Extraction and Purification of Invertase

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Name: Jeffrey S. Webster Date: 8th October, 2007 Course: BC21D ID No. : - Lab Partners: Paul Bair, Jermaine Levy, Rochelle Allen Aim To extract and purify the enzyme invertase, thereby allowing for subsequent study of the effects of the parameters of [enzyme], [substrate], pH, temperature, and inhibitors on invertase activity. Method As outlined in the BC21D lab manual. Effect of Temperature on reaction velocity Place 1.0 ml of sucrose and 0.8 ml of pH 4.7 acetate buffer into a test tube and incubate for 6 minutes in a water bath at 37 oC. Add 0.2 ml of yeast invertase and incubate at the same temperature for 6 mins. After incubation quench with 8.0 ml of 0.1M H2SO4 and mix thoroughly. Transfer 0.1ml aliquot of sample to a separate test tube and add 0.9 ml of H2O followed by 4ml of WSG and incubate at afore mentioned temperature for a period of 15 mins. The mixture is then assayed at 500nm. Procedure is to be repeated using temperature of 0o, 60o, and 80o C. Introduction Enzymes are single-chain or multiple chain proteins that act as biological catalysts with the inherent ability to promote specific chemical reactions in vivo, as well as in vitro. Like all catalysts enzymes work by lowering the activation energy required for the reaction to occur, this is achieved because enzymes facilitate the formation of the transition state from substrate to product. Enzymes have three distinctive characteristics: 1. High specificity The ability to select and thus promote a particular chemical reaction on a single or small number of structurally related molecules is a key aspect of enzyme mechanics. Invertase is the enzyme which catalyses the hydrolysis of the disaccharide sucrose, into the monomers of glucose and fructose; due to the high specificity of enzymes one would not expect invertase to catalyze the
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