Determination of km and V max of B- Amylase
To determination of km and V max of B-Amylase.
Theory: – B-amylase form higher plants catalyses the hydrolysis of the and 1- 4 glycoside linkage in the polysaccharides to give maltase. It is an example exoenzyme and therefore splits off maltose unit from free non reducing end of the chain.
If the activity of an enzyme is determined over a range of substrate concentration and graph is plotted. A curve similar to the rectangular hyperbola is obtained.
An equation relating activity [s] was first derived by Michaels Men ten.
Michaela men ten constant, km is a characteristic of each enzyme. It is independent of either enzyme or substrate concentration and many be obtained as the substrate concentration at high maximum velocity of reaction. Activity of B-amylase is determined by estimating the product (reducing sugar maltose) obtained. One of the regents used for estimating reducing sugar is 3, 5 DNA. In alkaline solution.
Product obtained after reduction is 3 – amino – 5 – nitro salicylic acid which is orange red in colour. The colour thus produced depends upon the concentration of reducing sugar and is measured calorimetrically at 540 nm.
Requirements: – 1) 5% stench solution. 2) DNS reagent.
3) 0.5% sodium chloride. 4) Buffer at PH 4.8
5) Amylase extract.
Procedure: – Proper two sets of test tubes (one set for sample and other for blank) as follows.
To one set of test tubes add 1ml of enzyme extract and incubate at 370C for 30 mins. After incubation add 1ml of DNS reagent, 1ml NAOH (bench reagent) and 2ml of water. To the second set add 1ml of DNS reagent, 1ml NaCH and then 1ml of enzyme extract. Add 2ml of distilled water to all the tubes. Heat both sets of test tubes in the boiling water bath for 10 mins. With a marble on top prevent loss of water by evaporation, cool the solution and dilute 1ml to 10ml and determine absorbance using respective blanks. Plot the graph of absorbance against [s] and determine km and Vmax.
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