Effect of concentration & biological variety on percentage activity of pectinase

How does the percentage activity of the enzyme – Pectinase in extracting juice from apples (Malus domestica) depends on the type of the apple – Red apple and Green apple and the percentage concentration of the pectinase enzyme used?

Connecting the concepts, I have studied to the observations I made has always been the factor to intrigue me and learn more. I have often seen my mother to prefer buying red apples instead of green apples and apple juice is a major part of our family diet especially during the time of pandemic. However, I was intrigued to know the reason behind preferring red apples over green apples to make apple juice. As studied during my IGCSE Biology class, using the enzyme pectinase to make juice is a common industrial practice. Based on my knowledge from Topic-2 in IB, I was well aware about the factors like temperature, pH and others which controls the enzyme action. The Lock & Key mechanism of enzyme action also suggests how reaching a saturation point limits the amount of enzyme-substrate complex and thus the concentration of substrate may become a limiting factor for the percentage activity of an enzyme. However, the factor that bothered me as how the concentration of enzyme may also be a decisive factor to influence percentage activity. A research on effect of the percentage concentration of ! −amylase and glucoamylase on the hydrolysis of red sorghum starch to glucose. Results shows that the optimum concentration of the enzyme was 0.27% and 0.20% for amylase and glucoamylase1 . This made my query clearer and I decided to explore the effect of concentration of enzyme on percentage activity of enzyme. However, connecting back to my personal observation, I wanted to investigate if the genetic variety or stage of ripening of the substrate has an effect on the enzyme action on it or not. Thus, I arrived at the research question stated above.

Pectinase is an enzyme that breaks down pectin, a polysaccharide found in plant cell walls. Pectin has a high molecular mass and is the major component of middle lamella and the primary cell wall. They are heteropolysaccharides containing residues of D-galacturonic acids linked with each other through 1,4- linkages. Pectinases refers to a class of enzymes which are again classified into various forms based on the action they do. The two major categories are2 – methylesterases; removes the methoxy groups of esters of galacturonic acids and polygalacturonases that breaks down the glycosidic linkages of between galacturonic acid residues. In industries, pectinases of varied combination are used along with other enzymes like cellulases and hemicellulases which works on substrates from fruits and maximize the production of fruit juices³ . Pectinase enzymes are commonly used in processes involving the degradation of plant materials, such as speeding up the extraction of juice from fruits, including apples and sapota. Apart from production of juice, pectinases are also used to make wine clarification, coffee concentration, degumming of plant fibres, protoplast fusion industry and many more4 . Fungi is the main producer of pectinases. Other organisms that are used as a natural source of pectinases also includes bacteria, insects, nematodes and protozoans⁵ . Commercially, the bacteria Aspergillus spp. is used for the production of pectinase⁶

The enzyme has a specific area / location where the substrate binds to it and this site is known as the active site of the enzyme. The working mechanism of an enzyme can be explained in two ways-

According to this theory by Fischer, the active site of an enzyme has a specific geometric shape and the substrate can bind to the enzyme only when it has a complementary geometric shape of the active site. This explains why the enzyme action is specific in nature and thus a particular enzyme can work on a definite substrate only.

According to this theory, the substrate induces the enzyme to modify its three-dimensional shape and thus re-orient the active site in such a way so that the substrate can fit into it. Sometimes, the substrate may have bonded to the enzyme but is unable to change the shape of the active site and thus cannot react. Whereas, there are cases, where the substrate is not large enough to influence the shape of the enzyme.

According to this mechanism, there are two major steps in the reaction between an enzyme(E) and a substrate (S)

Step-1 - Substrate binds with the enzyme at its active site to form a enzyme-substrate complex

\(E+S\rightarrow ES\)

Step-2 - The substrate is converted into products on the surface of the enzyme and thus it detaches from the active site releasing the free enzyme.

\(ES\rightarrow P+E\)

The major factors that have an impact on the activity of an enzyme are temperature, pH and substrate concentration.

Enzyme works the best at a particular pH and temperature which are considered as the optimum values. Beyond this, the enzyme loses its shape and thus the active sites are deformed. This is denaturation of the enzyme which inhibits the formation of enzyme-substrate complex and thus reduces the activity. As the concentration of substrate increases, more and more enzyme molecules bind with it and thus the activity of enzyme increases. However, it reaches a limiting stage where there are no more enzymes left to react with the substrate and thus the activity reaches a maximum constant point. The optimum pH and temperature of pectinases are reported to be 30⁰ C and 7.4 though the values may slightly differ depending on the source of pectin used.

Fibre content of green apple is approximately 22 % more than that of red apple¹⁰. Protein content of green apple is approximately 63 % more than that of red apple. Sugar content of green apple is comparatively less than that of red apple¹¹. Vitamin content of green apple is comparatively more than that of red apple. Iron and Potassium content of green apple is comparatively more than that of red apple. Quantity of antioxidant content in red apple is significantly high in red apple over green apple¹².

• There is no correlation between the activity of the enzyme pectinase and the percentage concentration of it.
• The effect of percentage concentration on the activity of the enzyme pectinase is same in red apple and green apple.

• There is a correlation between the activity of the enzyme pectinase and the percentage concentration of it.
• The effect of percentage concentration on the activity of the enzyme pectinase is not same in red apple and green apple.

Percentage concentration of pectinase

Pectinase solutions of percentage concentration (mass by volume)- 20.00%, 30.00%, 40.00%, 50.00% and 60.00% were used. These solutions were made by adding the specific amount of pectinase within 100 cm3 of distilled water. Pectinase is a water-soluble enzyme. A 20.00% solution will contain 20.00 g of enzyme within 100 cc of water. A digital mass balance was used to weigh the enzyme and a graduated measuring cylinder was used to add the required volume of water. Industrially, the pectinase solutions used are within the range of 30.00% to 50.00%. To get a more vivid data, a value lower than the range and a value higher than the range has also been added. The purpose of designing this variable is to understand how the change in enzyme concentration may have an effect on the activity of the enzyme or the yield of the process, measured in terms of the volume of the juice produced. As control, to understand the amount of juice made in absence of pectinase a 0.00% solution of pectinase (only distilled water) was used.

Another aim of the investigation is to understand if the effect of percentage concentration on enzyme activity depends on the type of the biological variety of the substrate or not. To do this, the test was carried out on both red and green apples. Though both of them belong to the same class yet they differ in the protein composition and developmental stages as well.

Percentage enzyme activity

The experiment aims to measure the percentage activity using the formula given below

percentage enzyme activity 

\(=\frac{(Volume \ of\ juice\ extracted\ with \ pectinase \ in\ ±\ 0.25cc)-(Volume \ of\ juice\ extracted\ without \ pectinase\ in\ ±\ 0.25\ cc)}{Volume \ of\ juice\ extracted\ without\ pectinase\ in\ ±\ 0.25cc} × 100\)

The enzyme is a biological catalyst. The efficiency of a catalyst is always determined by finding the outcome of a bio-chemical process with the catalyst and without it. Here, the process is production of juice and thus the activity has been measured in terms of a ratio of the volume of juice produced with the enzyme and without it. To obtain convenient data set and easier comparison, instead of using fractional values of the ratio, percentage values has been chosen. Thus, higher the value of the ratio, more effective is the enzyme in decomposing pectin and produce juice. A graduated measuring cylinder was used to measure the volume of the juice made.