Group 1: Aguila, Alog, Alejandro, Almajar, Angeles, Araño, Balictar, Buemio, De los Arcos, Escobillo, Manuel, Peña, Rabanal, Rivera,Ronquillo, Siazon,Sutingco,Tagalog, Tamayo, C. Uy, Yeo, Yu

Assessing Cellulose Digestion

 An enzyme that breaks down starch into simpler sugars Secreted by the salivary glands and the pancreas  A glycoside hydrolase Attacks  -1,4 glycosidic bonds

 Cellulose Major polysaccharide of glucose found in plants Serves a structural rather than a nutritional role An unbranched polymer of glucose residues joined by  -1,4 linkages Parallel chains interact through H-bonds to form fibrils

Cellulose Cannot be digested by humans - lack of necessary enzymes to break down  bonds Cellulase Enzyme that breaks down cellulose to fatty acids produced by microorgansm in the gut of ruminants and other herbivores Some bacterial metabolism of cellulose in the human colon

Assessing Cellulose Digestion

 Preparation of mixtures  Incubation – 60 minutes Tube 1 – freeze (-25 o C)  Preparation of Assay IKI test Benedict’s test

 IKI Test for presence of starch PositivePale Gray to Blue-black NegativeDiluted IKI (yellow)

 Benedict’s solution Test for presence of reducing sugars PositiveGreen to orange to reddish-brown NegativeNo color change

Tube No Additives Amylase, Starch, pH 7.0 buffer Amylase, Glucose, pH 7.0 buffer Amylase, Cellulose, pH 7.0 buffer Amylase, Cellulose Deionized Water Peptidase, Starch, pH 7.0 Buffer Bacteria, Cellulose, pH 7.0 Buffer Incubation Condition Frozen then incubated at 37 o C 37 o C IKI Test Benedict’s Test

Which tubes showed that starch or cellulose was still present? Tubes 4, 5, 6 tested positive for the IKI test. After adding the IKI, the color of the mixtures solutions changed to blue-black, which indicates the presence of starch.

Which tubes tested positive for the presence of reducing sugars? Tubes 1, 2, 3, 7 tested positive for the Benedict’s test. Benedict's reagent contains blue copper (II) ions (Cu2+) which are reduced to copper (I) (Cu+) by the reducing sugar in the mixture. As seen in the activity, the color of the mixtures in tubes 1, 2, 3, and 7 changed to reddish-brown after adding the Benedict solution, which indicates the presence of a reducing sugar.

What was the effect of freezing tube 1? Freezing was the only difference between tube 1 and 2. Freezing did not have any effect because their results for both tests were similar.

How does the effect of freezing differ from the effect of boiling? Freezing does not have any effect on enzyme activity. On the other hand, boiling restricts enzyme activity. Enzymes such as amylase are proteins and their secondary and tertiary structures are affected by temperature. An increase in temperature usually increases the rate of an enzyme-catalyzed reaction, until the enzyme becomes denatured by temperatures around 40C – 50 oC. Boiling involves temperatures until 100 oC, which restricts enzyme activity.

Does amylase use cellulose as a substrate? Hint: Look at the results for tube 4. Normally amylase breaks down starch to maltose. Tube 4 tested negative for the Benedict test, which indicates the absence of maltose. These results show that amylase does not have an effect on cellulose.

What effect did the addition of bacteria have on the digestion of cellulose? Bacteria digested the cellulose in tube 7. Tube 7 tested positive for the Benedict’s test which indicates that the cellulose has been broken down by the bacteria to a reducing sugar.

What was the effect of the different enzyme, peptidase, used in tube 6? Explain your answer, based on what you know about the substrate of peptidase. Peptidase did not have the same effect on starch as the other enzyme, amylase. Tube 7 tested positive for the IKI test, which indicates that it still contains starch. It also tested negative for the Benedict test, which indicates that t does not contain maltose. This shows that the starch in mixture was not broken down to maltose by the peptidase.

 Does amylase use cellulose as a substrate? Tube 4 shows evidence that amylase does not use cellulose as a substrate. Amylase is enzyme that digests starch, not cellulose. All amylases are glycoside hydrolases and act on α -1,4- glycosidic bonds. Cellulose has beta 1,4 linkages which can not be broken by amylase.

 Did freezing have an efect on the activity of amylase? Explain. Tube 1 shows that freezing does not have an effect on the activity of amylase because amylase was still able to break down starch into maltose. the enzyme only deactivates its activity, but it does not denature the enzyme. This means that the enzyme can still be reactivated to function.

 Do you think that the bacterial suspension contained the enzyme cellulase (an enzyme that digests cellulose)? Why or why not? The bacterial suspension in tube 7 probably contained cellulose because the cellulose was digested to maltose, as indicated by the positive Benedict’s test.

 What is the substrate of peptidase? Explain, based upon your results. Activity 3 shows that BAPNA is the substrate for peptidase. BAPNA is colorless and transparent in solution but in the presence of a protein digesting enzyme (like peptidase), it turns the solution to yellow. The intensity of the yellow in the solution reflects the strength of the reaction on the tube. A spectrophometer measures the intensity of the yellow and displays it as the optical density. As seen in activity 3, the optical density is increased in those solutions that contain both BAPNA and peptidase.

 Guyton, Arthur, and John Hall. Textbook of Medical Physiology. 11th ed. Philadelphia, Pennsylvania: Elsevier Saunders, Print.