Experimental investigation of factors...

You should be able to describe how the effects of pH, temperature, substrate and enzyme concentration can be investigated experimentally The following slides outline a number of procedures that can be adapted to investigate some or all of the key factors

Starch and amylase

Key points: Starch is a storage polysaccharide

composed of amylose and amylopectin

0:00 2:00 4:00

0:30 2:30

1:00 3:00 5:00

1:30 3:30 5:30

Amylase digests 14 glycosidic bond

produces maltose

4:30

Follow reaction by measuring time for

starch to disappear maltose to appear

Starch and amylase

Prepare starch agar and cut wells in agar with a cork borer Fill wells with amylase
vary pH or concentration of amylase

pH5 pH5

pH3 pH3

pH7 pH7

pH11 pH11

pH9 pH9

Incubate Flood agar with I/KI solution

clear zones around some of wells diameter proportional to activity

Well cut in agar and filled with enzyme solution buffered to pH indicated

Most of agar stained blue but clear zone around some wells when agar is flooded with I/KI

Invertase and sucrose

Key points Invertase (sucrase) is a carbohydrase

hydrolyses glycosidic bond produces glucose and fructose

Monitor reaction by appearance of glucose using Clinistix

reagent strip changes colour when 10-20 mg glucose 100cm-3 calculate rate from the time taken to reach this concentration

Trypsin and casein

Key points Casein is milk protein Trypsin is a protease enzyme

hydrolyses peptide bond

Colour standard for hydrolysed casein

Milk-trypsin mixture is compared to colour standard

Casein is milky white colour

when digested it is translucent time how long it takes for milk to clear

Catalase and H2O2

Cylinder of potato

Discs of potato

Crushed potato

Key points Catalase decomposes H2O2 to H2O and O2 Various ways to measure activity
froth height mass loss collect O2 by
upward displacement gas syringe

Group Activity
Outline a procedure to investigate the effect of a named factor (pH, temperature, enzyme concentration and substrate concentration) on enzyme action. You should provide a brief outline of:
methodology key variables (independent, dependent and control variables) how enzyme action is measured expected results/observations.

The following table should help you organise the material.

Effect of a named factor on a named enzymes activity

Method outline

Variables

Enzyme activity measure

Expected results