Starchy substances constitute the major part of the human diet for most of the people in the world, as well as many other animals. They are synthesized naturally in a variety of plants. Some plant examples with high starch content are corn, potato, rice, sorghum, wheat, and cassava. It is no surprise that all of these are part of what we consume to derive carbohydrates. Similar to cellulose, starch molecules are glucose polymers linked together by the alpha-1,4 and alpha-1,6 glucosidic bonds, as opposed to the beta-1,4 glucosidic bonds for cellulose. In order to make use of the carbon and energy stored in starch, the human digestive system, with the help of the enzyme amylases, must first break down the polymer to smaller assimilable sugars, which is eventually converted to the individual basic glucose units.
Because of the existence of two types of linkages, the alpha-1,4 and the alpha-1,6, different structures are possible for starch molecules. An unbranched, single chain polymer of 500 to 2000 glucose subunits with only the alpha-1,4 glucosidic bonds is called amylose. On the other hand, the presence of alpha-1,6 glucosidic linkages results in a branched glucose polymer called amylopectin. The degree of branching in amylopectin is approximately one per twenty-five glucose units in the unbranched segments. Another closely related compound functioning as the glucose storage in animal cells is called glycogen. which has one branching per 12 glucose units. The degree of branching and the side chain length vary from source to source, but in general the more the chains are branched, the more the starch is soluble.
Starch is generally insoluble in water at room temperature. Because of this, starch in nature is stored in cells as small granules which can be seen under a microscope. Starch granules are quite resistant to penetration by both water and hydrolytic enzymes due to the formation of hydrogen bonds within the same molecule and with other neighboring molecules. However, these inter- and intra-hydrogen bonds can become weak as the temperature of the suspension is raised. When an aqueous suspension of starch is heated, the hydrogen bonds weaken, water is absorbed, and the starch granules swell. This process is commonly called gelatinization because the solution formed has a gelatinous, highly viscous consistency. The same process has long been employed to thicken broth in food preparation.
Depending on the relative location of the bond under attack as counted from the end of the chain, the products of this digestive process are dextrin, maltotriose, maltose, and glucose, etc. Dextrins are shorter, broken starch segments that form as the result of the random hydrolysis of internal glucosidic bonds. A molecule of maltotriose is formed if the third bond from the end of a starch molecule is cleaved; a molecule of maltose is formed if the point of attack is the second bond; a molecule of glucose results if the bond being cleaved is the terminal one; and so on. As can be seen from the exercises in Experiment No. 3, the initial step in random depolymerization is the splitting of large chains into various smaller sized segments. The breakdown of large particles drastically reduces the viscosity of gelatinized starch solution, resulting in a process called liquefaction because of the thinning of the solution. The final stages of depolymerization are mainly the formation of mono-, di-, and tri-saccharides. This process is called saccharification. due to the formation of saccharides.
Since a wide variety of organisms, including humans, can digest starch, alpha-amylase is obviously widely
synthesized in nature, as opposed to cellulase. For example, human saliva and pancreatic secretion contain a large amount of alpha-amylase for starch digestion. The specificity of the bond attacked by alpha-amylases depends on the sources of the enzymes. Currently, two major classes of alpha-amylases are commercially produced through microbial fermentation. Based on the points of attack in the glucose polymer chain, they can be classified into two categories, liquefying and saccharifying.
Because the bacterial alpha-amylase to be used in this experiment randomly attacks only the alpha-1,4 bonds, it belongs to the liquefying category. The hydrolysis reaction catalyzed by this class of enzymes is usually carried out only to the extent that, for example, the starch is rendered soluble enough to allow easy removal from starch-sized fabrics in the textile industry. The paper industry also uses liquefying amylases on the starch used in paper coating where breakage into the smallest glucose subunits is actually undesirable. (One cannot bind cellulose fibers together with sugar!)
On the other hand, the fungal alpha-amylase belongs to the saccharifying category and attacks the second linkage from the nonreducing terminals (i.e. C4 end) of the straight segment, resulting in the splitting off of two glucose units at a time. Of course, the product is a disaccharide called maltose. The bond breakage is thus more extensive in saccharifying enzymes than in liquefying enzymes. The starch chains are literally chopped into small bits and pieces. Finally, the amyloglucosidase (also called glucoamylase) component of an amylase preparation selectively attacks the last bond on the nonreducing terminals. The type to be used in this experiment can act on both the alpha-1,4 and the alpha-1,6 glucosidic linkages at a relative rate of 1:20, resulting in the splitting off of simple glucose units into the solution. Fungal amylase and amyloglucosidase may be used together to convert starch to simple sugars. The practical applications of this type of enzyme mixture include the production of corn syrup and the conversion of cereal mashes to sugars in brewing.
Thus, it is important to specify the source of enzymes when the actions and kinetics of the enzymes are compared. Four types of alpha-amylases from different sources will be employed in this experiment: three of microbial origin and one of human origin. The effects of temperature, pH, substrate concentration, and inhibitor concentration on the kinetics of amylase catalyzed reactions will be studied. Finally, the action of the amylase preparations isolated from microbial sources will be compared to that from human saliva.
List of Reagents and Instruments
- Erlenmeyer flasks
- Graduated cylinder
- Pipets, 1ml, 10ml
- Test tubes
- Temperature bath
- Filter holder and filter paper
- Brookfield viscometer
- Bacterial amylase solution, 3000 SKB units/ml
- Fungal amylase powder, 40,000 SKB units/g. (Concentration of the fungal amylase solution to be used in class: 75g/l)
- Amyloglucosidase solution, 75 AG units/ml
- Human salivary amylase
- Corn starch
- HCl Stopping Solution, 0.1N HCl
- Iodine Reagent Stock Solution (in aqueous solution) See Note 1.
- Iodine: 5 g/l
- KI: 50 g/l
- Potassium Phosphate Buffers
- KH2 PO4 (monobasic phosphate) (FW=136.1)
- K2 HPO4 ·3H2 O (dibasic phosphate) (FW=228.23)
- CaCl2 ·2H2 O, 0.1M solution
- Reagents for the analysis of reducing sugars