Salivary Amylase

This report entitled “Enzymatic activity of Human saliva (Salivary amylase) against temperature” aims to know and observe the enzyme activity of the human saliva. The research only included the use of starch-agar as the medium to observe enzyme activity during the experiment. Five starch-agar plates were prepared and each were labeled 1, 2, 3, 4 and 5 respectively. One mL of saliva were placed in each starch-agar plate which was holed then incubated for 24 hours. The plate labeled 1 was stored with 0ºC. The plate numbered 2 was stored at 15ºC, plate 3 at 25ºC, plate 4 at 37ºC and plate 5 was stored at 45ºC. After incubation, drops of Iodine solution were poured into the surface of the plate. Qualitative data were gathered. The starch agar-plates were examined and the zone of activity was measured using a ruler.

Specifically, this investigation tested the following hypothesis: as the temperature increases, the zone of activity of the enzymes will become larger.

The study proved that the temperature is one of the factors that can affect the Enzymatic activity of human saliva, however when exposed to much higher than optimum temperature, will be denatured and the enzymatic activity will be lost. Further explanations and proofs that say temperature is a factor to consider in enzymatic activity of saliva will be typewritten on the next chapters of the study.

INTRODUCTION

Enzymes react differently when the temperatures of certain substances are changed. What we hope to show in this experiment is that enzyme reactions can be slowed down or sped up depending upon whether the substance is heated or cooled down. This is an important experiment in that we need to know how enzymes work and also how they are affected by variations in temperature and their surroundings. This information will prove to be interesting because it is most likely that these enzymes are going to be manipulated in some form or another. It is essential to find out how and why enzymes are slowed down or not so as to do more research in medical fields of study or things of that nature. I hope to prove from this experiment that heating up a substance will cause the enzymes in it to stop working, and also if cooled down to slow down the reaction of the enzymes in that substance. With this information in mind, I hope to set up in this study the effects of rising temperature to the enzymatic activity of human saliva.

An enzyme is a protein-based substance which serves as a catalyst in living organisms by regulating the rate of spontaneously chemical reactions. The rate of reaction does not solely depend on the free energy difference between the initial and final states, but instead on the actual path through which the reactants are transformed into products. Enzymes accelerate biochemical reactions by physically interacting with the reactants and products to provide a more favorable pathway for the transformation of one to the other. In fact, if a living organism is deficient in a particular enzyme, it could result in serious diseases and possibly death.

In Compton’s Encyclopedia enzymes are the crux of genetic engineering. There are many different ways that enzymes help in genetic engineering. Enzymes allow scientist to cut the chromosome of DNA into various desired length. Each time a particular restriction enzyme or set of restrictions enzymes is used, the DNA is cut into the same number of pieces of the same length and composition. At least 80 restriction enzymes are now known. When restriction enzymes are used along with other enzymes that tie together loose ends of DNA, it becomes possible to remove a bit of DNA from one organism’s chromosome and to insert it into another organism’s chromosome. Also in Compton’s Encyclopedia enzymes are referred to as a preserver.

Enzymes can also help in the increasing of shelf life for fresh fruits and vegetables. In a recent experiment scientist were called upon to help out in the problem of soft tomatoes. When the tomatoes were aloud to ripen on the vine the were found to be to soft for shipping. For this reason most store bought tomatoes are picked green, refrigerated while shipped, and then treated with ethylene gas to bring on the red color. The enzyme responsible for fruit softening is polygalacturonase. Scientist have taken the polygalacturonase gene and have inserted it, in reverse orientation, into tomato plants, giving the plants two genes for the enzyme. When the two genes meet, it leads to production of messenger RNAs that are complimentary to each other.

The two RNAs bind to one another and cannot be translated into the enzyme. This has a 90 percent decrease in the softening process. Under these conditions, the tomatoes are left to ripen on the vine, allowing the natural flavor to develop fully, and are then shipped without refrigeration. The production of this gene-altering tomato has changed a perishable fruit requiring an expensive refrigeration facility for shipping into a relatively sturdy fruit that can be made available to poorer populations.

MATERIALS AND METHODS

The methodology was divided into three parts; The preparation of materials, the preparation of test subjects prior to Incubation and the testing for enzymatic activity.

Preparation of Materials:

The starch-agar plates used in the study were obtained from National Sciences and Research Institute, Diliman, Quezon City. 3mm thick agar plates were then set aside to culture the test subject.

The Salivary amylase were procured from the proponent on the day of the
experiment to obtain accurate results.

Preparation of Test Subjects prior to Incubation:

Human saliva was prepared in a 50mL beaker. The starch-agar plates, about 3mm thick were inoculated with the saliva by putting it on the holes made by the use of the plastic straw. The amount of saliva which was transferred through a pipette in each of the 5 agar plates was 1mL. The plates were labeled 1, 2, 3, 4 and 5 respectively by the use of a glass marker. The starch-agar plates then were incubated for 24 hours at different temperatures. The plate labeled 1 was stored with 0ºC. The plate numbered 2 was stored at 15ºC, plate 3 at 25ºC, plate 4 at 37ºC and plate 5 was stored at 45ºC.

Testing for Enzymatic Activity:

After the Incubation period, the starch-agar plates were gathered. Qualitative observations were made. To make the zone of activity much more visible, drops of Iodine Solution were spread throughout the surface of the plates. Quantitative measurements were gathered using a ruler. To have the exact measurements of the zone of activity or the radius made by the enzyme, measure its length horizontally and vertically. Don’t rotate the agar plates to minimize errors. After measuring, get the average of its horizontal and vertical measures.

RESULTS

For the initial report after the incubation, the starch-agar plates were subjected to observation. The results showed that the agar plates didn’t show any foul odors even inoculated by the human saliva. The enzyme in plate number 4 had the largest zone of activity. The radius gradually increases as the number in the plate increase; however the fifth plate’s radius was smaller than that of the fourth. The table below shows the results of the procedure used in the study.

DISCUSSION

For the first starch-agar plate, the clearing zone was equal to 8mm. The second plate equaled 17mm. The third one got 26mm. The fourth one exhibited 31mm and the fifth one only got the same clearing zone as the second which is equal to 17mm. At 0ºC, enzyme action is low because the movement of molecules is low and so slow. This causes the collision frequency between the enzyme and the substrate to be low. Increasing the temperature speed up the movement of the molecules and thus the collision frequency increases, therefore enzymatic action also increases. Human bio-enzymes works best at 37ºC. As the temperature raises the shape of the enzyme changes and the enzyme becomes denatured. Temperature above 50ºC will denature most human enzymes.

The temperature range over which enzymes show activity is limited between the melting point (0ºC) and boiling point (100ºC) of water. If a temperature is too low, there can be no noticeable reaction rate since the enzyme is operating at a temperature far below its optimum. If the temperature at which the enzyme is operating at is well above 100ºC, then thermal deactivation can occur. Deactivation of enzymes may be irreversible or reversible. With this given results, I can hereby conclude that as the temperature increases, the zone of activity also increases however, when the temperature exceeds optimum, the enzyme will decrease its activity.

REFERENCES

  http://biology.clc.uc.edu/students/114-Fall96/chuck-da.htm  http://leavingbio.net/ENZYMES.htm#inhibitors
 http://www.worthington-biochem.com/introbiochem/Enzymes.pdf  http://www.wikipedia.com

 Miranda, S. R. and B. T. 1980. Essentials of Chemistry. Second Edition. pp 385-407.