Effect of Temperature on Rate of Hydrolysation of Pineapples

Sara Khan

Investigation to determine whether temperature affects the rate of hydrolysation of bromelain enzymes within pineapples when placed within protein substrates

Aim: To investigate the effects of increasing temperatures on pineapples, which contain the enzyme bromelain, and observe the affect on the rate of hydrolysation when placed within gelatine.

Scientific background: Gelatine is a common protein used in the manufacture of jelly. It consists mainly of collagen which is a fibrous protein present in all animals. The structure of a protein is made up of a numerous amount of amino acids joined together by the process of condensation as shown in the diagram below.

Figure 1.0

Proteins are polymers of amino acids, and these polymers or polypeptides are made up of amino acids in a specific sequence which determines the proteins function. This is known as the primary structure of a protein. The basic structure of these amino acids is shown below.

The R groups of the amino acids interact with each other, altering the chain to twist and fold into a three dimensional shape. The length of chains can coil into alpha helices or beta pleated sheets and this is known as the secondary structure. The polypeptide chain then folds to produce a specific 3D shape which forms the tertiary structure.

These proteins, as mentioned are made throughout the formation of peptide bonds between amino acid molecules. In order for the bonds formed in proteins to be broken down, the reverse of condensation must occur which is hydrolysis (amide hydrolysis for peptide bonds). The addition of water will allow for the peptide bond to break spontaneously, however this process is very slow and is usually facilitated by the use of enzymes. This rate of breaking the peptide bonds (rate of hydrolysation) can be increased by enzymes that are specific to proteins, called proteases. Enzymes are a form of globular proteins, where the polypeptide chains are folded into compact spherical shapes. This 3D shape of enzymes is crucial for them to be able to form enzyme substrate complexes. Enzymes contain a groove along their surface which is an active site that is specific to a particular substrate.

The lock and key principle suggests that the substrate binds to the active site of its specific shape to then form the enzyme substrate complex. There is also the induced fit principle which states that the active site itself moulds to fit the shape of the substrate.

When temperature is increased this affects the enzymes activity rate and the graph below illustrates this:

The above graph shows that with increasing temperature the enzyme’s rate of reaction increases as the molecules are given more kinetic energy, so they begin to collide more frequently and more enzyme substrate complexes are made, therefore the rate of reaction increases. This continues until a certain point around 40 degrees Celsius (optimum temperature) and then the increased temperature begins to denature the enzyme not allowing it to react as the active site structure becomes damaged and so the substrate is unable to bind.

This investigation has been based on the proteolytic enzyme bromelain which is extracted from the stem of pineapples. It is a natural mixture of two proteases that breaks down the peptide bonds within collagen.

Hypothesis:

The hypothesis for this investigation is that with an increase of temperature, the bromelain enzymes from fresh pineapple will catalyse the hydrolysis of the protein substrate i.e. the jelly, at a faster rate. However the rate of hydrolysation will then no longer increase due to the enzymes being denatured. For canned pineapples, the rate of hydrolysation will not increase at all.

Rationale:

From previous cooking experience, it has been gathered that whenever jelly is made and pineapples are placed within it, the jelly will never remain settled and it will no longer be firm as it would be when made without the pineapples inside it. Also, there has been a recent entrance of studies on bromelain and whether it is able to aid in digestion and reduce inflammation and also reduce some forms of cancer tumours due to its ability to catalyse the amide hydrolysis reaction. Both these points have initiated my interest towards this specific investigation. To further apply the biological principles of enzyme based reaction and amide hydrolysis, I decided to find an everyday example of such theory and to investigate it. The task I have chosen to approach the investigation with, will allow me to develop a suitable method for the task, carry out a process to collect useful data, analyse the data collected, and to be able to conclude and evaluate from it. I decided to use pineapples as this is the main commercial source for bromelain. When reading an encyclopaedia on pineapple nutrition, it was noticed that canned pineapples were said to be used within jelly rather than fresh pineapple solely due to the fact within the canning process, very high temperature methods are used to prevent the food from being spoiled before being contained. Thus, the bromelain is broken down within the pineapple. This urged to extend my investigation onto comparing the rates of hydrolysation of fresh pineapple and canned pineapples.

Preliminary Method: To begin with a pilot experiment will be set up to make sure the procedure being used will provide accurate enough results and that the concentrations, volumes etc are all suitable. Only fresh pineapples were tested upon in the preliminary experiment as the pilot was designed to just test the method. The same method will be used for testing the fresh and canned pineapples.

Method used: