Name

Name: Lau Jin Mei
Student ID: 19856471
Date: 13 September 2018
Practical: Biochemistry Of Amylase
Subject: Biology of Cell & Organism (BIO1CO)
Demonstrator: Dr Martin Grunert
ABSTRACT
This laboratory experiment investigated how salivary amylase enzyme activity on the structure of different polysaccharides affect the concentrations of reducing sugar. The experiment was conducted using starch, glycogen and cellulose solutions and reacted with diluted saliva and water in duplicated sets. The tubes were left for 30 minutes for reaction to take place and 3,5 di-nitrosalicylic acid (DNS) solution was added to each tube before heating for 5 minutes. It was hypothesized that cellulose has no reducing sugar while starch and glycogen have reducing sugar. Base on the results, there are reddish brown colour present in starch and glycogen solutions except for cellulose solution. Salivary amylase can hydrolyse the alpha bonds of polysaccharides but not beta bonds of cellulose. INTRODUCTION
Enzymes are highly specific in their action, they work only on the substrate with 3-D shape complementary to its active site. Chemical reaction takes place once the substrate is attached to active site and is converted to products (Lam, P. K., & Lam, E. Y., 2013). Saliva secreted by the salivary glands contains enzymes such as amylase, lysosome and lingual lipase (Becker, A, 2018). Amylase are found in two forms, alpha (?) – amylase and beta (?) – amylase. Alpha amylase is produced in the salivary glands and pancreas. It is an enzyme that hydrolyses ?-1,4-glucosidic bonds of polysaccharides to get glucose and maltose (“Vazhacharickal, P. J.”). Starch comprises of amylose which is a linear chain of D-glucose units joined by ?-1,4-glucosidic bonds and amylopectin are made of glucose units linked occasionally with ?-1,6-glucosidic bonds (Libretexts, 2016). Glycogen has similar structure as amylopectin but is more highly branched and has shorter chain (Libretexts, 2016). For cellulose, the glucose units are joined by ?-1,4-glycosidic linkages and is more extended than amylose (Libretexts, 2016). After enzyme reaction, 3,5 di-nitrosalicylic acid (DNS) solution is used for testing of reducing sugar due to presence of carboxyl group. Under alkaline conditions, DNS solution reacts with reducing sugar to form 3-amino-5-nitrosalicylic acid which turns reddish brown color (“Estimation of Reducing Sugars by DNSA method”). The aim of this experiment was to study how salivary amylase enzyme activity on the structure of different polysaccharides affect the concentrations of reducing sugar. It was hypothesized that cellulose has no reducing sugar while starch and glycogen have reducing sugar.

MATERIALS AND METHODS
1.0 ml of saliva was collected by spitting into a clean centrifuge tube and was diluted with 9.0 ml of water. 0.5 ml of glycogen, starch, cellulose and maltose solutions were pipetted into each of the 2.0 ml centrifuge test tube vials and repeated for another set. The polysaccharide solutions have been buffered to pH 6.8 to allow salivary enzymes to work. The first set of test tube vials was added with 0.5 ml of diluted saliva and second set with 0.5 ml of water. The samples were mixed thoroughly and allowed to stand for at least 30 minutes with occasional shaking for hydrolysis of polysaccharides. After 30 minutes, 1.0 ml of 3.5 di-nitrosalicylic acid (DNS) solution was added to each test tube vials and heated up in the digital dry bath for 5 minutes to stop enzyme reaction. The samples were removed after 5 minutes and changes were taken note.
RESULTS
49987201192530Cellulose + Saliva
Cellulose + Saliva
381001198245Glycogen + Saliva
00Glycogen + Saliva
17830801189990Maltose + Saliva
Maltose + Saliva
34975801189990Starch + Saliva
Starch + Saliva
Fig 1.1 Colour change of polysaccharides with diluted saliva after boiling for 5 minutes
left1370330Glycogen + Water
00Glycogen + Water
33832801383030Starch + Water
00Starch + Water
15925801390650Maltose + Water
00Maltose + Water
49225201390650Cellulose + Water
00Cellulose + Water

Fig 1.2 Colour change of polysaccharides with water after boiling for 5 minutes
DISCUSSION
Maltose is a negative control for the experiment to confirm that DNS indicator solution together with simple sugars produce reddish brown colour which is a strong positive result. This also ensures that they were no other variables present to give a false positive result. Duplicated tubes with polysaccharides with water was to check if any there is any breakdown of polysaccharides in the absence of salivary amylase. From Figure 1.1 and 1.2, the maltose produces a red colour after boiling when treated only with water and in diluted saliva. This implies that maltose has many reducing ends and the salivary amylase enzyme can hydrolyse the ?-1,4-glucosidic bonds of maltose. During hydrolysis of starch, enzyme amylase breaks bond to form maltose and further bond breaking by maltase releases individual glucose molecules (Lam, P. K., & Lam, E. Y., 2013). As glycogen is more highly branched and has shorter chain, it hydrolyses to glucose faster than starch. The alpha bonds of starch and glycogen are hydrolyzed by salivary amylase enzyme to form reducing sugar, giving rise to reddish brown colour as seen in figure 1.1. From figure 1.1, there is no colour change for cellulose. Human alpha amylase is unable to break down ?-1,4-glycosidic linkages of cellulose so it will be undigested. Undigested cellulose are fibers to help smooth working of intestine tract (“Carbohydrates – Cellulose”). Only herbivores like cows have symbiotic bacteria to produce cellulase that breaks down cellulose into monosaccharides (Cellulase, 2018). Source of error can be the diluted saliva were added to the polysaccharides at different timings, so the time taken for each enzyme reaction will be inconsistent. Therefore, diluted saliva should be added at the same time or start the timer once the polysaccharide solution is added with diluted saliva. We can only observe the colour change to determine whether there is reducing sugar present. Therefore, one potential way to improve the experiment is to use the spectrometer to measure the absorbance to determine the concentration of reducing sugar for each polysaccharide.
CONCLUSION
In conclusion, our salivary amylase enzyme is only capable of breaking alpha bonds. DNS indicator solution changes to reddish brown colour after starch, glycogen and maltose are hydrolysed to reducing sugars.

ACKNOWLEGEMENTS
I would like to thank my laboratory partner, Miren for doing the experiment with me, Laboratory Technician, Melvin for setting up the experiments and Dr Martin and Ms Kate for the demonstration.

REFERENCES
Lam, P. K., & Lam, E. Y. (2013). Biology matters. Singapore: Marshall Cavendish Education.

Becker, A. (2018, March 13). Names of the Enzymes in the Mouth & Esophagus. Retrieved from https://sciencing.com/names-enzymes-mouth-esophagus-17242.htmlVazhacharickal, P. J. (n.d.). GRIN – Behaviour of Salivary Amylase in Various Reaction Environments with Reference to Km and Vmax. An Overview. Retrieved from https://www.grin.com/document/367145
Libretexts. (2016, October 14). 14.7: Polysaccharides. Retrieved from https://chem.libretexts.org/Under_Construction/Textmaps_and_Wikitexts/MVC:_Chem_1406/Chapters/14:_Carbohydrates/14.7:_Polysaccharides
Estimation of Reducing Sugars by DNSA method. (n.d.). Retrieved from https://orbitbiotech.com/estimation-of-reducing-sugars-by-dnsa-method/
Carbohydrates – Cellulose. (n.d.). Retrieved from http://chemistry.elmhurst.edu/vchembook/547cellulose.html
Cellulase. (2018, August 04). Retrieved from https://en.wikipedia.org/wiki/Cellulase
APPENDIX
49517301252855Cellulose + Saliva
Cellulose + Saliva
16154401259205Maltose + Saliva
00Maltose + Saliva
901701264285Glucose + Saliva
Glucose + Saliva
34328101246505Starch + Saliva
Starch + Saliva

Fig 2.1 Diluted saliva with polysaccharides solution 49466501320800Cellulose + Water
00Cellulose + Water
34290001308100Starch + Water
00Starch + Water
17335501327150Maltose + Water
00Maltose + Water
1714501339850Glucose + Water
00Glucose + Water

Fig 2.2 Water with polysaccharides solution
49530001238250Cellulose + Saliva
Cellulose + Saliva
1143001262380Glucose + Saliva
00Glucose + Saliva
17068801264920Maltose+ Saliva
00Maltose+ Saliva
34290001250950Starch + Saliva
Starch + Saliva

Fig 3.1 DNS solution was added to the polysaccharides solution with diluted saliva
1714501398905Glucose + Water
00Glucose + Water
17272001386205Maltose + Water
00Maltose + Water
32702501398905Starch + Water
00Starch + Water
48577501405255Cellulose + Water
00Cellulose + Water

Fig 3.2 DNS solution was added to the polysaccharides solution with water