Lipolytic activity (AL) measurement

Jakub Szeląg

Author/User

Keywords: Lipolytic activity, lipase, hydrolysis

Equipment requirements:

- shaking bath (Elpin + model 357),

- homogenizator (Yellow Line DI 25 basic)

-titrator (Schott- Titroline)

- 0,066 M phosphate buffer (pH = 7,0)

- 40 % oil emulsion with 2 % polyvinyl alcohol (PVA)

- ethanol

- 0,05 M NaOH

Execution time: 1h for 1 samples

Description:

Substrate preparing

To prepare 100ml of 40% aqueous emulsion of rapeseed oil emulsion, weigh 2.5 g of polyvinyl alcohol (PVA) add the approx. 60 ml of distilled water. In order to dissolve the PVA in the water solution must be heated. After dissolving the polyvinyl alcohol solution has to be cooled, and then add distilled water to achieve 60 ml. Then, the PVA solution should be added 40 ml of rapeseed oil. Thus prepared blend should be homogenized for 10 min at 12 000 min -1.

Executing assignment:

Proper sample:

In order to prepare the proper sample weigh accurately in Pyrex glass bottles (25ml) 9,7-10,3mg

lipase preparation. Add to bottle 2.5 ml of phosphate buffer. Subsequently add substrate (40%

rapeseed oil emulsion stabilized with PVA). After addition of the substrate the sample should be

incubated in a bath shaken at 37 ° C for 30 minutes. Reactions were stopped by adding 10 ml of

ethanol.

Control sample

In order to prepare the proper sample weigh accurately in Pyrex glass bottles (25ml) 9,7-10,3mg lipase preparation. Add to bottle 2.5 ml of phosphate buffer and add 10ml of ethanol (to denaturate enzyme) Subsequently add substrate (40% rapeseed oil emulsion stabilized with PVA). After addition of the substrate the sample should be incubated in a bath shaken at 37 ° C for 30 minutes.

Counting lipolytic activity

a) Counting correction of NaOH volume resulting from the difference in weight of proper and control sample:

V SK=

mw∗V KmK

Where:VSK – value of corrected volume of NaOH solution, used for titration control sample [ml],mW - weight lipase preparation used in the proper sample [g],mK - weight lipase preparation used in the control sample [g],VK– volume of NaOH solution, used for titration control sample [ml].

b) Counting lipolytic activity (AL.):

AL=(VW−V SK )∗n

mW∗t

Where:AL – lipolytic activity [µkat/g]m W – weight of lipase used in proper sample [g],VW– volume of NaOH solution, which was used for titration proper sample [ml],VSK – value of corrected volume of NaOH solution, used for titration control sample [ml],t – time of reaction = 900 [sek], n – NaOH molarity = 0,05M (= 50 mM = 50 mol/ml).