BCH4160/2011:Notebook/Jigeshs Lab Notebook 2011/2011/11/08

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Contents

Enzyme Kinetics

The goal is to measure the kinetics of the digestion of starch by cellobiase and with and without urea and metaine monohydrate in solution.

Materials

In this experiment we used:

  • 1.2% starch stock solution
  • 9 molal stock urea solution
  • 10X stock resuspension buffer (pH 5)
  • Distilled water
  • Iodine
  • Cellobiase
  • UV spectrophotrometer

Procedure

Refer to Tiffany Byerly's enzyme kinetics protocol

Solution Prepartation

  1. Prepare X resuspension buffer from original 10X solution (pH 5)
    • Dissolved 16.7 mL original in 150mL distilled water
  2. Prepare 1.5% starch soltion
    • Dissolve 1.5g starch in 100mL buffer
  3. Prepare 4 molal (mol/kg) Betaine monohydrate
    • Dissovle 2.7g Betaine monohydrate in 5mL buffer
  4. Prepare 9 molal of urea
    • Dissovle 2.7g urea in 5mL buffer


Creating Baseline

Obtain baseline spectrum of 1.2% solution starch and iodine

  1. Dissovle 1mL 1.2% starch, 1mL buffer, and 5μL of iodine
    • Note: Final concentraions are 0.6% starch in solution.
  2. Measure Absorbance with UV specrophometer


Experiment 1

Purpose: Test solution with cellobiase without urea or betaine monohydrate.

  1. Add 1mL of 1.2% starch solution, 1mL of buffer and 5μL of iodine to a cuvetter. Shake well to mix.
  2. 10μL of cellobiase solution of cuvette. Quickly mix and place in UV spectrophotometer to measure absorbance.
  3. Repeate experiment for duplicates.


Experiment 2

Purpose: Test solution with cellobiase with .5 molal of betaine monohydrate and without urea.

  1. Add 1mL of 1.2% starch solution, 1mL of buffer and 5μL of iodine to a cuvetter. Shake well to mix.
  2. Add 0.25mL of betaine monohydrate (for final concentraion of .5 molal) to cuvette. Shake to mix.
  3. 10μL of cellobiase solution of cuvette. Quickly mix and place in UV spectrophotometer to measure absorbance.


Experiment 3

Purpose: Test solution with cellobiase with 1.0 molal of betaine monohydrate and without urea.

  1. Add 1mL of 1.2% starch solution, 1mL of buffer and 5μL of iodine to a cuvetter. Shake well to mix.
  2. Add 0.5mL of betaine monohydrate (for final concentraion of 1.0 molal) to cuvette. Shake to mix.
  3. 10μL of cellobiase solution of cuvette. Quickly mix and place in UV spectrophotometer to measure absorbance.


Experiment 4

Purpose: Test solution with cellobiase with 2.0 molal of betaine monohydrate and without urea.

  1. Add 1mL of 1.2% starch solution, 1mL of buffer and 5μL of iodine to a cuvetter. Shake well to mix.
  2. Add 1.0mL of betaine monohydrate (for final concentraion of 2.0 molal) to cuvette. Shake to mix.
  3. 10μL of cellobiase solution of cuvette. Quickly mix and place in UV spectrophotometer to measure absorbance.


Experiment 5

Purpose: Test solution with cellobiase with 3.0 molal of betaine monohydrate and without urea.

  1. Add 1mL of 1.2% starch solution, 1mL of buffer and 5μL of iodine to a cuvetter. Shake well to mix.
  2. Add 1.5mL of betaine monohydrate (for final concentraion of 3.0 molal) to cuvette. Shake to mix.
  3. 10μL of cellobiase solution of cuvette. Quickly mix and place in UV spectrophotometer to measure absorbance.


Experiment 6

Purpose: Test solution with cellobiase with 1.0 molal of urea and without betaine monohydrate.

  1. Add 1mL of 1.2% starch solution, 1mL of buffer and 5μL of iodine to a cuvetter. Shake well to mix.
  2. Add 0.222L of urea (for final concentraion of 1.0 molal) to cuvette. Shake to mix.
  3. 10μL of cellobiase solution of cuvette. Quickly mix and place in UV spectrophotometer to measure absorbance.

Results/Data

Contact

This experiment was done with Laura Lee and Brittany Webster.

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