Amylase

Fun with Amylase

An Enzyme that "Chews Up" Macromolecular Carbohydrate

As you probably already know from "common knowledge," STARCH is one of nature's major energy storage compounds. (Name another.*) Of course, such energy storage compounds are only useful in times of need IF they can be rapidly broken down and the stored energy released. In the case of starch (aka "amylose"), there are two enzymes that do this task. Both are named amylase. It is VERY important that you notice the suffixes -ose and -ase. Enzymes usually have names that end in -ase, and things containing sugars end with -ose (such as in dextrose, sucrose and amylose). Woe be to the confused!

Before we can intelligently differentiate the two types of amylases, we need to know something about the structure of the substrate ("food") of these amylases. What does amylose, or starch, look like? It is a macromolecule, and the way macromolecules are usually depicted is by taking a small sample out of the extensive chain of repeating units. That is why the dots are shown fore and aft of the molecule in this picture:

Now to talk about the two types of amylase. For starters, it may help you to think of enzymes as little PacMen that go around taking bites out of things. Now consider that there are two types of amylase PacMen. One has a mouth that likes to bite into the middle of the long chain of sugars that make up starch. Each bite makes a break in the chain. This PacMan is called alpha-amylase. More scientifically stated, alpha-amylase hydrolyzes saccharide bonds, which are those pointed at by the arrows in the figure, above. So imagine, a bunch of alpha-amylases randomly bouncing around (Brownian motion!) in among some extremely long starch molecules. Whenever a "mouth" - the enzymatic or active site - bounces against a saccharide bond, "snip", and the bond is broken (hydrolyzed as a water molecule is added "across" the saccharide bond). With that bond broken, the whole starch molecule is now in two pieces. The more "bites," the more and smaller pieces. To help you "identify" with alpha-amylase, you will be happy to know that most of you have lots of it in your saliva.**

The beta-amylase PacMan is very picky and can only "chew" on the ends of a starch molecule, and only on one end and not both. It can only chew on the "reducing" end of starch, and that is the end far off the righthand side of the figure above. When beta-amylase does its job, it bites of maltose units - in other words, two glucose units at a time. Hint: we are not gong to consider beta-amylase further other than to point out that one place it is found is in your stomach juices.(A lot of the technical stuff is included here for the teacher's benefit and for the smart students who have had a lot of chemistry. It's nice to see that what one learned in chemistry does have relevance in other sciences!)

Fun with Alpha-Amylase!

QUALITATIVE EXERCISE

Preparation of the substrate. Obtain a square of Bounty paper towels (some brands don't work), and lightly apply to both sides a light layer of spray starch. Hang this up by an edge to dry (about 30 minutes). After it has dried, cut off a square inch of it, and test it with the following iodine spray to make sure that you get a dark blue or black color. On the remainder of the square of starch-towel draw a dozen or so circles that are each about 2 inches in diameter. NEW! Try ordinary smooth stationery paper. It seems to have starch on it already! Do the next step immediately to check your paper!

Iodine Spray. Add about 500 ml of water to a cheap, plastic hand-pumped sprayer such as found in hardware stores or garden shops. To this add enough tincture of iodine from the pharmacist to make the solution somewhat orange. (After this, no one in the class should be susceptible to goiter for the next several months. Why?)

Amylase Survey.
1. Either the instructor will ask you to provide, or the instructor will provide a range of raw items from the supermarket. (Why 'raw?')
  - For positive controls, use either or both ginger root and/or banana.
  - What would you use for a negative control?
2. Near the circles on your starch paper write the names of the samples you will soon use.
3. Take a small bit of one of the samples, place it in the center of its designated circle and squash it onto the starch-towel. Since you will be using a lot of force, don't use squashers made of glass unless you enjoy bloodily impaling your hand.*** A good item to use is a very large nail (9 to 12 inches long, with a large flat head).
4. Do similarly with all the other samples from the supermarket. You might also want to add some of your own samples from a short fieldtrip.
5. After the last squashed sample has set for 5 or more minutes, go to a designate area to spray your towel lightly with the iodine spray. (No, it won't hurt your hands. Rinse hands with water.)
6. If a sample had amylase activity, the sprayed towel should reveal a white spot. Why?
*Fats and oils (triglycerides) are in a biochemical class called "lipids," The degradation pathway goes from triglycerides to fatty acids (Pathway 1), which are then broken down to acetyl-CoA's (Pathway 2) in preparation for entering the Krebs Cycle.
**Almost from the time when enzymes were first discovered, alpha-amylase was a favorite to demonstrate because not only was its starch substrate so easy to detect with the iodine, but also the enzyme itself was easy to obtain in crude form - human saliva has high activity. What with the rise of public health concerns, most jurisdictions have outlawed the use of human body fluids in classrooms. For this reason we have sought a non-animal source of high concentration amylase that has great activity with little preparation time needed by the teacher or laboratory manager. Such a source was found in the root of the ginger plant. These roots a usually available in supermarkets, and the preparation is easy. Another unexpectedly concentrated source is in the flowers of impatiens.
*** It may seem politically incorrect to use such ghoulish language in a science protocol, but this writer has found that by giving the students a graphic mental image of the consequences is a very effective method for having students learn by traumatic experience - without the experience!
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