IC398, Lipase

LIPASE, PURIFIED

NO. IC398

STANDARDIZED

 

DATE: _1 Dec 2009____LOT NO. ___64144____ANALYZED _1 Dec 2009_______

 

% PROTEIN __14.1_________ % LACTOSE _______85.9_________________

 

Units per mg of Protein __113.5___,  Units per mg of Product ____16.0____________

 

Lipase is purified from porcine pancreas by modifications of the method of Verger, et al., Biophys. Acta 188:272 (1969).  The lipase is standardized with lactose to a specific activity of 16,000 units per gram of solid.

 

ACTIVITY:

            16.0 units per mg of solid

            16,000 units per gram of solid

            One unit liberates 100 umoles of fatty acid per hour at pH 7.8 and 37°C using

            olive oil emulsion as substrate.

 

Store at 5°C or Freeze, -10 to -20°C.

Stable 9 to 12 months at 5°C ; 12 to 24 months at -10 to -20°C.

 

ADDITIONAL DATA

 

Units per mg of Protein as alpha Amylase ___0.90____________________.

 

Units per mg of Product as alpha Amylase  ______0.012________________.

 

RATIO alpha Amylase/ Lipase = _____0.00075_________________________.

 

One alpha Amylase unit forms one umole of reducing groups per minute at pH 6.9 and 37°C using soluble starch as substrate.

 

ORIGIN IS UNITED STATES OF AMERICA

 

 

 

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DETERMINATION OF LIPASE ACTIVITY, IC398

 

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, eg.,

                  water bath, constant temperature block.

            3.)  Class A micro buret, 2.00 ml having 0.1 ml subdivisions.

 

REAGENTS

All prepared using carbonate-free, all glass distilled, Type I 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

                   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; 0.0100 M NaOH, Standardized.

            6.)  0.1 M NaOH.

7.)  Enzyme:  Dissolve 1 mg/ml in .05 M Tris pH 8 containing 5 mM sodium deoxycholate.    

 

PREPARATION OF SUBSTRATE, 15.0 ml

Deliver in sequence 5.0 ml olive-gum acacia emulsion, 4.0 ml 1.5 M NaCl, 5.0 ml

0.015 M CaCl2 and 1.0 ml 0.054 M Sodium Taurcholate and mix well in the reaction vessel.

PROCEDURE

Equilibrate with stirring 15 ml of substrate to 37°C ± 0.2°C and titrate to a steady pH 8.0 with 0.1 M NaOH.  Add 0.002 to 0.020 mg of enzyme.  When the pH reaches 7.8 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 7.8 for 2 to 4 minutes and record the sample volume of titrant delivered per minute.

 

Determine a blank by repeating the titration procedure at 37°C on 15.0 ml of substrate without added enzyme.  The blank volume of titrant should be less than 0.10 ml during the 2 to 4 minutes.  Record the blank volume of titrant delivered per minute.

CALCULATION OF SPECIFIC ACTIVITY.

 

Units       =      (Sample Vol - Blank Vol) x 0.0100 x 1000

                                     mg                                           mg enzyme

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We hereby certify:

 

a)         The Lipase is of animal origin only.

b)         The Lipase was derived only from animals which were slaughtered in the United States in USDA inspected abattoirs where the animals were subjected to ante- and post mortem inspection and the animals were fit for human consumption or use in laboratory studies.

c)         The United States if free from foot and mouth disease and rinderpest.

 

 

Signed  :__Carmen Schatz______Dated: ______27 May 2009_____________