How do a range of temperatures of 5, 15, 25, 35, 45, 55, 65℃ affect bromelain activity
with a 0.0167M copper sulfate inhibitor, measured by percent loss in mass of gelatine
gummy bears?
1. Exploration and Personal Engagement
1.1 INTRODUCTION
Enzymes are generally proteins that act as catalysts in speeding up the rate of reactions, being continuously used and not
destroyed. They are significant to many chemical reactions such as respiration, digestion, muscle function and more, making them
essential for life and survival. Specifically, bromelain is a proteolytic enzyme found in pineapple which has been promoted as a dietary
supplement in order to lessen swelling and inflammation after injury or surgery (NCCIH). Besides its medicinal properties,
bromelain can be used as a meat tenderizer, and it possesses the ability to catalyze the breakdown of proteins into amino acids, one
example being collagen, which in the processed form is gelatine. Collagen is found in mammal’s connective tissues, and being a
fibrous protein, can be broken down through hydrolysis using enzymes like bromelain. However, these processes can be delayed by
inhibitors, which can bind to enzymes and reduce the likelihood of substrates and enzymes colliding. Examples of an inhibitor of
bromelain are not only bromein, but metal ions such as Cu2+, Ca2+, (Kaur et al.) and more. Among the numerous factors that can
affect enzyme activity, the influence of temperature was explored in this experiment. Essentially, this factor is a crucial aspect of life
that determines biological activity and growth as different organisms thrive at distinct temperatures, and maintaining temperatures is
a significant part of physiological homeostasis.
Personal Interest
As bromelain has been correlated to many medicinal properties, such as reducing inflammation or aiding digestion, I was
intrigued by investigating its properties. Extreme temperatures may inhibit enzyme activity, so I was also curious how inhibitors
would influence enzyme activity and potentially affect optimum temperatures, thus, I combined my curiosity in this with my
interest in bromelain. This led me to explore bromelain activity in this experiment through a range of temperatures when
involved with an inhibitor.
Research Question
How do a range of temperatures of 5, 15, 25, 35, 45, 55, 65℃ affect bromelain activity with a 0.0167M copper sulfate inhibitor,
measured by percent loss in mass of gelatine gummy bears?
Background Information
Bromelain is a group of sulfhydryl proteolytic enzymes with cysteine proteases found in the stem and fruit of Ananas
comosus, commonly known as pineapples. It is known to have an optimum temperature ranging from 35- 55℃ (Martins et al.)
and pH of 3-8 in the fruit, whilst it is slightly different in the stem with an optimum temperature of 50-60℃ and pH of 6-7
(Manzoor et al.). Bromelain is a biocatalyst, which provides an alternate pathway for reactions to occur by requiring lower
activation energies. One such reaction is protein hydrolysis, where proteins are broken down into amino acids through disrupting
peptide bonds between nitrogens and carbons. Specifically, the protein of gelatine from the original Haribo Goldbear gummy
bears was utilized as the substrate in this experiment. Gelatine is a form of collagen, a fibrous protein found in the skin, tendons,
and bones of animals, in which its primary structure of the amino acid sequence consists of glycine-X-hydroxyproline, (X being
any of 17 different amino acids) where every third amino acid is glycine. The chains of collagen are tightly bound due to glycine’s
small size (Wu and Crane). As bromelain enzymes come into contact with this substrate, the active sites of the enzyme specifically
bind to the triple helices of collagen which allows hydrolysis to be catalyzed. This degradation of the secondary structure further
breaks down the inner peptide bonds of the tertiary structure, by cleaving the fragments into the peptides and amino acids
(Wagermaier and Fratzl). Nonetheless, this process can be delayed or halted after a period of time in the presence of an inhibitor
of bromelain. One example which was explored in this paper was Cu (II) ions from CuSO4. Copper ions are significant to cell
physiology such as in mitochondrial respiration, iron absorption and more (Tapiero et al.) and can also function as cofactors of
1
, enzymes. Bromelain has been found to be inhibited noncompetitively by Cu (II) ions (Kaur et al.) essentially meaning that the
metal ions bind to the enzyme-substrate complex which prevents bromelain from catalyzing the reactions further (Jakubowski).
Temperature is the measurement of hotness or coldness, which is an intensive property being independent of the
quantity of matter like mass or volume (The Editors of Encyclopaedia Britannica). There are multiple scales utilized to measure
temperature involving the Fahrenheit, Celsius and Kelvin scales. Enzymes possess their own unique optimum temperature where
reactions can more efficiently occur, however, ranges far from the optimum can affect the enzyme structure by altering the active
site and denaturation. Therefore, temperature can affect enzyme activity, however, more research is needed to identify how
optimum temperatures can change when enzymes are exposed to an inhibitor.
Justification
The inhibitor of copper sulfate was utilized in this experiment, yet other inhibitors and how temperature affects enzyme activity
in this circumstance can be investigated further to understand bromelain activity in different conditions. Determining its activity
in the presence of different inhibitors may be beneficial in finding potential foods to avoid when intaking this enzyme for
medicinal purposes, at what temperatures which bromelain is the most active, and if this fits with the human body temperature.
Predicted effect of IV on DV
Within the temperature range of 5, 15, 25, 35, 45, 55, 65℃, the percent loss in mass will increase until 55℃ as bromelain’s
optimum temperature is found at 50℃ (Martins et al.), and will greatly decrease at lower temperatures and at 65℃ as bromelain
retains activity until 60℃ (Martins et al.). The optimum temperature may be higher than with CuSO4 as it inhibits bromelain
(Kaur et al.), which would mean the temperature may need to be increased to fasten the reaction as inhibitors slow the enzymatic
reaction down.
1.2 METHODS
Variables and Controlled Factors
Table 1.1: Variables and Controlled Factors
Variable Manipulation, Measurement, Control Monitoring
- Temperatures 5, 15, 25, 35, 45, 55, 65℃ were established using a digital
water bath for hot temperatures and ice baths for cold temperatures which
Independent Temperature were checked using a thermometer to ensure the correct level.
- Controlled measurements in each trial ensured mass loss derived from
bromelain activity and not temperature.
- Initial and final mass was recorded to calculate % loss in mass of gummy
𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝑚𝑎𝑠𝑠−𝑓𝑖𝑛𝑎𝑙 𝑚𝑎𝑠𝑠
bears using the formula: 𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝑚𝑎𝑠𝑠
· 100%
Dependent % loss in mass of
gelatine gummy bears - Initial mass was measured before the solution was poured.
- Final mass was measured an hour after gummy bears were inserted in the
water bath and the solution was poured out.
- The same flavour of “green apple” was used as the chemical composition of
Gummy bear flavour different gummy bear flavours are different, affecting enzyme activity and
mass loss.
Controlled
- A controlled amount of 10mL of pineapple juice for each independent
The amount of variable/trial was measured using the same 10mL graduated cylinder. 200g
pineapple juice for each of pineapple and 75mL water was also kept the same for each batch.
independent variable - This helped minimize variation in the amount of catalyzation possible as
and trial different volumes would result in a range of enzymes used across trials.
2
with a 0.0167M copper sulfate inhibitor, measured by percent loss in mass of gelatine
gummy bears?
1. Exploration and Personal Engagement
1.1 INTRODUCTION
Enzymes are generally proteins that act as catalysts in speeding up the rate of reactions, being continuously used and not
destroyed. They are significant to many chemical reactions such as respiration, digestion, muscle function and more, making them
essential for life and survival. Specifically, bromelain is a proteolytic enzyme found in pineapple which has been promoted as a dietary
supplement in order to lessen swelling and inflammation after injury or surgery (NCCIH). Besides its medicinal properties,
bromelain can be used as a meat tenderizer, and it possesses the ability to catalyze the breakdown of proteins into amino acids, one
example being collagen, which in the processed form is gelatine. Collagen is found in mammal’s connective tissues, and being a
fibrous protein, can be broken down through hydrolysis using enzymes like bromelain. However, these processes can be delayed by
inhibitors, which can bind to enzymes and reduce the likelihood of substrates and enzymes colliding. Examples of an inhibitor of
bromelain are not only bromein, but metal ions such as Cu2+, Ca2+, (Kaur et al.) and more. Among the numerous factors that can
affect enzyme activity, the influence of temperature was explored in this experiment. Essentially, this factor is a crucial aspect of life
that determines biological activity and growth as different organisms thrive at distinct temperatures, and maintaining temperatures is
a significant part of physiological homeostasis.
Personal Interest
As bromelain has been correlated to many medicinal properties, such as reducing inflammation or aiding digestion, I was
intrigued by investigating its properties. Extreme temperatures may inhibit enzyme activity, so I was also curious how inhibitors
would influence enzyme activity and potentially affect optimum temperatures, thus, I combined my curiosity in this with my
interest in bromelain. This led me to explore bromelain activity in this experiment through a range of temperatures when
involved with an inhibitor.
Research Question
How do a range of temperatures of 5, 15, 25, 35, 45, 55, 65℃ affect bromelain activity with a 0.0167M copper sulfate inhibitor,
measured by percent loss in mass of gelatine gummy bears?
Background Information
Bromelain is a group of sulfhydryl proteolytic enzymes with cysteine proteases found in the stem and fruit of Ananas
comosus, commonly known as pineapples. It is known to have an optimum temperature ranging from 35- 55℃ (Martins et al.)
and pH of 3-8 in the fruit, whilst it is slightly different in the stem with an optimum temperature of 50-60℃ and pH of 6-7
(Manzoor et al.). Bromelain is a biocatalyst, which provides an alternate pathway for reactions to occur by requiring lower
activation energies. One such reaction is protein hydrolysis, where proteins are broken down into amino acids through disrupting
peptide bonds between nitrogens and carbons. Specifically, the protein of gelatine from the original Haribo Goldbear gummy
bears was utilized as the substrate in this experiment. Gelatine is a form of collagen, a fibrous protein found in the skin, tendons,
and bones of animals, in which its primary structure of the amino acid sequence consists of glycine-X-hydroxyproline, (X being
any of 17 different amino acids) where every third amino acid is glycine. The chains of collagen are tightly bound due to glycine’s
small size (Wu and Crane). As bromelain enzymes come into contact with this substrate, the active sites of the enzyme specifically
bind to the triple helices of collagen which allows hydrolysis to be catalyzed. This degradation of the secondary structure further
breaks down the inner peptide bonds of the tertiary structure, by cleaving the fragments into the peptides and amino acids
(Wagermaier and Fratzl). Nonetheless, this process can be delayed or halted after a period of time in the presence of an inhibitor
of bromelain. One example which was explored in this paper was Cu (II) ions from CuSO4. Copper ions are significant to cell
physiology such as in mitochondrial respiration, iron absorption and more (Tapiero et al.) and can also function as cofactors of
1
, enzymes. Bromelain has been found to be inhibited noncompetitively by Cu (II) ions (Kaur et al.) essentially meaning that the
metal ions bind to the enzyme-substrate complex which prevents bromelain from catalyzing the reactions further (Jakubowski).
Temperature is the measurement of hotness or coldness, which is an intensive property being independent of the
quantity of matter like mass or volume (The Editors of Encyclopaedia Britannica). There are multiple scales utilized to measure
temperature involving the Fahrenheit, Celsius and Kelvin scales. Enzymes possess their own unique optimum temperature where
reactions can more efficiently occur, however, ranges far from the optimum can affect the enzyme structure by altering the active
site and denaturation. Therefore, temperature can affect enzyme activity, however, more research is needed to identify how
optimum temperatures can change when enzymes are exposed to an inhibitor.
Justification
The inhibitor of copper sulfate was utilized in this experiment, yet other inhibitors and how temperature affects enzyme activity
in this circumstance can be investigated further to understand bromelain activity in different conditions. Determining its activity
in the presence of different inhibitors may be beneficial in finding potential foods to avoid when intaking this enzyme for
medicinal purposes, at what temperatures which bromelain is the most active, and if this fits with the human body temperature.
Predicted effect of IV on DV
Within the temperature range of 5, 15, 25, 35, 45, 55, 65℃, the percent loss in mass will increase until 55℃ as bromelain’s
optimum temperature is found at 50℃ (Martins et al.), and will greatly decrease at lower temperatures and at 65℃ as bromelain
retains activity until 60℃ (Martins et al.). The optimum temperature may be higher than with CuSO4 as it inhibits bromelain
(Kaur et al.), which would mean the temperature may need to be increased to fasten the reaction as inhibitors slow the enzymatic
reaction down.
1.2 METHODS
Variables and Controlled Factors
Table 1.1: Variables and Controlled Factors
Variable Manipulation, Measurement, Control Monitoring
- Temperatures 5, 15, 25, 35, 45, 55, 65℃ were established using a digital
water bath for hot temperatures and ice baths for cold temperatures which
Independent Temperature were checked using a thermometer to ensure the correct level.
- Controlled measurements in each trial ensured mass loss derived from
bromelain activity and not temperature.
- Initial and final mass was recorded to calculate % loss in mass of gummy
𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝑚𝑎𝑠𝑠−𝑓𝑖𝑛𝑎𝑙 𝑚𝑎𝑠𝑠
bears using the formula: 𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝑚𝑎𝑠𝑠
· 100%
Dependent % loss in mass of
gelatine gummy bears - Initial mass was measured before the solution was poured.
- Final mass was measured an hour after gummy bears were inserted in the
water bath and the solution was poured out.
- The same flavour of “green apple” was used as the chemical composition of
Gummy bear flavour different gummy bear flavours are different, affecting enzyme activity and
mass loss.
Controlled
- A controlled amount of 10mL of pineapple juice for each independent
The amount of variable/trial was measured using the same 10mL graduated cylinder. 200g
pineapple juice for each of pineapple and 75mL water was also kept the same for each batch.
independent variable - This helped minimize variation in the amount of catalyzation possible as
and trial different volumes would result in a range of enzymes used across trials.
2
