University of KwaZulu-Natal Biochemistry Practical 1: Starch Isolation & Hydrolysis PDF

UNIVERSITY OF KWAZULU-NATAL (WESTVILLE CAMPUS) DISCIPLINE OF BIOCHEMISTRY BIOC202: BIOENERGETICS AND INTEGRATED METABOLISM PRACTICAL 1: STARCH ISOLATION & HYDROLYSIS 1 PRACTICAL 1: STARCH ISOLATION AND HYDROLYSIS Background The most important stor...

UNIVERSITY OF KWAZULU-NATAL (WESTVILLE CAMPUS) DISCIPLINE OF BIOCHEMISTRY BIOC202: BIOENERGETICS AND INTEGRATED METABOLISM PRACTICAL 1: STARCH ISOLATION & HYDROLYSIS 1 PRACTICAL 1: STARCH ISOLATION AND HYDROLYSIS Background The most important storage polysaccharides in nature are starch in plant cells and glycogen in animal cells. Starch contain two types of glucose polymer, amylose and amylopectin. The first, amylose consists of long unbranched chains of D-glucose units connected by α(1 -4) linkages. By contrast, the glycosidic linkages joining successive glucose residues in amylopectin chains are α(1-4), but the branch points, occurring every 24 to 30 residues, are α(1-6) linkages. Glycogen is an animal polysaccharide similar in structure to amylopectin. The two compounds differ in terms of the number of branch points, which occur more frequently in glycogen (branches every 8 to 12 residues). Saliva is a mixture derived from the secretions of three pairs of glands: the parotid, submaxillary and sublingual, as well as from small mucous glands scattered diffusely over the mucosa of the mouth. Hydrated starch and glycogen are attacked by the endosaccharidase α-amylase, which is present in saliva and pancreatic juice. Starch and glycogen molecules can be heavily hydrated as they have many exposed hydroxyl groups available to hydrogen bond with water. Hydration of the polysaccharides occurs during heating, which is essential for efficient digestion of starch. The amylase present in saliva and pancreatic juices hydrolyses the internal α(1-4) linked D-glucose units in polysaccharides randomly. At a pH of about 6-7 and in the presence of chloride ions, α-amylase catalyses the hydrolysis of starch to the disaccharide maltose, the trisaccharide maltotriose and α-limit dextrins, which had an average of 8 glucose units with one or more α(1- 6) glycosidic bonds. This enzyme cannot hydrolyse the branch points, α(1-6) glycosidic links, present in starch, nor are the α(1-4) bonds of glucose units that serve as branch points hydrolysed. β-Amylase found in plants removes maltose units stepwise from the non-reducing end of starch, it is an exoenzyme and its products are β-maltose and β-limit dextrins. The β amylase cannot pass the branch points, α(1-6) glycosidic bonds, present in starch. Test principle In neutral or acid solution, iodine forms absorption complexes with amylose, amylopectin glycogen and with dextrin. Polysaccharides and their degradation products yield a characteristic colour when treated with a solution of iodine. 1. Starch yields an intense blue colour which is due to the formation of a starch-iodine complex. The blue-coloured starch-iodine complex is formed by the winding of left handed helix of amylose around clusters of iodine atoms. Amylose exists in the form of a left-handed helix which contains 6 glucosyl residues per turn of the helix. Branched polysaccharides, with an interrupted helix (e.g. amylopectin) yield less intensely coloured complexes. 2. Highly branched polysaccharides (e.g. glycogen) with short helical segments due to extensive branching yield only pale reddish brown complexes with iodine. NaCl addition intensifies this colour reaction as it fulfils the optimum ionic requirement of the reaction. 3. Dextrin yields the same pale colour as glycogen. 4. Maltose and glucose do not make complexes with iodine and simply retain normal iodine colour. 2 The factors affecting optimum reaction conditions and the helical nature of starch are likely to reduce the intensity of the reactions between starch and iodine. i) Excessive heating is likely to denature the α-helical coils in amylose such that iodine clusters are displaced from within the left handed helices back into solution which would be pale to colourless. ii) Recall that the interaction between iodine and starch require the optimum pH of close to neutral-acidic. Thus if the solution was excessively acidified (add H2SO4) or made alkaline (NaOH), interaction between starch and iodine would not occur. iii) Addition of sodium thiosulfate (Na2S2O3), would result in disappearance of the blue-black colour since iodine would take part in the ffg reaction: I2 + 2S2O3 → 2I + S4O6 → I2S4O62- Iodide Tetrathyonate Please note that this practical manual has been compiled so that you understand experimental detail. However, this practical will NOT be carried out in the lab this year. Reagents ♦ 0.01M Iodine ♦ 1% Glycogen ♦ NaCl powder ♦ 10% NaOH ♦ 1% Thiosulphate ♦ Benedict’s solution ♦ Starch (Isolated from Potato) ♦ Salivary amylase (from your saliva) !CAUTION!! : a) Iodine --> Avoid contact with skin and eyes. Harmful if inhaled. b) NaOH ---> Corrosive. Avoid contact with skin. 3 PART 1: Isolation of Starch from Potato a) Collect freshly ground potato (±20g) and strain through cheese cloth into a 400ml beaker as shown in the picture below. b) Add 50 ml water to the pulp on the cloth and collect the filtrate in the beaker. c) Add 50 ml water to the beaker and allow the starch granules to settle to the bottom. d) Decant/remove the supernatant fluid. Repeat the above washing process of the starch once more. e) Suspend the starch in a total volume of about 25 ml water. f) Pour this mixture slowly with stirring into about 25 ml of boiling water, and continue the boiling for 3 minutes. If the solution is extremely viscous, water may be added such that the preparation may be conveniently poured or pipetted. This step will help the starch to dissolve. g) You will use this solution in the second and third parts of this practical, 2) Iodine test and 3) Starch Hydrolysis by Salivary Amylase. Straining potato through cheese cloth: Final solution will look as follows: 4 PART 2: Iodine test a) Add the following to 3 clean test tubes; Tube 1 - 3ml of water (negative control) 2 - 3ml of previously prepared starch solution (from Part 1) 3 - 3ml of 1% glycogen b) Now add 4-5 drops of 0.01 M iodine solution dropwise to each tube. What colour will you expect to see? 5  c) Add a little of NaCl (half a spatula) to tube 3 that contains glycogen + iodine. Note how NaCl intensifies the colour reaction since NaCl fulfils the optimum ionic requirement of the α-amylase action. d) Divide the blue-black iodine-starch mixture of tube 2 roughly into 3 tubes and treat as follows: Tube 2A - warm gently Tube 2B - add a few drops of 10% NaOH Tube 2C - add a few drops of 1% thiosulfate solution What do you think will happen in each of these tubes? Explain the reasons for all the results. This explanation is in the power-point presentation. PART 3: Starch hydrolysis by salivary amylase Lastly you will digest or hydrolyse the starch that was isolated from potato using the enzyme salivary amylase. Progress and products of this hydrolysis will be followed over time using both the iodine reaction and Benedict’s reagent. The iodine reaction will give an indication of the amount of starch present. The Benedict’s test will indicate presence of maltose which is a product of starch hydrolysis. The Benedict's test identifies reducing sugars (monosaccharides and some disaccharides), that have free ketone or aldehyde functional groups. Thus, the action of α-amylase can be followed by observing the time taken to reach the point at which the reaction mixture no longer gives a positive colour with the iodine reaction, the achromic point which is the time interval required for total digestion of starch by salivary amylase. 6 a) Measure 30 ml of the remaining isolated starch solution (from Part 1) into a test tube and incubate in a 37-40°C water bath. This warm starch solution should be used for the rest of the experiment. b) In a second test tube, collect a few ml of saliva (containing salivary amylase) in a clean test tube and incubate in a 37-40°C water bath. c) Add the saliva to the starch solution. Mix and START timing. d) At 2 minute intervals, transfer a few drops from the starch-saliva solution to a well containing iodine solution. A negative (yellow) iodine test indicates complete breakdown of starch by salivary amylase. The point or time at which this is achieved is called the achromic point. Watch the following - Video 1: Hydrolysis of Starch using Salivary Amylase https://www.youtube.com/watch?v=zsOs3v8Z-P4 e) To confirm complete digestion of starch, perform the Benedicts test as follows: i. Transfer 1 ml of the starch-saliva solution to a test tube containing Benedict’s reagent. ii. Heat the test tube for a few minutes. iii. Formation of a red precipitate indicates presence of glucose or maltose. Watch the following – Video 2: Iodine and Benedicts Test after Starch Hydrolysis https://www.youtube.com/watch?v=ISf6ClTbg78 Watch the following (Video 3) to understand the Effect of temperature and pH on Digestion of Starch by Salivary Amylase. https://www.youtube.com/watch?v=iaqxTaXit9M 7