Effect Of Concentration And 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.

• Use safety gloves and safety masks.
• Hair must be tied while performing the experiment.
• Use laboratory coats.
• The knife was handled with care to avoid any unwanted accident.
• The electric switches were not touched with wet hands.

Optimum quantity of apple was purchased and none of it has been wasted during the exploration.

• No heating procedure has been introduced in this exploration which reduced the chances of evolution of harmful gases that might affect the environment.
• The apples considered in this exploration were chosen in such a way that no buds were attached with the twig of the apple. As the bud would develop into a fully grown tree, thus the buds needed to be protected as an environmental consideration.
• The seeds of the apples were conserved and planted after the end of the experiment to follow the standard protocols of disposing green waste.

• A watch glass was placed on the top pan mass balance.
• The reading of the balance was adjusted to 0.00 ± 0.01 g.
• Using a spatula, the pectinase powder was transferred from the reagent container to the watch glass until it reads 20.00 ± 0.01 g.
• The weighed mass of pectinase was transferred from the watch glass to a 100 cc glass beaker.
• Both the watch glass and the spatula were washed with distilled water and the washings were collected in a glass beaker.
• Distilled water was added till the mark of 100 cc using a graduated measuring cylinder.
• A glass rod was used to stir the solution and obtain a clear 20.00 % solution of pectinase.
• All of the above steps were repeated with 30.00 ± 0.01 g, 40.00 ± 0.01 g, 50.00 ± 0.01 g and 60.00 ± 0.01 g of pectinase powder to make 30.00 %, 40.00 %, 50.00 % and 60.00 % pectinase solutions respectively.

• A piece of apple was peeled off and cut into pieces using a knife and a chopping board.
• The cut pieces were transferred into a container of mixer grinder in the container where the container is only half occupied with the apple pieces.
• The mixer blender was switched on and operated at Level 1 for 3 minutes and at Level 2 for 2 minutes. A stop-watch was used to monitor these timings. The lid of the container was held tight by pressing it with the palm when the blender was operated.
• The puree thus obtained was then transferred into a 100cc beaker.
• All of the above steps were repeated for a piece of red apple.

• A 100 cc clean and dry glass beaker was taken.
• The beaker was placed on a top pan digital mass balance.
• The reading of the balance was adjusted to 0.00 ± 0.01 g using the ‘Tare’ option.
• The apple puree of green apple was added to the beaker until the balance reads 5.00 ± 0.01 g.
• The beaker was taken away from the balance.
• Using a graduated measuring cylinder, 20.00 ± 0.25 cc of 20.00 % pectinase solution was added to the beaker.
• A buffer sachet of pH=7.2 was taken and the entire sachet was added to the beaker.
• Using a glass rod, the puree was mixed uniformly with the solution added to obtain a homogenous suspension.
• The stop-watch was started.
• As soon as the stop-watch reads 60.00 ± 0.01 minutes, the content of the beaker was filtered using a Whatmann-41 filter paper, a funnel and the filtrate was collected in a graduated measuring cylinder.
• The reading of the graduated measuring cylinder was noted down.
• Steps 1-10 were repeated for 9 more times to collect data in ten trials.
• Steps 1-11 were repeated for the other pectinase solutions- 30.00%, 40.00%, 50.00% and 60.00%.
• Steps 1-12 were repeated for red apple puree.

• The pectinase solution made was light brown in color. As the % concentration increased, the color was deeper.
• There was a change in the color and viscosity of the content in the beaker containing apple puree and the pectinase solution. With the passage of time, the solution became denser and the viscosity reduced.
• The apple puree was found to settle down at the bottom after a while of adding the pectinase solution.

Sample Calculation (when percentage concentration of pectinase is 0.00%)

Mean Volume of juice extracted \(=\frac{sum\ of\ volume\ of\ juice\ made\ in\ all\ trials}{Number \ of \ trials}\)\(=\frac{26.00\ +26.50\ +\ ...+\ 26.50}{10}\)= 26.15 cm³

Standard Deviation in volume of juice extracted =\(\sqrt\frac{\sum(trial\ value\ -mean\ value)^2}{Number \ of\ trials}\)

\(=\sqrt\frac{(26.15-26.00)^2+(26.15-26.50)^2\ +...\ +\ (26.50-26.50)^2}{10}\)= 0.34 cm³

Percentage Uncertainty =\(\frac{absolute\ uncertainty\ of\ volume\ juice}{mean\ volume\ of\ juice\ extracted}\)× 100 =\(\frac{0.25}{26.15}\)× 100 = 0.19%

Sample Calculation (for red apple when the percentage concentration of pectinase is 20.00 %)

Percentage activity of pectinase =\(\bigg(\frac{Experimental\ value\ -\ Controlled \ value}{Controlled \ Value}×100\bigg)\%\)

\(=\frac{27.85-26.15}{26.15}\)× 100 = 6.50 %

From Graph 1, the variation of percentage activity of the enzyme – ‘pectinase’ has been studied with respect to the concentration of pectinase used in the trial, for two different varieties of apple, i.e., red apple and green apple. In the graph, the percentage concentration of pectinase (independent variable) has been plotted along the X – axis and the percentage activity of pectinase has been plotted along the Y – axis. The main findings of the graph are:

• In case of red apple, it has been observed that with an increase in the percentage concentration of pectinase from 20.00 % to 60.00 %, the percentage activity of pectinase has increased from 6.50 % to 23.33 % in red apples and from 1.79 % to 9.16 % for green apples. In both cases, there is an increase in the percentage activity of the enzyme used along with the increase in the % concentration of the enzyme used. This allows us to claim that higher the concentration of enzyme used, more the volume of juice extracted from both the varieties of apples.
• Furthermore, the equation of trend obtained in case of red apple is: y = 0.4169) − 1.646 whereas, the equation of trend obtained in case of green apple is: y = 0.0.1781 ) + 0.846, where, y indicated percentage activity of pectinase, and ) indicated percentage concentration of pectinase. In both cases, the value of gradient is positive which again confirms that the volume of juice extracted and thus the activity of the enzyme increases with the increase in the % concentration of the enzyme used.
• However, comparing the gradient shows that the gradient is more in case of red apples and less in case of green apples. The gradient indicates the rate at which the % activity increases with the increase in the % concentration of the enzyme used. The gradient is 0.41 in case of red apples and 0.17 for green apples. This claims that the increase of the percentage activity with the increase in the % concentration of enzyme is more significant in case of red apples than that in case of green apples.
• For all values of x axes, the values of y axes for the red apple are higher than that for the green apples. This again confirms that the volume of juice extracted from red apples is more than that extracted from green apples irrespective of the % concentration of enzyme used.
• For both red and green apples, the line passes through the origin. This is because in both cases, at 0.00% pectinase solution (control), the value of volume extracted experimentally and the controlled value is same. Thus, the value of percentage activity of the enzyme will be 0.00% as there is no enzyme used in this case.
• There are both vertical and horizontal error bars indicated for all the data points where the vertical error bars indicate the uncertainties associated with the data points in terms of the percentage activity while the horizontal error bars indicate the uncertainties associated with the values of percentage concentration of enzyme used. The graph shows that in most of the data points, the horizontal error bars are longer than the vertical error bars which shows that the error in the values of the concentration of enzyme used is little more significant that the error in % activity of the enzyme.

An ANOVA test was done to understand if there is any significant difference between the way the % concentration of pectinase have an impact on the % activity of the enzyme. As the two groups, red and green apples are mutually exclusive and are independent of each other, an ANOVA test (Analysis of Variance) was done as a tool of inferential statistics.

Sample Calculation

\(\large\sum\)a₁=6.50 +11.09 + 15.11 +19.12 +23.33 =75.15

The mean of percentage activity of pectinase in Red Apple and Green Apple would be equal

H_O: μ_{Red Apple} = μ_{Green Apple}

The mean of percentage activity of pectinase in Red Apple and Green Apple would be unequal

H_O: μ_{Red Apple} = μ_{Green Apple}

Number of type of apples (a) = 2

Total number of observation (N) = 10

Number of observation for each type of apple = 5

df _Between =a-1=2-1=1

df _Within = N- a =10-  2 = 8

df _total = N-1=10-1=9

From the ANOVA test table, it was obtained that the critical value for df _Between= 1, and df _Within = 8 is 5.3177.

SS_Between\(=\frac{\sum(\sum a_I)^2}{n}-\frac{T^2}{N}\)

\(\frac{75.15^2+... \ .\ .+31.39^2}{5}\)

\(=\frac{(75.15 \ +{...}_{...}\ +\ 31.39)^2}{10}\)

=1326.57 -1135.08

=191.49

SS_{Within =\(\Huge\sum\)y^{2-\(\frac{\sum(\sum a_{I})^2}{N}\)}}

=(6.50² + 11.09² + ^...

+23.33² + 1.79²

+5.10^{2 + ...}

+9.16²)

-1326.57

=1534.69-1326.57

=208.12

SS_{rotal=\(\Huge\sum\)}y^₂ -\(\frac{T^2}{n}\)

=1535.69 -1135.08

=400.61

MS_{Between =\(\frac{SS_{Between}}{df_{Between}}\)}

_{\(=\frac{191.49}{1}\)}

=191.49

\(\frac{208.12}{8}\)

 _{=\(MS_{Within}\ \frac{SS_{Within}}{df_{Within}}\)}

= 26.02

\(F\frac{MS_{Between}}{MS_{Within}}\)=\(\frac{191.49}{26.02}\)

= 7.36

Since, the value of F (7.36) is greater than the critical value (5.32), the Null Hypothesis would be rejected.

Graph-1 clearly shows that there is an increase in the % activity of the enzyme with the increase in % concentration of the pectinase solution used. Thus, null hypotheses -1 is rejected and the alternate hypotheses 1 is accepted. The statistical analysis also shows that the mean of the two groups – red and green apple are significantly different from each other. This again confirms that effect of increase in % concentration on % activity of the enzyme pectinase is not the same in case of red and green apples. Thus, null hypotheses -2 is rejected and alternate hypotheses -2 is accepted.

A research report on the use of pectinases to decompose the pectin in agricultural waste products¹³ reports that the change in concentration of the pectinase used changes the percentage activity of pectinase positively; as the concentration of enzyme used increases the percentage activity of the enzyme also increases. However, it also depends on certain other factors like incubation period, temperature and many more. Thus, the results in this investigation is in alignment with a literature reference.

As the percentage concentration of pectinase solution increases, there are more enzyme molecules to bind with the substrate pectin and thus higher number of pectin-pectinase complexes are formed. This eventually causes more hydrolysis of glucuronic acid linkages of pectin decomposing more amount of pectin in the cell walls. Thus, more volume of juice is extracted. This increase of experimental value of juice extracted in presence of the enzyme eventually increases the percentage activity of the enzyme used. Pectin is a carbohydrate. Red apples are at an advanced stage of ripening than the green apples. Thus, the carbohydrate content of red apples is more than that in green apples. Red apples have been reported to have 0.14% of pectin (by mass) in their pulp while the value for green apples is 0.09% only. Thus, it is clear that there is more amount of pectin in red apples than green apples. This explains why for all values of % concentration of enzyme, the volume of juice extracted from red apples is more than that for green apples.

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

• It has been observed that with an increase in the percentage concentration of pectinase from 20.00 % to 60.00 %, the percentage activity of pectinase has increased from 6.50 % to 23.33 % in red apples and from 1.79 % to 9.16 % for green apples. Higher the concentration of enzyme used, more the volume of juice extracted from both the varieties of apples.
• The increase of the percentage activity with the increase in the % concentration of enzyme is more significant in case of red apples than that in case of green apples.
• The volume of juice extracted from red apples is more than that extracted from green apples irrespective of the % concentration of enzyme used.
• The qualitative observations that the viscosity of the content in the beaker was increasing with the increase in the percentage concentration of the enzyme used also shows that the data collected is in coherence with the qualitative data as apple juice is viscous in nature. Thus, more the juice extracted, more viscous the mixture of the puree and the pectinase solution is.
• Both the null hypotheses have been rejected and both the alternate hypotheses has been accepted.
• The increase in the percentage activity of the pectinase with the increase in the concentration of the pectinase solution is mainly due to the increase in the number of enzyme-substrate complex formed.
• The red apples show a better result with the increase in the percentage concentration of the pectinase due to a higher carbohydrate content and thus more amount of pectin in it in comparison to green apples.

• The range of independent variable obtained was complete and extensive. It included the values of independent variable possible from the minimum limit to the maximum limit at a fixed interval. This has made the exploration more coherent and reliable for every value of the independent variable.
• The values of the volume of juice extracted for both the apple has been taken by determining the average of ten trials to ensure that any methodological limitation or human error introduced during the experimental procedure could be reduced making the exploration more coherent and reliable.
• The values of standard deviation obtained for each trial of volume of juice extracted is significantly less and could be neglected. This makes the exploration more coherent as there is less dispersion in the data resulting in incurrence of less error in the data set.
• The quantitative data and the qualitative data show the same result like increase in activity with concentration as shown in Graph-1 and increase of viscosity as a qualitative observation.

All the red apples or green apples considered during the experiment involving red apples or green apples respectively are assumed to have same genetic composition. The apples taken for the experiment are not verified whether they are genetically similar or not. If each and every red apple or green apple considered during the experiment involving percentage activation of pectinase for red apple or green apple respectively are not genetically similar, then the enzyme action in those apples would be different from the others. This would introduce a significant error in the exploration. To nullify this error, genetically engineered apples should be obtained which would have same pH, and other specific parameters of apple for the exploration. However, in absence of such samples, apples bought from the same farm and preferably grown in the same plant can be used.

To explore the unanswered of the similar topic, one of the best topics to work on would be to determine the effect of pH of Pectinase on Red Apple and Green Apple determined using percentage activity of Pectinase. The exploration methodology is stated hereafter: Equal weight of red apple, measured using a digital weighing machine should be obtained from the market place provided that bud attached with the twig or seed should be conserved and planted later on. Pectinase solution was prepared with varied pH using buffer tablets. The apple was then soaked with different pectinase solutions of different pH but same concentration for a fixed amount of time. The time was measured in stopwatch. Thereafter, the apple was smashed and juice was extracted. The percentage activity of pectinase was again measured. The research question of the exploration could be framed as: “How does the percentage activity of the enzyme Pectinase in extracting juice depends on the pH of the enzyme taken and the type of apple (Malus domestica) – Red apple and Green apple used?

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