Amylase, Lactase, Protease, and Catalase Enzymes
. h t t p : / / W W W . S C I E N C E - P R O J E C T S . C O M / . . . . . . .

E n z y m o l o g y

.....(At this point, your instructor will give you a brief description of what a catalyst is. Be prepared to take home some big words such as "activation energy" and "free energy" or "enthalpy." Make sure your instructor gives you the story of "Thermodynamics for Preschoolers." This whole matter is so extremely complicated that even preschoolers can understand it! It's merely encumbered with an incomprehensible esoteric vocabulary. If anything, eschew obfuscation!)

..... This laboratory set is presented in two separate days. On the first day you will become acquainted with several common enzymes from very common sources. On the second day, you will delve much more deeply in the "kinetics" of one of the enzymes studied the first day. In the past, the first day was not given, and very frequently the students felt as if they had been thrown into the sea without a life-jacket. They foundered trying to sort out the concept of enzymes from analytical techniques. This year we are going to give you swimming lessons first, by looking at three enzymes that are readily available: alpha-amylase, lactase, and catalase. A fourth enzyme is one found in the product called "Bean-O", which eliminates the "gas" when you eat beans. However, as no good qualitative tests have been found for that enzyme, we will only discuss it so that you will at least know beans about it!

--- Alpha-Amylase ---

.....Most people have lots of alpha-amylase in their saliva, which starts the process of breaking down starch.. We are going to test each of ourselves for its presence. Anyone who does not have salivary amylase will be asked to stand before the class to see if any interesting linked-genetic characteristics are apparent.

.....Here is a very short section of a starch molecule showing only five glucose links in it. Starch can be thousands or millions of glucose-links long. In solution, the long strands form helices. Chemists have long known that iodine likes to fit itself into those helices and make a very dark blue or black color. If amylase snips away at the long strands (see the arrows, below), helices cannot form and therefore no dark color can form. Thus the "iodine test" is a good one for determining whether or not there is amylase present.

A figure showing a short section of a starch molecule and where alpha-amylase cleaves it.

....."QUALITATIVE" EXPERIMENT:.... Get a large test tube and "bomb" some spit into the bottom of the tube. Don't laugh if you didn't hit the mark on the first shot. Laughter will prevent you from hitting the mouth of the tube on your second try! Next add about one inch of the starch solution; swirl to mix. Allow the tube to sit for about 15 to 30 minutes (during which time move on to the look at some other enzymes). Finally add a few drops of iodine solution. Oops! Did you run a control without spit?! What color does the control become upon addition of the iodine solution? (Starch solution: add 1 gm of starch to 100 ml of cold tap water; make a smooth slurry; bring to a boil with CONSTANT swirling to dissolve [not unlike making gravy] then add extra 100 ml of cold water; mix. // iodine solution: to 1 ml of water, add 1 gm of KI; completely dissolve; then add 1 gm of black crystalline iodine; mix to dissolve; then add 400 ml of water. / Object here is to have starch solution concentrated enough to give positive iodine test, yet dilute enough so that saliva can break the starch all down completely within a few minutes so as to give negative test if amylase is present.)

.....Now you will be let on in a little biochemist's secret. When may you begin feeding your baby cereal, etc.? "Dr. Spock" says to wait two months. If you feed the cereal too early, the baby gets constipated. What is behind this is that newborns do not produce amylase. Check it out: Someday, put some of your baby's drool into a tube and test for its ability to breakdown starch. The baby won't be able to! Each few days, retest the baby. Finally after perhaps 4 or 5 weeks, the baby will test positive. THEN you can feed cereal to the baby. Shovel cereal down the hatch in the evening. The baby will sleep most of the night. Finally you and your spouse will get a good night's sleep, too. And that brings TECHNOLOGY right into your home!!!

Safety note: Normally the use of human fluids in labs is frowned upon. Hepatitis and HIV must be controlled. In this experiment, the iodine sterilizes the system of any bacteria or viruses! Therefore, just deal with your own tube(s) until the iodine is added. You can't catch what you've already got.

--- Lactase ---

.....You have heard about lactose intolerance. Such people must either not consume any lactose (milk sugar), or artificially break it down in the milk, ice cream or cheese before they eat those things. Do you know anyone who is lactose intolerant?

.....Let's look at the human ingestion of lactose. Back in cave-days, the only time a person would ever ingest lactose would be when they were infants and getting milk from their mothers. Thereafter in their lives milk was never consumed. Only with the invention of agriculture and animal husbandry has milk become readily available to adults. Do you know of any cultures that still rarely have milk products available to adults? Did you ever get ice cream in a Chinese restaurant? Many people of Oriental or African descent are extremely lactose intolerant as adults. They often get violently nauseated upon eating a spoonful of lactose. As a matter of fact, most of you would feel a bit strange after a tablespoon of lactose. You probably aren't violently lactose intolerant, but actually lactose tolerant, but still not lactose degraders. Babies are lactose degraders because in that period of their development genes are turned on that lead to the production of lactase - the enzyme that splits the disaccharide we call lactose.

A figure showing a lactose molecule's being hydrolyzed by the enzyme lactase into two monosaccharides - glucose and galactose.

.....Lactose is a disaccharide with one glucose sugar molecule bound to one galactose sugar molecule. Once lactose is split, our bodies readily metabolize the glucose and galactose products. Now, can you think of any other developmental stage in which a person produces lots of lactase? When else in a human's lifetime is there lots of lactose in the body? The nursing mother, of course, makes lactose to put into her milk to feed her baby. She uses lactase to catalyze the reverse reaction: glc + gal -> lactose. Later, the baby takes it in the opposite direction: splits the lactose, and the glucose mostly is metabolized for energy, and some of the galactose goes into making brain material. Hence, generally the more intelligent the mammal, the more lactose in mother's milk.

The word "mammal" derives from "mamma" or "ma",
which are among the very, very few universal words.

.....Let's watch lactase work. Alas, it is hard to observe lactase work on lactose, unless you want to taste the concoction to see if it gets slightly sweeter with time. Lactose is barely sweet. Galactose is a just a tad sweeter; and glucose is about half as sweet as table sugar (sucrose). (But people frown on tasting things in the lab because we never know what was in the beaker or flask previously.) But there is another way to see lactase work. Suppose we had a chemical that was somewhat similar to lactose, but that when split, one portion becomes a colorful dye. We thus wish to use an ANALOG of lactose. A commonly used one is ONPG (ortho-nitro-phenyl-galactoside). This analog has a "ONP" instead of the monosaccharide glucose. When the bond is snipped between the ONP and the Galactose, the resulting ONP is a bright yellow water-soluble dye. Thus, as lactase works on zillions of ONPG molecules in a solution, the solution slowly turns yellow as more and more of the ONPG molecules are snipped in half.

A figure of ONPG's being hydrolyzed by lactase into two molecules - galactose and a yellow ONP.

....."QUALITATIVE" EXPERIMENT:.....Break off a very small piece of a lactaideŽ pill. Drop it into a test tube and add about a half inch of water; swirl to dissolve. Add six drops of the ONPG solution. Note how fast the solution turns yellow. (Do not taste this solution as the yellow ONP is toxic.) (Teachers: This must be made fresh for each lab. ONPG solution: Take out a bottle of ONPG powder from freezer; allow it to warm before opening so as to prevent condensation from forming INSIDE the bottle. Weigh out 80 mg and dissolve in 10 ml of water. Dispense into several small tubes for student use.)

--- Bean-O ---

.....This commercial preparation is a mixture of several enzymes which break down carbohydrates. Bean and some other "gassy" foods contain a great deal of complex carbohydrates that our bodies own enzymes cannot touch. Thus those carbohydrates pass through the stomach unchanged and then go into the intestines. When their finally arrive at the bacteria-filled large intestine, the bacteria find that they can degrade this carbohydrates And as they do the degraded polysaccharides are converted into CO2 and methane. Those two gasses are what give people embarrassing urges and bouts of borborigmi (look that one up!), tummy cramps and even worse.

--- Catalase ---

.....The primary reaction catalyzcd by catalase is the decomposition of H2O2, to form water and oxygen.

2 H2O2 --> O2 + 2 H2O

.....Were aliens to land on earth and take off their space suits, they would probably gasp and die from the oxygen in our atmosphere. Oxygen is, of course, a strong oxidizer. Furthermore, most metabolism in the presence of atmospheric oxygen leads to the production of hydrogen peroxide. It is the peroxide that kills. Earth-life has figured out two ways of getting around this toxic problem of "peroxide." One is not to make it. The other is to have an enzyme that destroys it. The bacteria that are in cheese use the first strategy. Almost all of the rest of us make catalase, which we use to split peroxide into a water molecule and an oxygen atom.

....."QUALITATIVE" EXPERIMENT:.... Take a few drops of the suspension that is marked yeast, and add it to a milliliter of 10% hydrogen peroxide. Note the fizzing! That's the oxygen gas split from the peroxide by the enzyme. Does your saliva contain catalase? (Don't be a dummy: do NOT put the peroxide solution in your mouth; put some spit in a tube and add the peroxide!) How about your partner's blood? (Yes, it does! - so you don't need to assault your partner!) // "40 volume" hydrogen peroxide (=20%) can be purchased from your nearest beauty supply store.

.....Now for a "QUANTITATIVE" EXPERIMENT on catalase after reading how just a little thinking about what you have just done with catalase has ramifications covering large sections of biology.

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