Laccase efficiency - ABTS
Laccase is a protein belonging to the multicopper oxidase family. Laccase uses laccase redox mediators,
such as 3-ethylbenzothiazoline-6-sulfonate (ABTS) or 1-hydroxybenzotriazole (HBT), to form the laccase
mediator system (LMS)
ABTS, the non-phenolic dye, will be oxidized by laccase into a more stable cationic radical state, so in the
reaction below (Fig1). ABTS will lose two electrons and become a divalent cationic radical, and the product
will
appear blue-green. At this time You can measure the absorbance. And the absorbance can be read at 420nm.
Therefore, by measuring the absorbance of the blue-green product and calculating its Km value, we can know
the laccase enzyme activity, because the darker the blue-green color becomes during the reaction time, it
means the greater the degree of ABTS oxidation by laccase, so you can know the laccase activity will be
bigger.[1][2]
Fig 1. ABTS reaction
Hydrophobic binding efficiency
To ensure the stable adsorption of hydrophobin onto commercially Color catchers (PET papers), we designed
several
methods to assess the binding capacity between hydrophobin and the blotting membranes. The following are the
tests we designed:
1. WCA (Water Contact Angle) test is performed to verify whether the displayed HFBI could alter the surface
hydrophobicity of the Color catcher:
The WCA test is the commonly used test in laboratories to determine the wettability of materials, if WCA is
smaller that 90°, the material is hydrophilic, if WCA is larger than or equal to 90°, the material is
hydrophobic.(Fig.2)[3]
2. Fluorescence microscopy analysis:
To confirm the binding efficacy using fluorescence, a specific segment of the gene, GFP (Green Fluorescent
Protein), was incorporated into the gene design. This inclusion allows the produced protein to exhibit a
green fluorescence. Leveraging this feature, hydrophobin was applied to Color catchers and immersed in
non-flowing water. Observations were made at hourly intervals to record the rate of detachment.
Fig 2. (a) If 0˚ < q < 70˚, representing a hydrophilic material. (b) If 70˚
< q <
110˚, representing a hydrophobic material.
Protein Complex
The purpose of this part is to make sure if our protein complex is formed as expected. In the first place,
we've confirmed the successful production of proteins qualitatively by using SDS-PAGE. The gel
electrophoresis reveals it clearly [fig3].In addition, these 3D structures of the produced protein can
help us predict whether proteins assemble correctly. Unfortunately, our proteins seem structurally difficult
to assemble into a stable complex [fig4]. However, due to the impending wiki freeze, we were
slightly running out of time. It resulted in that we couldn't perform quantitative measurements on the
protein complex. But in order to complete our experiment, we're planning to employ the bicinchoninic
acid
(BCA) assay as our quantitative measurement in the future.
Such assay depends on the conversion of Cu2+ to Cu+ under alkaline conditions. The
Cu+ is then
detected by reaction with BCA. The reaction results in the development of an intense purple color with an
absorbance maximum at 562 nm. Since the production of Cu+ in this assay is a function of protein
concentration and incubation time, the protein content of our samples may be determined
spectrophotometrically by comparison with known protein standards. Then we can get the concentration of our
protein and complete the quantification.
Fig 3. gel electrophoresis
Fig 4. Illustration of Failed Protein-Protein Docking;
The intended interaction between the 'Tag' (Red) and 'Catcher' (Pink) did not occur,
leading to a failure
in protein-protein docking. The entities are visualized as not aligning or binding in the anticipated
manner, reflecting the complexities and challenges inherent in designing protein-protein interactions.[4]
Color catcher (PET paper)
Color catcher (PET paper) is a common way for decolorization. We tried to improve our experiment by
combining color
catcher and our protein together, and we looked forward to see a better result after this step. Hence, at
the beginning of our experiment, we estimated the function of two well-known color catcher (PET paper)
brands, "Dalli" and
"Dr. Beckmann", by measuring their absorbance.
We used the spectrophotometer,made in our dry lab, to measure the absorbance. We used the textile dyes (red,
black,
blue and yellow), we recieved from Shin Kong textile factory and added them in ddH2O in case of other
unexpected
interference. Then, we set the spectrophotometer to 580nm. After that, we measured their absorbance once
per hour. In order to make the data precise, we prepared three tubes of dyed water. The data we showed in
the forms is the average of the three tubes.
The following is our result, the data inside the form is the transmittance of the dyed water and the same
one after we put color catcher inside. (Fig 5.)
After our experiment, we can point out the two colors catcher perform almost the same in red and yellow.
The brand "Dr. Beckmann" has a better effect in blue after 2 hours, and "Dalli" perform much better than
"Dr. Beckmann" in
black dye. As a result, we knew that both of them have their advantages, and we were looking forward to try
our protein to combine with these color catchers(PET papers) in the future.
Fig 5. Absorbance of different color catcher (PET paper):
Textile Dyes ( Acid dye: yellow, blue; Disperse dye: red; Azo dye: black )
