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.
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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