionized water.

First of all the following reagents are pipette out in suitable containers.⁵

	Test (in ml)	Blank (in ml)
Deionized water	2.50	2.50
Buffer (reagent 1)	1.00	1.00
Olive oil (reagent 2)	3.00	3.00

All the above reagents are mixed properly and balanced with the temperature being maintained at 37°C. Then 1 ml lipase enzyme solution is added. It is then mixed and incubated at 37°C for 30 minutes. Immediately after starting the incubation 1ml of enzyme solution is pipette out into a 50 ml Erlenmeyer flask marked blank and stored at 0-4°C.

After 30 minutes the test solution is transferred to a 50 ml Erlenmeyer flask & the blank solution also. It is then labelled as Blank. Then 3 ml 95% ethanol (3^rd reagent) is added. The solution is mixed properly and then 4 drops of Thymolphthale indicator is added to both test and blank solutions. A 25 ml burette is used for titration. The solutions are titrated with Sodium hydroxide until a blue colour appears. Titration is done with 0.1 ml graduations.

Calculations

NaOH= (Volume in ml of Reagent No.5 used for test) – (Volume in ml of Reagent No.5 used for blank)

1000= conversion factor from milliequivalent to microequivalent

2= time conversion factor from 30 mins to 1 hour (unit definition)

Df= dilution factor

1= volume in millilitre of enzyme used⁶

Units: one unit will hydrolyze 1.0 microequivalent of fatty acid from a triglyceride in 1 hr at ph 7.2 at 37°C

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CONCLUSION

Final Assay Concentration: In 7.5 ml reaction mixture, the final concentrations are 26.7 mm Tris, 40% olive oil and 500-1000 units Lipase.

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REFERENCES

Svendsen A.;” Lipase protein engineering”. 2010: 223-228.
Afonso C., Tulman E., Oma E., Kutish G; “The Genome of Melanoplus sanguinipes entomologists”.: 73(1): 533-52.
Girod A., Wobus C., Zadori Z., Ried M., Leike K., Kleinschmidt J.”The VP1 capsid protein of adeno associated virus type 2 is carrying a phospholipase A2 domain required for virus infectivity “. J Gen Virol. 83: 973-8
Winkler FK., Hunziker W.,” Structure of Human Pancreatic Lipase”. 1990: 343(6260): 771-774
Hasan F., Shah A., Hameed A., “Methods for detection and characterization of Lipases”. A Comprehensive Review-Biotechnical Advance. 2009: 27;782-798
“Reagents Chemicals ACS Specification”., 1993:8^th edition.;95

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