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  Section: Plant Lab Protocols
 
 
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Methodology for Enzymes

 
     
 
Amylases
(a-1,4glucan 4-glucanohydrolase EC 3.2.LI and a-1,4 glucan maltohydrolase EC 3.2.1.2)
 
Starch degrading enzymes - universally distributed - act on glycogen and related polysaccharides. a-amylase causes endo-cleavage of substrates and hydrolyses a-1,4 linkages in a random manner. It has the ability to by-pass a-1,6 branch points. The viscosity reduction of the substrate is fast but the production of reducing sugars is slow.
 
b-amylase hydrolyses alternate bonds from the non-reducing end of the substrate. The enzyme degrades amylose, amylopectin or glycogen in an exo-or stepwise fashion by hydrolysing alternate glycosidic bonds. The end product is b-maltose. b-amylase is incapable of bypassing branch points i.e., 1,6-glycosidic linkages in amylopectin and glycogen. This results in about 55% conversion of amylopectin to maltose. The other product is a large limit dextrin. The viscosity reduction of the substrate due to/J-amylase action is slow but the production of reducing sugars is fast.
 
 
Principle
The reducing sugars produced by the action of a- and/or b-amylase react with dinitrosalicylic acid and reduce it to a brown colored product, nitroaminosalicylic acid.
 
 



Materials
Sodium acetate buffer, 0.1M pH 4.7
Starch, 1% Solution
   Prepare a fresh solution by dissolving 1g starch in 100mL acetate buffer. Slightly warm, if necessary.
Dinitrosalicylic Acid Reagent (refer experiment No. 1.3)
40% Rochelle Salt Solution (Potassium Sodium Tartrate)
Maltose Solution
   Dissolve 50mg maltose in 50mL distilled water in a standard flask and store it in a refrigerator.
Extraction of Amylases
   Extract 1g of sample material with 5-10 volumes of ice-cold 10mM calcium chloride solution overnight at 4°C or for 3h at room temperature. Centrifuge the extract at 54,000g at 4°C for 20 min. The supernatant is used as enzyme source.
Extraction of b-Amylases (free and bound)
   The free b-amylase is extracted from acetone defatted sample material in 66mM phosphate buffer (pH 7.0) containing 0.5M NaCl. The extract is centrifuged at 20,000rpm for 15min. The supernatant is used as a source of free b-amylase.
   The pellet is then extracted with phosphate buffer containing 0.5% 2-mercaptoethanol. The clear extract is used as source of bound b-amylase. All operations are carried out at 4°C.
 
 
Procedure
1.
Pipette out 1mL of starch solution and 1mL of properly diluted enzyme in a test tube.
2.
Incubate it at 27°C for 15min.
3.
Stop the reaction by the addition of 2mL of dinitrosalicylic acid reagent.
4.
Heat the solution in a boiling water bath for 5min.
5.
While the tubes are warm, add 1mL potassium sodium tartrate solution.
6.
Then cool it in running tap water.
7.
Make up the volume to 10mL by addition of 6mL water.
8.
Read the absorbance at 560nm.
9.
Terminate the reaction at zero time in the control tubes.
10.
Prepare a standard graph with 0-100mg maltose.
 
 
Calculation
A unit of a- or b-amylase is expressed as mg of maltose produced during 5 min incubation with 1% starch.
 
 



Notes
The extraction procedure given is suitable for cereal grains. There are a variety of extraction procedures used for the purpose depending upon the source material. For instance, the plant tissue is extracted in precooled 20% aqueous glycerol and the filtrate is used as enzyme source of amylases.
 
 
References
1.  Peter Bernfield (1955) In: Methods of Enzymology (Eds Colowick, S and Kaplan, N O) Academic Press New York 1,149.
2.  Krugen,JE (1972) Cereal Chem 49 379.
3.  Niku-Paavola, M L, Nummi, M, Kachkin, A, Daussant, J and Enari, T M (1972) Cereal Chem 49 580.

 
     
 
 
     




     
 
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