Salgado, Jan Joseph V.

MEB-11

5/7/2020

Find a research paper about the characterization of an enzyme. Look for two methods used in the
characterization of the enzyme. Discuss these methods in detail. (draft)

Purification and Characterization of Melanogenic Enzyme Tyrosinase from

Button Mushroom

Kamal Uddin Zaidi,1 Ayesha S. Ali,2 and Sharique A. Ali2

1 Molecular Biotechnology Laboratory, Centre for Scientific Research & Development, People’s
University, Bhopal 462010, India 2 Department of Biotechnology, Saifia College of Science, Bhopal
462001, India

Abstract

Melanogenesis is a biosynthetic pathway for the formation of the pigment melanin in human skin. A
key enzyme, tyrosinase,catalyzes the first and only rate-limiting steps in melanogenesis. Since the
discovery of its melanogenic properties, tyrosinase has been in prime focus and microbial sources of
the enzyme are sought. Agaricus bisporus widely known as the common edible mushroom, it’s taking
place in high amounts of proteins, enzyme, carbohydrates, fibers, and low fat contents are frequently
cited inthe literature in relation to their nutritional value.

In the present study tyrosinase from Agaricus bisporus was purified by ammonium sulphate
precipitation, dialysis followed by gel filtration chromatography on Sephadex G-100, and ion exchange
chromatography on DEAE-Cellulose; the enzyme was purified, 16.36-fold to give 26.6% yield on total
activity in the crude extract and final specific activity of 52.19U/mg. The SDS-PAGE electrophoresis
showed a migrating protein band molecular weight of 95 kDa. The purified tyrosinase was optimized
and the results revealed that the optimumvalues are pH7.0 and temperature 35∘C.Thehighest activity
was reported towards its natural substrate, L-DOPA, with an apparent Km value of 0.933 mM.This
indicated that tyrosinase purified from Agaricus bisporus is a potential source for medical applications.
Assay of Tyrosinase Activity.

The tyrosinase activity assay was performed as reported by Sung and Cho spectrophotometrically,
measuring conversion of L-DOPA to red colored oxidation product dopachrome. The initial rate of
reaction is proportional to concentration of the enzyme.An aliquot containing tyrosinase was incubated
for 5min at 35∘C at time zero, 1mL of L-DOPA solution (4mg/mL) for measured at 475 nm. After
incubation for additional 5min, the mixture was shaken again and a second reading was determined and
was measured for 3 minutes.The change in absorbance was proportional to enzyme concentration. One
unit of enzyme corresponded to the amount which catalyzed the transformation of 1 𝜇mol of substrate
to product per min under the above conditions and produced 1.35 changes in absorbance. Specific
activity was expressed as enzyme unit per milligram of protein.The protein content of the enzyme was
determined by the method of Lowry, with bovine serum albumin as standard.

(Method 1) Spectrophotometric Enzyme Assays

The spectrophotometric assay is the most

common method of detection in enzyme assays.
The assay uses a spectrophotometer, a machine
used to measure the amount of light a substance's
absorbs by calculating the appearance of product
or disappearance of substrate concentrations.
The spectrophotometric assay is simple, non-
destructive, selective, and sensitive.

During a spectrophotometric assay, the operator

follows the course of an enzyme reaction by measuring the changes in the intensity of the light absorbed
or scattered by the reaction solution. Most tests use the UV/visible (UV/vis) spectroscopy as the
detection method, which usually falls into the wavelength range of 100-1100 nm. If the light is in the
visible region, meaning the wavelength of 400-700 nm or 360-900 nm, the color of the assay can be
visibly captured by the naked eyes. Therefore, this type of tests is also called colorimetric assays.

• When the UV/vis spectroscopy is performed to determine the reaction rate or catalysis
efficiency, the reaction solution presents color or brightness drifts from reactants to products,
and such drifts can be detected by the UV/vis spectrometer.
• The rate constant of the enzyme reaction can be quantified by measuring the UV/vis absorbance
spectrum over a period of time.
 • Often as a first step, the optimal wavelengths for detecting all species involved in the reaction
will also be determined. The optimal wavelength should clearly indicate the difference between
the reactant and product, while interferences from other chemicals stay minimal.
• When measuring the enzyme reaction, the sensitivity of the detector and stability of the whole
spectroscopy system are critical to reliable measurement.
o The sensitivity of the detector guarantees detection of subtle changes in light intensity
of an enzyme reaction.
o The stability maintains low fluctuation and noise levels
o Jointly, the two aspects of the instrument control the signal-to-noise ratio, and high-
quality instruments can test enzyme activities in far more cases than an average
instrument.

Sodium Dodecyl Sulfate-Polyacrylamide Gel (SDS-PAGE) Electrophoresis of Purified

Tyrosinase.
SDS-PAGE was performed using a 12% separating gel and 4% stacking gel. The samples were heated
for 5min at 100∘C in capped vials with 1% (w/v) SDS in the presence of 𝛽-mercaptoethanol.
Electrophoresis was performed at a 125V for 4 h in Tris-HCl buffer of pH 8.3. After electrophoresis,
proteins in the separating gel were made visible by staining with Coomassie Brilliant Blue250. The
standards used to make a plot of log molecular weight versus mobility of the protein band were
lysozyme (20 kDa), myoglobin (26 kDa), carbonic anhydrase (38 kDa), ovalbumin (46 kDa), glutamate
(62 kDa), bovine serum albumin (91 kDa), 𝛽-galactosidase (120 kDa), and myosin (200 kDa).

(Method 2) Enzyme Purification by Electrophoresis

Enzyme purification is important in
acquiring the information regarding
structural and functional properties of
the enzyme and to foretell its
applications. The objective behind
deciding the strategy for purification is
to obtain the greatest possible yield of
the desired enzyme with the highest
catalytic activity and the greatest
possible purity.
Electrophoresis is an electrokinetic process which separates charged particles in a fluid using a field of
electrical charge. It has become a basis of a number of analytical techniques, which can be used in
enzyme separation by size, charge, or binding affinity. The type of electrophoresis used in the study
was Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

• SDS-PAGE is a widely used method to analyze and isolate proteins. During the electrophoresis,
proteins would move towards the positive electrode, with the migration rate representing a
positive correlation to the molecular weight of the protein.

• In SDS-PAGE, proteins are separated in a palyacrylamide gel based on their molecular weight.
Proteins are amphoteric molecules,which means they have both positive and negative charges.
To make them move in a single direction, a uniform negative charge is created on them. When
the proteins are mixed with SDS, they acquire a uniform net negative charge.

• SDS is a detergent having a negative charge, therefore it is an anionic detergent, it denatures

the native proteins by disturbing the non-covalent forces.

• The non-covalent forces include hydrogen-bonding, hydrophobic and ionic interactions which
are responsible for the three-dimensional structure of a native protein

• The denatured linear protein molecules are loaded onto the polyacrylamide gel (PAGE) which
is made by polymerizing the acrylamide monomers.

• PAGE has two phases: a stacking gel and a separating gel.

o Under an applied electric field, the stacking gel concentrates the SDS-loaded linear
protein molecules

o Under an applied electric field the separating gel separates the proteins on the basis of
molecular weight.

• After the run, PAGE gel is placed in a Coomassie Brilliant Blue

R250 dye solution for staining for a few hours and is de-stained to
visualize the separated protein molecules as bands.

Polyacrylamide gel electrophoresis of

tyrosinase
References:
1. Purification and characterization of Melanogenic enzyme tyrosinase from Button
mushroom. (2014, August 14).
Retrieved from: https://www.hindawi.com/journals/er/2014/120739/
2. Sodium Dodecyl sulfate polyacrylamide gel electrophoresis. (n.d.).
Retrieved from: https://sciencedirect.com/topics/biochemistry-genetics-and-molecular-
biology/sodium-dodecyl-sulfate-polyacrylamide-gel-electrophoresis
3. Spectrophotometric enzyme assays. (n.d.).
Retrieved from: https://www.creative-enzymes.com/resource/spectrophotometric-
enzyme-assays_5.html