yeastinvertaselab - CHEMISTRY 365 BIOCHEMISTRY Team...

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CHEMISTRY 365 BIOCHEMISTRY Team Project #1 ISOLATION AND PURIFICATION OF YEAST b -FRUCTOFURANOSIDASE References: 1) Melius, Paul J. Chem. Educ. , 1971 , 48(11) , pp 765-766. 2) Bollag, D. M.; Edelstein, S.J. Protein Methods Wiley-Liss: New York, 1991. 3) Whitaker, J. R. Anal. Chem. 1963 , 35, pp 1950- 1953. This project involves the analysis of a yeast extract for b -fructofuranosidase specific activity, and then purification of the extract by organic solvent precipitation and by gel permeation chromatography 1 . b -fructofuranosidase (EC , (aka invertase, saccharase or sucrase), has a molecular weight of 270 kD and catalyzes the hydrolysis of the a 1 b 2 linkage of sucrose yielding an equimolar mixture of glucose and fructose. Protein precipitation as a purification technique has already been discussed. Gel permeation chromatography refers to a technique which separates molecules based on their molecular size and shape, and therefore their molecular weight. Under appropriate conditions, this technique may be used to separate mixtures of large biomolecules or to remove low molecular weight components from solutions of large molecules. The technique may also be used to estimate the molecular weights of unknown macromolecule fractions from plots of log molecular weight versus distribution coefficient 3 . Large molecules will be excluded from the porous gel beads, travel through the channels between beads, and be eluted first. Small molecules are able to penetrate the gel beads, have a much larger volume in which to travel, and are eluted later. The degree of purification is found from comparing the specific activity of the redissolved precipitate, and the specific activity of the chromatography fractions, to the specific activity of the initial extract.
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This note was uploaded on 11/27/2011 for the course CHM 365 taught by Professor Thomaszamis during the Spring '06 term at FSU.

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yeastinvertaselab - CHEMISTRY 365 BIOCHEMISTRY Team...

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