Isolation and Testing Activity of B-Fructofuranosidase

B-Fructofuranosidase is formed in yeast and catalyzes the hydrolysis of sucrose to glucose and fructose. Enzyme is inverters of sucrose. Substrate is non reducing sugar and product formed is a reducing therefore can be easily identified.

During isolation, it is necessary to avoid loss of activity due to denaturizing of enzyme. Inactivation may be minimized by avoiding extreme of PH keeping the enzyme at 0 deg C. Extreme physical conditions must be avoided. While attempting enzyme isolation. Physical rather than chemical methods are used generally low temperature and PH close to neutral are preferred.

Isolation of enzyme: Place 1.5gm of yeast in a mortar and add about 2 gm of sand. Add 2ml of toluene and grind the mixture, Add 15ml of water in 3ml portions grinding the mass thoroughly during 15mins. Centrifuge for 10mins. Pipette out the supernatant fluid which will be clear or milky depending on hydrated or dehydrate yeast is used.

Precipitation by alcohol: Add 4 times the volume of 95% alcohol to the yeast extract and allow the mixture to stand until the ppt is formed centrifuge the suspension and dissolve the residue in water.

Test the solution for activity as follows:-
To 5ml of enzyme solution add 2ml of buffer of PH .2 and 1ml of fresh 1% sucrose solution. Mix well the contents of tube and allow to stand for 30 mins. Make the solution just alkaline with 1m Na2CO3 and perform Benedicts test [2ml of above solution + 2ml Benedicts reagent + 0.5gm of Na2CO3 and heat Red ppt. indicates +ve

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Chromatographic Separation Of Purin Bases

How to Do Chromatographic Separation Of Purin Bases
Chromatography is an analytical method and technique of separation in which the ingr. Are separated on basis of their difference in migration in a system of two phases. One is fixed on a stationary phase and other is a mobile phase. The difference in migration rates of ingr. Are based on different adsorption partition on exchange molecular sieving effect chief subst. to be separated.
The stationary phase is solid absorbent or a lia. Feed on a solid carrier. Mobile phase travel through stationary phases it carries with it ingr. From sample at different rates. The technique is widely used for separation of amino acids, lipids, alkaloids etc. TLC of Purina bases viz. adenine and Uralic stationary phase: silica gel supported as glass plate.


Mobile phase: - n-butane, n-methanol, H2O, NH3 in ratio 60:20:2:1.
a) Purina bases show weak flu ore séance in U.V.light.
b) Spray of 0.2% cosign in ethanol sate. C mercuric chloride which forms a complex with bases is appearing as black spot in U.V.light.


Procedure:- Preparation of plate: Prepare 10% slurry of silica gel in H2O and spread it evenly on a glass plate and allow it to set – dry it at 1100c and wol.


Soln of chamber: - Preparation a solvent ys. And pour about 20 ml in a TLC chamber and put a pica of filter paper in it close in air tight container keep it from nearly 30 mins.

Preparation of sample: - Prepare 1% w/w soln of adenine and Uralic in hot water and sy of them.
Spotting: Spot about 0.05 ml of each sample and each std. soln on TLC and dry in air at r.t.note that spots don’t spread two much are sufficient apart.
Developing: - Pat the plate. In previously sold chamber and allow to develop till the solvent runs to a distance of 20 cms. Mark the solvent front, take out the plates and keep as flat sur. Dry in air.
Location: - Observe the plates as such under UV or spray the plate c reagent. Allow to stand for 15 min and observe under UV the spots and calculate RP value.


Observation:-
Dist. Traveled by A = 10 cms.
Dist. Traveled by B = 8 cms.
Dist. Traveled by solvent = 15 cms.


Rf = Dist.travelled by samples .
Dist.travelled by solvent front.


Therefore Rf of A = 10/15 = 0.66


Rf of B = 5 /15= 0.53


Result:-
Rf of A = 0.66
Rf of B = 0.53

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