(a-1,4glucan 4-glucanohydrolase EC 3.2.LI and a-1,4 glucan maltohydrolase EC 126.96.36.199)
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.
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.
» 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.
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.
||Pipette out 1mL of starch solution and 1mL of properly diluted enzyme in a test tube.
||Incubate it at 27°C for 15min.
||Stop the reaction by the addition of 2mL of dinitrosalicylic acid reagent.
||Heat the solution in a boiling water bath for 5min.
||While the tubes are warm, add 1mL potassium sodium tartrate solution.
||Then cool it in running tap water.
||Make up the volume to 10mL by addition of 6mL water.
||Read the absorbance at 560nm.
||Terminate the reaction at zero time in the control tubes.
||Prepare a standard graph with 0-100mg maltose.
A unit of a- or b-amylase is expressed as mg of maltose produced during 5 min incubation with 1% starch.
The extraction procedure given is suitable for cereal grains. There are a varietyof 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.
1. Peter Bernfield (1955) In: Methods of Enzymology (Eds
Colowick, S and Kaplan, N O) Academic Press New York 1
2. Krugen,JE (1972) Cereal Chem
3. Niku-Paavola, M L, Nummi, M, Kachkin, A, Daussant, J and Enari, T M (1972) Cereal Chem