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Sunday, November 22, 2009

How to estimate beta -Amylase

B-amylase from higher plants catalyses the hydrolysis of 1, 4 – glycosides bands to maltose. It is an examples i.e. splits maltose units from non-reducing end. When a branch point is reached, the hydrolysis ceases giving limit dertin. The name B-amylase is given because the initial product of hydrolysis is B-maltose which lutes on comes in equilibrium with maltose and forms an equilibrium mixture. The activity of enzyme is determined by estimating the reducing sugar produced. The amylase activity is expressed in terms of mg. of lactose liberated in 3 mins. By 1 ml of enzyme solution at optimum PH and temperature.

Reagent: - 1% starch solution [substrate], 5% homo genet of moony [enzyme]. DNS reagent, Buffer PH 4.8, Maltase Standard solution [1mg / ml].

Procedure: - Standard curve.
Prepare a series of test tubes with varying conc. [0 – 2 mg] of maltose. Make up volume to 3 ml. Add of DNS reagent and heat on coater bath for 10 mins. Add 5 ml water to all tubes and determine absorbance. Plot the graph of absorbance against concentration of maltose.

Assay of B-Amylase: - Incubate 1 ml of enzyme solution with 0.5ml of starch and 0.5 ml buffer stop the reaction at the end of 3 mins. By adding 2 ml DNS reagent. Heat the solution in a boiling water bath for 10 mins. Cool and read absorbance (after proper dilution) against blanks.
5% extract of germinating moog.
Effect of varies Ions on Amylase Activity study of effect of Mg++, Zn++, Pb++ and Cl icons on amylase activity.

Many enzymes are stimulated by the presence of simple molecules, ions. These are enzyme stimulants i.e. cultivators. Many compounds have the ability to combine with certain. Enzyme and then block catalysis by that enzyme. Such comps are called inhibitors.

Reagent: - 1) Enzyme (5% homogenate of moog) 2) Buffer of PH 4.8
3) 0.5 % Nacl 4) H.starch (substrate)
5) 4 % znso4 6) 2 % Pb (No3)2
7) 4 % Mg So4

Procedure: - Prepare series of tubes as follows add 1 ml of enzyme extract to all tubes except blank. Incubate all tubes at 370 c for 20 mins. Add 1 ml of DNS reagent and 1 ml of Nacl to all tubes. Add 1 ml of enzyme extract to blank. Heat all tubes on a boiling water bath for 10 mins. Read absorbance at 540 nm.

Result: - The effect of ions on B-amylase activity from moong is
Activators are Cl - & Mg ++
Inhibitors are Pb ++ & Zn++

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