enzyme lipase experiment method

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Determination of lipase activity

Lipase acts on triacylglycerols and releases fatty acids. Determination of its activity is calculated by titration of free fatty acids.

Substrate preparation

• A buffer solution containing Na2HPO4/NaH2PO4 50 mM, pH=7, is prepared.
• Next an olive oil emulsion (40 ml of olive oil are added to 60 ml of gum arabic – emulsifier solution 5%, w/v) is prepared and the mixture is homogenized in laboratory homogenizer.
• The substrate of the enzyme is composed of 50 ml of olive oil emulsion in 45 ml of buffer solution.

Determination of enzymatic activity

• 0.5 ml of crude enzyme solution is added into 9.5 ml of substrate.
• The mixture is incubated in a shaker for 1 h at T=28 °C
• This is followed by titration of the mixture with NaOH solution 50 mM until pH=9

A unit of activity of lipase is the quantity of the enzyme that releases 1 μ mole of fatty acids in 1 h at T=28 °C.

Unit of Microbiology, Department of Biology, University of Patras

Designed by http://www.microbiology.biology.upatras.gr .

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