DETERMINATION OF LIPASE ACTIVITY (46)
Equipment
1. Research quality expandable scale pH meter equipped with a glass-body combination electrode having Ag/AgCl reference electrode.
2. Stirred reaction vessel (25 ml) within a constant temperature device, e.g., water bath, constant temperature block.
3. Class A micro buret, 2.00 ml having 0.01 ml subdivisions.
Reagents
All prepared using carbonate-free, all glass distilled, Type 1 water
1. Olive oil-Gum acacia emulsion. Slowly dissolve 20.6 gm gum acacia in 150 ml water. Add 25 ml reagent grade, low acidity olive oil and blend for 10 minutes at low speed Bring to 250 ml with water.
2. 1.5 M NaCl
3. 0.015 M CaCl2
4. 0.054 M Sodium Taurcholate
5. Titrant; 0.010 M NaOH, standardized
6. 0.1 M NaOH
7. Enzyme: dissolve 1 mg/ml in 0.5 M Tris pH 8 containing 5 mM sodium deoxycholate
Preparation of Substrate, 15.0 ml
Deliver in sequence 5.0 ml olive oil-gum acacia emulsion, 4.0 ml 1.5 M NaCl, 5.0 ml 0.015 M CaCl2 and 1.0 ml 0.054 M CaCl2 and 1.0 ml 0.054 M Sodium Taurcholate and mix well in the reaction vessel.
Procedure
Equilbrate with stirring 15 ml of substrate 25ºC ± 0.2ºC for Lipase, EPC No.L397, and EPC No.HL938, and to 37°C ± 0.3ºC for Lipase EPC No.LC452 and EPC No.IC398. Titrate to a steady pH 8.5 with 0.1 M NaOH. Add 0.002 to 0.004 mg of enzyme. When the pH reaches 8.0 start a timer and deliver titrant (0.0100 M NaOH) having the outlet from the buret ca. 5 mm below the surface of the stirring reaction mixture. (It is convenient to use silicone tubing from the buret tip to reaction mixture.) Maintain a constant pH 8.0 for 2 to 4 minutes and record the sample volume of titrant delivered per minute. Determine a blank rate by repeating the titrant in procedure on 15.0 ml of substrate without added enzyme. The blank volume of titrant should be less than 0.10 ml during 2 to 4 minutes. Record the blank volume of titrant delivered per minute.
Calculation of Specific Activity
Units = (Sample Vol-Blank Vol) x 0.01200 x 1000
mg mg Enzyme