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  Section: Plant Lab Protocols
 
 
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Methodology for Enzymes

 
     
 
Pectin Methyl Esterase
(Pectin pectyl hydrolase EC 3.1.1.11)
 
Pectin methylesterase (PME) (Pectase or pectinesterase) deesterifies pectin into pectic acid and methanol and thus makes it susceptible to precipitation by calcium or other polyvalent cations. The roots, stem, leaves and fruits of most higher plants contain PME.
 
 



Principle
Since PME produces methanol and free carboxyl groups in pectic acid, either methanol or the carboxylic group may be measured as a function of the enzyme.
Pectin + H2O
PME
Pectic Acid + CH3OH

The measurement of methanol is cumbersome. The increase in free carboxyl groups can be easily followed titrimetrically while a constant pH in the reaction mixture is maintained. The procedure given below is based on this principle.
 
 



Materials
Solid salt mixture of NaCl and EDTA disodium salt 10:1, (w/w)
Salt Mixture Solution
   Dissolve 7.5g of the above mixture in 100mL water.
Pectin
   Prepare 2% (w/w) pectin solution in 1.5M NaCl. From this stock solution, dilute accordingly to get 1% solution in 0.1M NaCl.
0.1N or 0.02N NaOH. For low enzyme activities use dilute alkali.
Enzyme Extract
   To about 30g macerated tissue add 2.25g NaCl-EDTA salt mixture and stir for 15 min and centrifuge. Collect the supernatant. Again stir the residue with 20mL of salt mixture solution for 15min, centrifuge and collect the supernatant.  Repeat the extraction step.  To the supernatant collected each time, add 100mL cold ethanol and collect the precipitate. Pool the precipitate, dissolve in 1.5M NaCl and use as enzyme source.
 
 
Procedure
1.
Take 100mL of pectin (1%) solution in a 500mL beaker and add 30mL of 1M NaCl and 60mL distilled water.
2.
Place it in a water bath at 30°C. The water bath should be maintained on a magnetic stirrer cum hot plate so that the solution can be stirred continuously.
3.
Place a pH meter adjacent to the magnetit stirrer and dip the electrode in the reaction mixture.
4.
Bring the reaction mixture to pH 7.5 by adding 0.1N NaOH with continuous mixing.
5.
Now add 10mL of the enzyme solution and set the timer on.
6.
Maintain a constant pH 7.5 of the reaction mixture by continuous addition of 0.1N NaOH for a definite period of time.
7.
Measure the alkali consumption as a function of time.
8.
Run a blank using the inactivated enzyme. The enzyme is inactivated by placing it in boiling water for 10 min.
 
 
Calculation
One unit of pectin methylesterase activity is that amount of enzyme; which releases one milliequivalent of carboxyl groups per min per g sample. Milliequivalent of carboxyl group is calculated by multiplying titre value with the normality of the alkali used.
 
 


References
1.  Hobson, G E (1964) Biochem J 92 304.
 
     
 
 
     




     
 
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