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  Section: Plant Lab Protocols
 
 
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Methodology for Enzymes

 
     
 
Polygalacturonase Activity
(Poly 1,4-a-D-galacturonide glycanohydrolase EC 3.2.1.15)
 
Polygalacturonase (PG) has been implicated in the softening of many fruits such as tomatoes, peaches, pears, avacados and mangoes. The enzyme is essentially a hydrolase and the mode of action is attributed to two enzymes exo PG (PG II; EC 3.2.1.15) and endo PG (PGI). Exo PG acts by removing galacturonic acid units from the non-reducing end of polygalacturonic acid (FGA) while endo PG hydrolyses PGA randomly, giving rise to short chain oligogalacturonic acids which can ultimate be hydrolyzed to a mixture of tri-, di-, and monogalacturonic acids, depending on the source of the enzyme considered. Fruits such as peaches, tomatoes, papayas and cucumbers were found to have both enzymes. Avacado and carrot, on the other hand, have only one PG enzyme each, PGI and PGII respectively.
 
In assessing fruit ripening process and shelf life of fruits, assay of polygalacturonase serves as a good indicator. PG activity is also measured in diseased plants, since a large number of pathogens have been reported to produce pectic enzymes to macerate the plant tissues.
 
PG activity can be assayed by both reductometric and viscometric methods. Since viscometric method is not specific for PG and certain other enzymes may also contribute to viscosity reduction, the reductometric method is described.
 
 
Principle
Polygalacturonases I and II are capable of releasing reducing groups from the substrate polygalacturonic acid or pectic acid. The quantum of reducing groups released is the measure of the activity of the enzyme. The reducing groups in the assay mixture are analyzed by the conventional arsenomolybdate method. An enzyme unit is defined as that amount which catalyzes the formation of 1mmol of reducing group per h.
 
 



Materials
Polyethylene Glycol
0.2% Na2S2O5 (Sodium Disulphite or Sodium metabisulfite)
0.5M NaCl
PM-10 Membrane
Sephadex G-100
0.15M NaCl
0.2M Tris-Acetate buffers - pH 4.5 and 5.5
0.01MCaCl2
1% solution of polygalacturonic acid in Tris Acetate buffer (PGA I)
Enzyme Extract
Slice the plant sample (100g) and blend in 100mL cold H2O containing 12g polyethylene glycol 4000 and 0.2g Na2S2O5. Centrifuge the slurry at 8000g for 20 min at 2°C. Discard the supernatant. Disperse the sediment in 2L cold water containing 0.2% Na2S2O5 and centrifuge. Repeat the washing step and suspend the insoluble residue in 2L of 0.5M NaCl. Stir the mixture in the cold for 2h and centrifuge. Concentrate the supernatant by ultrafiltration to 30mL using a PM-10 membrane.
Use this concentrate as enzyme source for PG assay. If separation of PGI and PGII is desired proceed as given below.
Prepare a column of Sephadex G 100 (5x90 cm) and equilibrate with 0.15M NaCl. Apply 30mL of the enzyme concentrate in 0.15M NaCl to the top of the column. Elute the column with 0.15M NaCl and collect 20mL fractions. The peaks 17 and 27 from this column show PGI & PGII ac­tivities respectively. Pool the fractions under each peak, concentrate by ultrafiltration to 10mL and use as enzyme source.
 
 
Procedure
1.
The assay mixture for PGI contains
0.2mL enzyme in 0.15M NaCl
0.2mL 0.2M Tris acetate buffer pH 4.5
0.1mL 0.01M CaCl2

0.5mL 1% solution of PGA I
2.
Prepare a blank for each sample by boiling the reaction mixtures before the addition of substrate.
3.
Incubate for 1h at 37°C
4.
Stop the reaction by heating at 100°C for 3min.
5.
Take 0.5mL of the reaction mixture and analyze for reducing sugar by Nelson-Somogyi method.
 
For assaying PGII - Use the buffer pH 5.5 instead of pH 4.5 for the reaction mixture as well as for preparing the substrate.
 
 
Calculation
Calculate the mmol of sugar units released and find out the enzyme units per mg of protein as per the definition given in the principle section individually for PGI and PGII.
 
 



Notes
1.  If PGA is not available use pectic acid or its salt as the substrate. However, the activity will be only 40% of the activity observed with PGA.
2.  An alternative procedure for extraction and assay for PG is given below.
     Blend 500g of cleaned and sliced plant sample with 500mL of cold 50mM potassium phosphate buffer, pH 7.5 containing 6% (w/v) (NH4)2SO4 0.5% (v/v) Triton X-100 and 1.5% (w/v) polyvinyl pyrrolidone (PVP). Stir the homogenate for 30 min and centrifuge for 30 min at 11,000rpm at 2°C. Discard the pellet and precipitate PG from the supernatant with (NH4)2SO4 to 80% saturation. Gummy material may float, if the sample is a fruit. Stir for 1h and then filter through cotton wool. Dissolve the residue in 150mL of 1% NaCl solution. Filter the slurry through a Whatman No. 1 filter paper, prelayered with PVP. Dialyze the filtrate against 1% NaCl overnight. Centrifuge at 15,000rpm for 20min at 2°C to clarify the dialysate. Concentrate to 1/3 volume by ultrafiltration using YM-10 membrane with 50 psi pressure of the compressed air.
     Determine PG activity with the reaction mixture containing 0.5mL of enzyme source, 0.5mL of 50mM Tris-acetate buffer (pH 5.2) and 1mL of 1% sodium polypectate. The substrate solution should also be at/>H 5.2. For reagent blank heat 0.5mL of enzyme in a boiling water bath for 3min before adding the buffer and the substrate. Incubate both at 37°C for 24h. Terminate the reaction by heating the tubes in a boiling water-bath for 3min. Measure the reducing group in an aliquot of the mixture by the Nelson-Somogyi method. Define one unit of enzyme as the amount of enzyme which produces 1mmol reducing groups per milliliter of enzyme per 24h. Specific activity is expressed as units of enzyme per milligram protein.
3.  Use galacturonic acid as standard for measuring the reducing groups through the Nelson-Somogyi method.
 
 
References
1.  Pressey, P and Avants, J K (1976) Phytochem 15 1349.
2.  Ghazahl, HM and Leong,CK (1987) FoodChem 24 147.
 
     
 
 
     




     
 
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