A detailed study on Enzymes chemical reactions

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Enzymes were discovered by a German chemist Eduard Buchner near the terminal of the nineteenth century. He had been seeking to pull out a fluid for medicative usage from barm, nevertheless, the barm infusion kept traveling bad. He so decided to add sugar to the barm, nevertheless, the barm converted the sugar into intoxicant, which is besides known as agitation. Buchner investigated into this and shortly found out that populating cells were non responsible for this agitation and that it was caused by the fluid that was seeking to be extracted from the barm. The word enzyme was coined for the active ingredients in the juice that promoted agitation. Although enzyme literally means “ in barm ” , it is now nevertheless being used as the corporate noun for several 100s of compounds that have shown to hold a catalytic action on specific chemical reactions.

Enzymes are biological or organic accelerators made up of protein. They catalyse ( increase/decrease the rate of ) chemical reactions without themselves being chemically changed at the terminal of the reaction. It can hence be used repeatedly and so is effectual in little sums. They basically work by take downing the activation energy of the reactions and hence leting the reaction to topographic point at a quicker rate. In enzymatic reactions, the molecules are the start of the procedure are called substrates, and the born-again molecules, the merchandises.

Properties of enzymes:

Enzymes have the undermentioned belongingss:

Enzymes alter the rate of chemical reactions without themselves being chemically changed at the terminal of the reaction.

Enzymes are really powerful. Since enzymes are really specific, a little sum of an enzyme is capable of catalyzing a immense chemical reaction.

Enzymes are affected by temperature. Enzymes are inactive at low temperatures. Increasing the temperature increases the activity of the enzymes. There is an optimal on the job temperature at which certain enzymes work best. This is usually between 37-42 grade centigrades. However, a high temperature, anything above 45 degree centigrades usually destroys the active sites of the enzymes and causes it to denature. This for good amendss the enzyme and they become functionless.

Enzymes are affected by pH. Certain enzymes work best in acidic conditions whereas certain enzymes function better in alkalic conditions. For illustration, pepsin plants best in the tummy where the pH is below 7, nevertheless enteric enzymes work better in coditions of pH of above 7.

Some enzymes may necessitate a compound to be bound to them before they can catalyze chemical reactions. These compounds are called co-enzymes.

Enzymes can work in either waies. Metabolic reactions are reversible and the way in which the reaction goes depends on the sums of substrate and merchandises present. The reaction will continue from left to compensate until an equilibrium is reached between the substrates and merchandises. Besides, if there is a big sum of merchandises, so the contrary reaction starts and hence causes the merchandise to be split up until once more equilibrium is established.

Lock and Key & A ; Induced Fit Hypothesis:

Although enzymes have a big size, nevertheless, they merely have a little part that is functional. This is known as the active site. Active sites can be described as depressions on the surface of the enzyme. Merely a few of the aminic acids of the enzyme molecule make up this active site ; the balance are used to keep its overall 3-dimensional form. The active site is the site where the substrate binds onto the enzyme and merely substrates with a peculiar molecular form will hold any opportunity to adhere efficaciously with the peculiar enzyme. This is the ground why enzymes are specific in their actions as they can merely adhere to specific substrate molecules.

Another thought which arose from the lock and cardinal hypothesis is the induced fit hypothesis that suggests that the enzyme alters its form somewhat to guarantee that the enzyme molecules bind tightly with the substrate molecule. However, one time the merchandise or substrate leaves the active active site, the active sight realigns itself to its original signifier.

Now, holding discussed enzymes by and large, we shall travel onto discoursing the enzymes more specific to this experiment. The enzyme being used in this probe is Neutrase. Neutrase is a bacterial peptidase which is produced from a bacterial strain called Bacillus Amyloliquefaciens. Protease is an enzyme which catayses the hydrolysis of proteins into polypeptides/amino acids. In worlds, the digestion of protein chiefly starts in the tummy with Pepsin in the gastric juice where the proteins are converted into polypeptides. Protein digestion is represented in this experiment by the fact that when the proteins in the milk are digested, the solution turns from opaque cloudy white to a diaphanous solution.

In an enzyme controlled reaction such as that of protein digestion in milk, an addition in the concentration of enzyme will take to an increased reaction rate. This is due to the fact that when there are more enzyme molecules present, there will be a greater opportunity of more of the enzyme molecules clashing with the substrate and hence increasing the frequence of the hits. This increased frequence of hits will assist to organize an enzyme-substrate composite more quickly.

Purpose:

The purpose of this experiment is to see if there is a negative correlativity between the enzyme concentration and the rate of digestion of the protein content in the milk. Increase in enzyme concentration taking to diminish in the rate of digestion of protein content.

Hypothesis:

H1 – There is a positive correlativity between the enzyme concentration and the rate at which the protein content in the milk digests. ( Increase in rate of reaction ) .

H0 – There is no correlativity between the enzyme concentration and the rate at which the protein content in the milk digests. ( No affect on rate of reaction ) .

Equipment:

Equipment

Justification For Use

Marvel Powdered Milk

The beginning of protein upon which the enzyme to work on.

Enzyme ( Neutrase )

The enzyme which digests the protein content in the milk.

Sodium Phosphate Buffer ( pH 6.4 )

In order to guarantee the pH of the solution remains changeless for optimum working of the enzyme.

Test Tubes And Test Tube Rack

Test Tube: To keep the enzyme, buffer and milk pulverization solution.

Test Tube Rack: To keep the trial tubing.

Measuring Cylinder

To mensurate out the volume of distilled H2O.

Measuring Scale

To mensurate out the mass of the milk pulverization.

Pipette And Pipette Filler

To mensurate out accurately the volume of the enzyme.

Beakers

To keep the H2O and the trial tubing incorporating the enzyme, buffer and milk pulverization solution.

Stopclock

To clip the continuance it takes for the enzyme to wholly digest the protein content in the milk.

Syringe

To add the solution of the enzyme, buffer and milk pulverization into the curvettes.

Colorimeter

To mensurate the light absorbancy values.

Preliminary Experiment:

A preliminary experiment was conducted ab initio in order to prove whether the method intended for usage was flawless or non. However, through the conductivity of this experiment, a figure of defects were noticed and therefore dealt with to bring forth a more unflawed and sound experimental process.

In the preliminary experiment, it was decided that the temperature of the solution incorporating the enzyme would be kept changeless by utilizing a Bunsen burner. However, fluctuation in temeperature were traveling to be apparent and therefore doing inaccuracies. Consequently, it was decided to utilize a thermostatically controlled H2O bath to maintain the temperature invariable.

Besides, when utilizing the tintometer, I decided to put the solution inside the curvette and so put it onto the tintometer and record the values over clip until it reached 0. However, this would give inaccurate consequences based on the fact that when the solution was removed from the H2O bath and placed inside the curvette, the temperature would diminish over clip and hence affect enzyme activity. As a consequence, it was decided that the solution be removed from the H2O bath and placed into the curvette at regular intervals. After every 20 seconds, a new curvette would be used with solution removed from the H2O bath and so placed onto the tintometer to enter the light absorbancy values.

Method:

Measure out 10.00 gms of milk pulverization utilizing an electronic mass balance into a beaker. Then step out 200 cm^3 of distilled H2O utilizing a measurement cylinder. Add the H2O into the beaker incorporating the milk pulverization and utilize a spatula to stir the solution good.

Now, different concentrations of enzymes are to be prepared.

To do a 1 % concentration solution of enzyme, add 1cm^3 of the Neutrase into a trial tubing utilizing a pipette. Then add 99cm^3 of distilled H2O measured out utilizing a measurement cylinder. Shake the trial tubing incorporating the solution ( 100cm^3 ) good.

To do a 2 % concentration solution of enzyme, add 1cm^3 of the Neutrase into a trial tubing utilizing a pipette. Then add 98cm^3 of distilled H2O measured out utilizing a measurement cylinder. Shake the trial tubing incorporating the solution ( 100cm^3 ) good.

Use the same method as above to do 3 % ,4 % and 5 % solutions. Place each of the trial tubing into the trial tubing rack.

Add Sodium Phosphate Buffer ( 6.4 pH ) to each trial tubing.

Place one of the trial tubing incorporating the enzyme solution and any one of the milk solution trial tubing into the H2O bath for 5 proceedingss.

After 5 proceedingss, instantly pour the enzyme solution into the trial tubing incorporating the milk solution.

Use a colorimiter to look into the sum of light go throughing through the solution with clip. Press ‘R ‘ to reset the tintometer ( use the ruddy filter ) as ruddy visible radiation is transmitted the best and this will give accurate readings.

After every 10 seconds, topographic point some solution from the trial tubing into the a curvette and the curvette onto the tintometer and record the consequence.

Variables:

The exclusive independent variable in this experiment is the concentration of the Neutrase solution which ranges from ( 1-5 ) % .

Other variables which could impact the probe have been tabulated as follows:

Variable

How may a alteration in this variable affect the informations?

How will it be controlled?

Type of milk used.

Some milks will hold more or less protein molecules present than others which alters the sum of substrate molecules being available to organize enzyme-substrate composites.

This will be controlled by guaranting that the same milk pulverization is used throughout the experiment.

pH of the solution.

Enzymes work better in solutions of different pH. The activity of the enzyme depends upon the pH of the solution ; if optimum pH solution is used, the enzyme would work better than if non-optimum pH was used.

This will be controlled by guaranting that a pH buffer ( Sodium Phosphate – pH 6.4 ) is used. This will understate any alterations in the pH of the solution and guarantee that the optimal pH is kept changeless for efficient working of the enzyme.

Volume of Neutrase solution used.

A larger volume would of enzyme ( Neutrase ) soltuion would ensue in a greater sum of enzyme-substrate composites and hence increasing the rate of the reaction.

Neutrase solution volume will be kept changeless by exactly mensurating the volume required utilizing a pipette and pipette filler.

Temperature of the solution.

Temperature alters the rate of enzyme activity and a higher temperature would ensue in greater enzyme activity. However, anything usually above 45 degree centigrades would take to denaturing of the enzyme.

Using a thermostatically controlled H2O bath will keep a changeless temperature. The temperature would be kept changeless at the optimal on the job temperature for the enzyme.

Volume of milk used.

A larger volume of milk would ensue in a greater sum of enzyme-substrate composites being formed as there is a greater figure of protein present and therefore increasing the rate of the reaction.

Milk volume will be kept changeless by exactly mensurating the volume required utilizing a pipette and pipette filler.

Health & A ; Safety Regulations:

Wearing a lab coat inside the research lab.

Wearing safety goggles to protect the eyes from chemicals.

Wearing plastic baseball mitts when managing the enzyme and milk solution and to forestall taint.

Keeping the trial tubing in a rack to forestall inadvertent breakage or spilling.

Bunsen burners will be ensured that they are kept on xanthous fire when non in usage.

Consequences:

Time ( s )

Colorimeter reading for

0.0 % Neutrase concentration ( arbitrary units )

Colorimeter reading for

1.0 % Neutrase concentration ( arbitrary units )

Colorimeter reading for

2.0 % Neutrase concentration ( arbitrary units )

Colorimeter reading for

3.0 % Neutrase concentration ( arbitrary units )

Colorimeter reading for

4.0 % Neutrase concentration ( arbitrary units )

Colorimeter reading for

5.0 % Neutrase concentration ( arbitrary units )

( 1 )

( 2 )

( 3 )

( 1 )

( 2 )

( 3 )

( 1 )

( 2 )

( 3 )

( 1 )

( 2 )

( 3 )

( 1 )

( 2 )

( 3 )

( 1 )

( 2 )

0

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

20

2.5

2.5

2.5

1.9

1.7

1.7

1.5

1.6

1.6

1.4

1.5

1.4

1.0

1.1

1.0

0.7

0.5

40

2.5

2.5

2.5

1.5

1.6

1.5

1.1

1.2

1.3

1.1

1.1

1.4

0.5

0.5

0.6

0.2

0.1

60

2.5

2.5

2.5

1.3

1.4

1.4

0.8

0.8

1.3

0.5

0.7

1.2

0.3

0.1

0.2

0.0

0.0

80

2.5

2.5

2.5

1.0

1.1

1.1

0.4

0.5

1.2

0.2

0.4

0.6

0.0

0.0

0.0

0.0

0.0

100

2.5

2.5

2.5

0.8

0.8

0.8

0.2

0.1

1.2

0.0

0.1

0.0

0.0

0.0

0.0

0.0

0.0

120

2.5

2.5

2.5

0.6

0.6

0.6

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

140

2.5

2.5

2.5

0.3

0.2

0.2

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

160

2.5

2.5

2.5

0.0

0.0

0.0

0.0

0.0.

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

* ( 1 ) – repetition figure 1

Average of the consequences:

Time ( s )

Average tintometer reading for

0.0 % Neutrase concentration ( arbitrary units )

Average tintometer reading for

1.0 % Neutrase concentration ( arbitrary units )

Average tintometer reading for

2.0 % Neutrase concentration ( arbitrary units )

Average tintometer reading for

3.0 % Neutrase concentration ( arbitrary units )

Average tintometer reading for

4.0 % Neutrase concentration ( arbitrary units )

Average tintometer reading for

5.0 % Neutrase concentration ( arbitrary units )

0

2.5

2.5

2.5

2.5

2.5

2.5

20

2.5

1.8

1.6

1.4

1.1

0.6

40

2.5

1.5

1.2

1.2

0.5

0.1

60

2.5

1.4

1.0

0.8

0.2

0.0

80

2.5

1.1

0.7

0.3

0.0

0.0

100

2.5

0.8

0.2

0.0

0.0

0.0

120

2.5

0.6

0.0

0.0

0.0

0.0

140

2.5

0.2

0.0

0.0

0.0

0.0

160

2.5

0.0

0.0

0.0

0.0

0.0

Statistical trial:

I have chosen to utilize the Spearman ‘s Rank Correlation to utilize as a statistical trial.

The Spearman ‘s Rank Correlation is a technique used to prove both the way and strength of the relationship between two variables. It uses the statistic R ‘s which autumn between the scope of -1 and +1.

Spearman ‘s Rank:

% Concentration of Neutrase

Mean Light Absorbency ( Arbitrary Units )

Rank % Concentration of Neutrase

Rank Light Absorbency

Difference ( D )

Difference2 ( D2 )

5.0 %

0.05

6

4.0 %

0.06

5

3.0 %

0.06

4

2.0 %

0.1

3

1.0 %

0.3

2

0.0 %

2.5

1

A

Rs = 1- ( 6Sigma d2/n3-n ) =

Decision:

The consequences of the experiment prove that the H1 hypothesis is right. There is a positive correlativity between the enzyme ( Neutrase ) concentration and the rate at which the protein in the milk digests. In other words, increasing the concentration of the enzyme increases the rate of reaction.

The consequences show that an addition in the concentration of the enzyme leads to a descrease in the clip taken for the solution to travel clear. This is due to the fact that when the concentration of the enzyme is increased, there are more active-sites nowadays on the enzymes onto which the substrate binds onto. This creates more and more enzyme-substrate composites. The higher concentration of enzyme increases the kinetic energy of the molecules and increases the frequence of the hits between the enzyme and substrate molecules. A lower concentration of enzymes agencies there would be fewer active sites and hence, a slower rate of reaction.

However, if we kept increasing the concentration of the enzyme, one would detect that the line would get down to level off because all of the active sites on the enzymes would be occupied and therefore, increasing the concentration would hold no consequence on the rate of the reaction.

Evaluation:

Detecting the consequences, one can easy bring forth the decision that there is a positive correlativity between the enzyme concentration and the digestion of the protein content. Even though, the set of consequences achieved are non to the full accurate and therefore, there are anomalousnesss. However, betterments can be made to understate inaccuracies and undependability in the experiment.

One of the factors that could hold affected the experiment could hold been human reaction mistake in timing. Besides, the starting of the stop-clock for each of the tubings could hold been different and this could hold resulted in undependable consequences. To avoid this, nevertheless, an automatic stop-clock could be used.

Another factor that could hold affected the experiment could hold been the motion of the reacting mixture invariable. The commixture between the substrate ( Protein in the milk ) and the Enzyme ( Neutrase ) could non hold been same in each tubing. This could hold resulted in inaccuracies in the consequence as the molecules in the tubings with the better commixture would hold greater kinetic energy and hence, the reaction rates would be quicker as more and more enzyme-substrates would be formed quicker. To avoid the extent of blending in each tubing from impacting the consequences, I will guarantee that following clip, there will be no stirring of the mixtures when solutions are added in any manner so that this manner, it will be changeless ( no commixture ) for each tubing.

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