EXTRASCTION AND CATALYTIC ACTION OF POLYPHENOL OXIDASE ON

PIGMENT FORMATION FROM MUSHROOM CAP AND STALK

INTRODUCTION

Enzymes are biological catalyst which increases the rate of a reaction by lowering the activation
energy requirement, thereby allowing a thermodynamically feasible reaction to occur at a
reasonable rate in the absence of thermal activation (Hardin et al, 2012). They are made of
proteins with unique properties. Living cells perform a multitude of chemical reactions very
rapidly because of the participation and involvement of substances called enzymes (van den
Burg, 2003). The materials with which the catalyst reacts with is known as a substrate and it is
the one modified during the chemical reaction to form a new product. The product can either be
joint molecules of the substrate or broken down molecules of the substrate thus the classification
of enzymes into two groups based on this. . Enzymes are also used for clinical purposes, they are
the preferred markers in the various disease states such as cancer and neurodegenerative
disorders. Hardin et al, (2012) shows that they provide insight into the diseases process by
diagnosis, prognosis and assessment of response therapy.

Polyphenol oxidase (PPO) is an example of an enzyme that catalyze the oxidation of phenolic
compounds using molecular oxygen. The reactions catalyzed by polyphenol oxidase are
hydroxylation and oxidation reactions. In case of oxidation reactions, polyphenol oxidize can
catalyze reactions resulting in black, brown and red pigments (polyphenols) which are the cause
of fruit coloring (Mayor, 2006). According to Zeikus (2001), “There are mainly three types of
polyphenol oxidases classified according to their substrate specificities and mechanism of
actions. These are; tyrosinase, catechol oxidase and laccase.” Real life applications of
polyphenol oxidases include the flavor enhancement of coffee, tea and cocoa production, and
determination of food quality (Burton, 1994). In medicine, they have several uses in treatments
of Parkinson’s disease, phenylketonuria and leukemia whereas in wastewater treatment, they are
used for the removal of phenolic pollutants from wastewaters. Van den Burg (2003) stated that,
“Other clinical importance of polyphenol oxidases is to monitor levels of L-3, 4-
dihydroxyphenylalanine (L-DOPA) which is an enzyme substrate that is made and used as part
of the normal biology of humans.”
Since L-DOPA attracted notice as a specific drug for Parkinson's disease, reports concerning its
microbial production have appeared. In addition, this enzyme is responsible for the formation of
melanin (Claus and Decker, 2006).Therefore, the aim of the experiment was to isolate
polyphenol oxidase (tyrosinase) from mushrooms and then study its ability to catalyze pigment
formation.

MATERIALS AND METHOD

The mushroom cap and stalk tissue were collected and separated, of which 2g of each was
weighed out and used. Each weighed sample was grinded using a mortar and pestle, 5mL of
extraction buffer. One group consisting of two members worked on the cap while the other
worked on the stalk and later shared results. Mushroom samples were grinded to a fine
suspension with a pestle using the buffer solution of which small volumes were added at a time.
After preparing the extracts, the suspensions were emptied into two different empty centrifuge
tubes kept in an ice bucket then the pestles and mortars were rinsed with the remaining 3mL of
the extraction buffer. All the extracts were centrifuged at the same time at 15000rpm for
10minutes at 4°C and the supernatant was collected into a clean sample bottle then placed on ice.

1.5mL of assay buffer (100mM phosphate (NaH2PO4) buffer (pH 6)), 0.3Ml of 4mM L-DOPA
and a certain amount of water to make the total volume up to 2.95 mL were measured and added
into a 3.0mL spectrophotometer test tube using different pipettes for each solution. They were
mixed and placed inside the spectrophotometer set at 475nm which was the zero- time reading to
measure the amount of light absorbed or transmitted through a solution. 0.05mL of enzyme
extract was added to the spectrophotometer test tube containing the assay buffer and L-DOPA
and change in absorbance was recorded every 30 seconds over the next 10 minutes. This process
was repeated using 0.5mL, 0.7mL, 0.9mL and 1.2mL of 4mM L-DOPA using different
spectrophotometer test tubes.
DISCUSSION
From the results, there are two graphs, Figure 1 for the stalk and Figure 2 for the cap of a
mushroom. These graphs are line graphs that show an increase in absorbance over time. The cap
recorded higher absorbance as compared to the stalk, on average. This could be explained that
since the mushroom cap has a better exposure to the sun rays hence ultraviolet radiation, it would
have higher melanocytes as compared to the mushroom stalk. The results shows that both the
mushroom stalk and cap had the highest absorbance at 1.2ml L-DOPA. The absorbance differed
with different concentrations. The more the concentration the higher the absorbance but
mushroom stalk results somehow had a disagreement, i.e. At the concentration of 0.5 ml L-
DOPA, the absorbance was much larger than at 0.7ml and 0.9ml L-DOPA. This might be due to
errors that were committed during the process, because the spectrophotometer machines were
shared some tubes were left out for some time to allow for others to finish and hence leading the
variance in the results because according to Ruijssenaars and Hartmans, (2004 )‘The longer the
exposure, the higher melanin production and darker skin, which serves as a protective
mechanism from the harmful ultraviolet radiation’
Sadava et al (2012) shows that increasing the amount or concentration of the enzyme will speed
up the reaction, as long as there is substrate to bind to it as such increasing the amount of the
polyphenol oxidase extract would have result in the reaction speeding up hence the change in
absorbance would have increased. Enzymes catalyze and participate in reactions, but are neither
reactants nor products; therefore they are never exhausted in chemical reactions (Copper, 2000),
because polyphenol oxidase is not a reactant in the reaction, L-DOPA can increase and
polyphenol oxidase remains the same, and an increase in product hence absorbance observed.
Once all of the substrate is bound to the enzyme, the reaction will no longer speed up since there
will be nothing for additional enzymes to bind to.
In human beings, the role of L-DOPA reflects similarly to this experiment with melanin
synthesis by polyphenol oxidase. In melanin synthesis, L-DOPA is the precursor cell, and its
concentration level increases the melanin content in the skin (Claus and Decker, 2006). Similar
pigments in potatoes cause the process of peeled potatoes turning brown due to the ability of
polyphenol oxidase to form pigments in plants in the presence of aromatic compounds that are
substituted by PPOs. The activity of plant PPOs can cause browning or bleaching in certain fruit
or vegetables. PPOs contain copper and react with oxygen and the phenolic compounds in plants
to generate reactive quinones (Queiroz et al, 2008).
These quinones frequently continue reacting with each other and nearby proteins to generate
brown pigments. Such pigments result in the enzymic browning of the fruits and vegetables,
often rendering the food unfit for human consumption.
When potatoes are immersed, the formed pigments tend to fade and formation of new pigments
is slowed because there is little molecular oxygen available to react as compared with air
(Queiroz et al, 2008).
CONCLUSION
It was concluded that as the concentration of L-DOPA increases the change in absorbance also
increases and more polyphenol oxidase activity was present in the mushroom cap as compared to
that of mushroom stalk. The highest absorbance in both the stalk and cap was recorded at 1.2mL
of L-DOPA.

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