Enzyme concentrations using trypsin enzume and casein solution

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Enzyme concentrations using trypsin enzyme and casein solution


To investigate the effect of a reduction in enzyme concentration on the rate of reaction, in this case the breakdown of protein by protease enzyme.

The protein substrate we are using is casein (found in milk which makes the milk cloudy).  The protease enzyme, trypsin, breaks down the protein (polypeptide).  Trypsin is an enzyme found in the small intestine, and is produced in the pancreas.  These enzymes woks best in alkaline conditions.  



The higher the enzyme concentration, the faster the protein trypsin (substrate) will be broken down.  The protein solution would turn clearer when enzymes are more concentrated.  The site of the reaction occurs in an area on the surface of the protein (protease) called the active site. At low enzyme concentration, there is great competition for the active sites and the rate of the reaction is slow.  With a higher concentration, there are more active sites so there is more of a chance of collisions between enzymes and the substrate molecules.  The rate of reaction and rate of colliding can proceed at a faster rate.


  • 1% trypsin
  • 5% casein solution (milk powder)
  • 10 Boiling tubes
  • Labels
  • Syringes (measure small volumes 1cm3 and 5cm3)
  • Water bath - 30°  
  • Colorimeter – measure transmission
  • Distilled water
  • Stop clock
  • Pipettes


  1. In five of the boiling tubes, different concentrations of trypsin are going to be used.  By using a syringe measure out: -
  • 1st boiling tube - 10ml of trypsin by 0.0ml distilled water – 10% trypsin
  • 2nd boiling tube – 7.5ml of trypsin by 2.5ml distilled water – 75% trypsin
  • 3rd boiling tube – 5.0ml of trypsin by 5.0ml distilled water – 50% trypsin
  • 4th boiling tube – 2.5ml of trypsin by 2.5ml distilled water – 25% trypsin
  • 5th boiling tube – 0.0ml of trypsin by 10ml distilled water – 0.0% trypsin

Label each boiling tube with the correct concentration.

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  1. Put 10ml of the substrate casein into the five remaining boiling tubes.  Again label each boiling tube.  

  1. Put all 10 boiling tubes into the water bath, for the temperature to acclimatize.  

  1. Within the first minute, empty the casein into the first boiling tube.  After every minute, repeat this step for the other 4 boiling tubes.  

  1. Every 5 minutes take a sample out the boiling tube, using a pipette and fill up the cuvette.

  1. Taking care in not touching the ridged sides, place it into the ...

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** Research and rationale There is little evidence of any background research to justify the investigation. There are no references listed. Appropriate biological principles are not discussed with reference to the investigation. Planning A testable hypothesis has been stated and some biological knowledge used to explain the prediction. Some of the key variables to be controlled are described but additional details could be provided. The apparatus is listed but not all justified. A range of concentrations is identified but there is no indication as to why this range was chosen. It would have been better f the candidate had repeated the collection of data and then compared these to class results. The attempts to assess safety are superficial. Implementing It appears that the apparatus was used competently. The data was not recorded in a suitably headed table. Units and headings were both missing. Analysing The trends and patterns in the data were recognized and commented on but the term rate of reaction was used inappropriately. The explanations were sound and related to basic biological knowledge. The graph was not available to view. Summary tables were not presented and there seems to be no recorded statistical information such as the calculation of a standard error. Evaluation The paragraph under the heading of evaluation was not an evaluation but a conclusion. Communicating The layout of the report was acceptable but data was not presented effectively using SI units where appropriate. There were minor spelling and grammatical errors but technical terms were used appropriately throughout. There were no references used or evaluated.