IB Biology SL Internal Assessment
Research question: How does immobilisation of pectinase, through
entrapment of pectinase in calcium alginate beads, affect the hydrolysis of
pectin in apple (Malus domestica) and pear (Pyrus pyrofolia) juice measured
in terms of change in absorbance using a Vernier colorimeter over a time
period of 20 minutes?
May 2020
, Biology Internal Assessment
Research question: How does immobilisation of pectinase, through entrapment of
pectinase in calcium alginate beads, affect the hydrolysis of pectin in apple (Malus
domestica) and pear (Pyrus pyrofolia) juice measured in terms of change in absorbance
using a Vernier colorimeter over a time period of 20 minutes?
Background Information
Enzymes are proteins that act as biological catalysts and increase the rate of reaction without
undergoing any change to themselves. Each enzyme is substrate specific, and binds to the active site
of a particular substrate. As seen in the graph below, enzymes lower the activation energy (Ea) of a
reaction, leading to an “increase in the number of reactant molecules that can overcome the activation
barrier and form the product.” (Openstax)
Enzymes are used for industrial purposes to catalyze certain
reactions. They are often immobilised so they can be
separated from the products of the reaction, thereby
avoiding contamination. (Allott et al. 2014). Enzymes can
be immobilised through attachment to an inert substance to
constrain their mobility (ibid). In this experiment, the
technique of entrapment was used to form calcium alginate
Source: Enzymes and Reaction Rates,
www2.nau.edu/lrm22/lessons/enzymes/enzymes beads. The process of entrapment involves the
.html
“incorporation of enzymes into the lattices of a semi-permeable gel or enclosing the enzymes in a
semi-permeable polymer membrane” (Patel et al). Immobilisation of enzymes by entrapment is one
of the most popular methods of enzyme immobilisation, and matrices like polyacrylamide, agar-agar
and alginate are used to immobilise enzymes by entrapment (ibid).
Background Information
Pectin is a polysaccharide found in the cell walls of certain fruits and vegetables, including apples,
pears and berries. Pectin often contributes to the viscosity and turbidity of fruit juices. However, the
production of clear fruit juice requires the removal of aggregated particles in the juice. Pectinase,
according to Jadaun et al., “hydrolyses the glyosidic bonds present between the galacturonic acid
monomers, decreasing pectin’s water holding capacity.” This results in higher juice yield, and aids
in removing turbidity. Pectinase converts pectin into galacturonic acid and simpler, soluble sugars.
, At the same time, although the enzyme pectinase is widely used in fruit processing industries to
hydrolyse pectin in fruit juices, the process is limited by the “short-term operational stability of the
enzyme and its low yield.” (Patel et al.). Pectinase is often immobilised to overcome these
constraints, resulting in higher juice yield and reusability.
My interest in the biological mechanisms associated with turbidity removal arose while making
apple juice at home. I realised that home-made apple juice is much thicker and more turbid than
store-brought, bottled juices. I was curious about the industrial processes employed by companies in
fruit juice clarification. Furthermore, given the surge in the fruit juice market, I wondered if there
was an environmentally friendly alternative for turbidity removal. After conducting research on the
topic, I learnt that pectinase immobilisation posed several advantages in this regard, due to its
reusability and catalytic efficiency.
Pilot study
Several preliminary studies were conducted to determine the feasibility of the method employed in
the experiment and effectively operationalise the variables.
The hydrolysis of pectin was measured by change in absorbance using colorimetric analysis. The
wavelength of 465 nm was chosen for colorimetric analysis for the three experimental conditions as
this was the wavelength at which maximum absorbance was observed.
To narrow down on the fruits used in the experiment for the preparation of the substrates, research
on the average pectin levels of fruits and vegetables was conducted. According to Wichtl et al. in
2004, average pectin levels of apples and pears were 1–1.5%, and 1.4% respectively. Pectin levels
for other fruits, like cherries and guavas were significantly lower, at 0.4% and 0.2%. Apples and
pears were chosen due to their high pectin concentration, because this would allow for greater surface
area for pectinase to act upon. A mass of 50 g was chosen for both pears and apples because this was
determined to be adequate for observable experimental changes to occur.
Furthermore, studies with varying concentrations of sodium alginate, at 1%, 1.5%, 3% and 6% were
conducted to determine the optimal concentration of sodium alginate. A concentration of 1.5% was
selected to obtain the “desired hardness” for immobilised pectinase beads. (Wang et al.) In addition,
preliminary studies with syringes, burettes and drippers were performed to determine the most
Research question: How does immobilisation of pectinase, through
entrapment of pectinase in calcium alginate beads, affect the hydrolysis of
pectin in apple (Malus domestica) and pear (Pyrus pyrofolia) juice measured
in terms of change in absorbance using a Vernier colorimeter over a time
period of 20 minutes?
May 2020
, Biology Internal Assessment
Research question: How does immobilisation of pectinase, through entrapment of
pectinase in calcium alginate beads, affect the hydrolysis of pectin in apple (Malus
domestica) and pear (Pyrus pyrofolia) juice measured in terms of change in absorbance
using a Vernier colorimeter over a time period of 20 minutes?
Background Information
Enzymes are proteins that act as biological catalysts and increase the rate of reaction without
undergoing any change to themselves. Each enzyme is substrate specific, and binds to the active site
of a particular substrate. As seen in the graph below, enzymes lower the activation energy (Ea) of a
reaction, leading to an “increase in the number of reactant molecules that can overcome the activation
barrier and form the product.” (Openstax)
Enzymes are used for industrial purposes to catalyze certain
reactions. They are often immobilised so they can be
separated from the products of the reaction, thereby
avoiding contamination. (Allott et al. 2014). Enzymes can
be immobilised through attachment to an inert substance to
constrain their mobility (ibid). In this experiment, the
technique of entrapment was used to form calcium alginate
Source: Enzymes and Reaction Rates,
www2.nau.edu/lrm22/lessons/enzymes/enzymes beads. The process of entrapment involves the
.html
“incorporation of enzymes into the lattices of a semi-permeable gel or enclosing the enzymes in a
semi-permeable polymer membrane” (Patel et al). Immobilisation of enzymes by entrapment is one
of the most popular methods of enzyme immobilisation, and matrices like polyacrylamide, agar-agar
and alginate are used to immobilise enzymes by entrapment (ibid).
Background Information
Pectin is a polysaccharide found in the cell walls of certain fruits and vegetables, including apples,
pears and berries. Pectin often contributes to the viscosity and turbidity of fruit juices. However, the
production of clear fruit juice requires the removal of aggregated particles in the juice. Pectinase,
according to Jadaun et al., “hydrolyses the glyosidic bonds present between the galacturonic acid
monomers, decreasing pectin’s water holding capacity.” This results in higher juice yield, and aids
in removing turbidity. Pectinase converts pectin into galacturonic acid and simpler, soluble sugars.
, At the same time, although the enzyme pectinase is widely used in fruit processing industries to
hydrolyse pectin in fruit juices, the process is limited by the “short-term operational stability of the
enzyme and its low yield.” (Patel et al.). Pectinase is often immobilised to overcome these
constraints, resulting in higher juice yield and reusability.
My interest in the biological mechanisms associated with turbidity removal arose while making
apple juice at home. I realised that home-made apple juice is much thicker and more turbid than
store-brought, bottled juices. I was curious about the industrial processes employed by companies in
fruit juice clarification. Furthermore, given the surge in the fruit juice market, I wondered if there
was an environmentally friendly alternative for turbidity removal. After conducting research on the
topic, I learnt that pectinase immobilisation posed several advantages in this regard, due to its
reusability and catalytic efficiency.
Pilot study
Several preliminary studies were conducted to determine the feasibility of the method employed in
the experiment and effectively operationalise the variables.
The hydrolysis of pectin was measured by change in absorbance using colorimetric analysis. The
wavelength of 465 nm was chosen for colorimetric analysis for the three experimental conditions as
this was the wavelength at which maximum absorbance was observed.
To narrow down on the fruits used in the experiment for the preparation of the substrates, research
on the average pectin levels of fruits and vegetables was conducted. According to Wichtl et al. in
2004, average pectin levels of apples and pears were 1–1.5%, and 1.4% respectively. Pectin levels
for other fruits, like cherries and guavas were significantly lower, at 0.4% and 0.2%. Apples and
pears were chosen due to their high pectin concentration, because this would allow for greater surface
area for pectinase to act upon. A mass of 50 g was chosen for both pears and apples because this was
determined to be adequate for observable experimental changes to occur.
Furthermore, studies with varying concentrations of sodium alginate, at 1%, 1.5%, 3% and 6% were
conducted to determine the optimal concentration of sodium alginate. A concentration of 1.5% was
selected to obtain the “desired hardness” for immobilised pectinase beads. (Wang et al.) In addition,
preliminary studies with syringes, burettes and drippers were performed to determine the most
