How to determine enzyme activity rate contant Michaela men ten constant KM and maximum velocity of reaction, or maximum enzyme activity V max of a enzyme example B- Amylase

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.

How to estimate content of RNA in a given sample by orcinol method

Estimation of RNA by Orcinol Method
To estimate RNA in a given sample by orcinol method.

When a solution of RNA is treated with orcinol. Reagent, it gets hydrolyzed to component base, phosphate and rebase reduce. Further heating with orcinol produces bluish green complex in presence of Fe+3.

Requirement: - Std. soln of RNA [0.05 mg/ml] sample solution of RNA, fresh orcinol reagent.

Procedure: - Mix 2 ml of sample and standard solution of RNA with 3 ml of orcinol reagent. Heat on boiling water bath for 30 mins. Cool and determine extinction at 665 nm. Against oreinol blank.

Calculation: - Conc. of sample = (Conc. Of standard / Absorbance of std.)x Absorbance of sample
                              

= 0.05 /0.249* 0.448


= 0.8945 mg / ml.

The conc. of RNA in given sample was found to be 0.8945 mg/ml.

Isolation of casein from Milk

How casein is Isolated from Milk

Casein is the main protein found in milk and is present at a concentration of about 3-5 gm / 100 ml. It is actually a heterogeneous mixture of phosphorus containing proteins and not a single compound.
Most protein show a minimum solubility at their iso electric point and this principle is used to isolate casein by adjusting the PH of the milk to 4.6 its isoelectric PH. Casein is also insoluble is ethanol and this property is used to remove unwanted fat from preparation.

Take  Milk, 1% acetic acid, ethanol, ether, narrow range PH paper.

Procedure: - Transfer 25 ml of given milk in 250 ml beaker. Add 25 ml distilled water in it. Add 1% acetic acid solution, carefully from sides of beaker, till PH of solution is adjusted to 4.6 (check with PH paper). Digest solution for half an hour on water bath. Decant the super natunt. Wash the ppt in the beaker (10-12 times) with boiling water, decanting the super natunt each time. Transfer the ppt. To a small beaker and cover it with. 95% alcohol (15 ml) and stir vigorously for 10- 15 mins. Filter ppt. through watman filter paper no.1 and press the rpt.within the fold to remove alcohol. Pep eats extraction with alcohol till casein obtained is finely, divided and free from fat. Dry ppt of carse in an ovea at 110 C.