Uppsala iGEM 2019

Proof of concept Protocols

Enzyme assays 2
Protocol #1: ​FOX-Assay 2
Protocol #2: ​ABTS-assay 2
Protocol #3: Manganese Peroxidase 3
Protocol #4: Enzymatic assay for Manganese Peroxidase 4

Lignin assays 4
Protocol #5: Fractionation of Lignin 4
Protocol #6: Native-PAGE 5
Protocol #7: SDS-PAGE 6
Protocol #8: Fractionating lignin in high pH 7
Protocol #9: Degrading lignin with HRP extracted from horse-radish 7
Protocol #10: Sonicating lignin 8
Protocol #11: Degrading lignin with enzyme and Tris-buffer 8
Protocol #12: Degrading lignin with HRP with continuous addition of hydrogen peroxide 9
Protocol #13: Degrading lignin with HRP 9
Protocol #14: Lysation of yeast pellet with enzymes 10
Enzyme assays

1. FOX-Assay
Purpose:
The oxidase that was used in the lab, was able to produce hydrogen peroxide. In
order to measure the amount of the enzyme, an assay was performed to detect the
amount of hydrogen peroxide that the enzyme produced. The assay is modified from
Thermo Fisher. The presence of H​2​O​2​ allowed for detection of whether the desired
oxidase was present. In this assay, hydrogen peroxide oxidizes ammonium ferrous
(​Fe​2+​) ions into ammonium ferric (​Fe​3+​) ions. The ammonium ferrous ions were in a
H​2​SO​4 solution.
​ This would be tested using a solution of sorbitol and xylenol orange.
Method:
Working reagent (WR) was made of:
Reagent A (25mN ammonium ferrous(II) sulfate and 2.5 M H​2​SO​4​)
Reagent B (100mM sorbitol, 125 μM xylenol orange in water)
The WR was made of 1 volume Reagent A and of 100 volume Reagent B (1:100). 1
mL of WR was prepared for each sample. The samples were prepared in cuvettes by
adding 10 volumes of WR to 1 volume of sample, for example the ratio 1 mL of WR
was added to 100 μL of sample. The sample that was used was undiluted
supernatant from ​P. Pastoris​. The cuvettes with the samples and WR was incubated
in room temperature for 20 min and then measured in a spectrophotometry at 560
nm.

2. ABTS-assay
Purpose:
The peroxidase horseradish peroxidase is able to break down parts of the lignin, so
a substrate was needed that had phenolic rings and could resemble lignin. The
substrate used was 2,2'-Azino-bis(3-Ethylbenzthiazoline-6-Sulfonic Acid) (ABTS).
The method is a continuous spectrophotometric assay. The assay is modified from
Sigma.
Method:
Reagent 1: Prepare a 100 mM Potassium Phosphate buffer, pH 5.0, by making a
13.6 mg/mL solution in purified water using Potassium Phosphate, Monobasic.
Adjust the pH of this solution to pH 5.0 using 1M KOH.
Reagent 2: (substrate) Prepare 9.1 mM ABTS by making a 5.0 mg/mL solution in
Reagent 1 using ABTS. Check the pH of this solution and adjust to pH 5.0 as
necessary. Prepare Fresh.
Reagent 3: Prepare a 0.3% (w/w) Hydrogen Peroxide Solution (H​2​O​2​) from a stock
solution of hydrogen peroxide. Prepare the solution in purified water using Hydrogen
Peroxide 30% (w/w) Solution. Prepare Fresh.
Reagent 4: (diluent) Prepare a 40 mM Potassium Phosphate Buffer with 0.25% (w/v)
Bovine Serum Albumin and 0.5% (v/v) Triton X-100, pH 6.8.
The sample was undiluted for the ​P. Pastoris​ produced enzymes and diluted 1:200
for the bought HRP.

Tabel 1: Reagent and setup for the ABTS assay

Reagent Test Blank

Reagent 2 (substrate) 2.9 mL 2.9 mL

Reagent 3 (H​2​O​2​) 0.1 mL 0.1 mL

Reagent 4 (diluent) - 0.05 mL

Enzyme sample 0.05 mL -

Put the reagent 2 into the cuvette and let the substrate incubate in room temperature
for 5 min. Then add reagent 3 and reagent 4 or the enzyme, depending on what
sample is measured. Make the measurement of the sample in the cuvette, in a
spectrophotometry for 3 minutes at 405 nm.

Calculate the units/mg solid with:

ΔA405 nm /min(test)−ΔA405nm /min(blank)*3.05*DF
36.8*0.05
DF=Dilution factor of enzyme
3.05= Final volume(in milliliters) of reaction
36.8=millimolar extinction coefficient of oxidized ABTS at A405 nm
0.05= volume (in milliliters) of enzyme used

3. Manganese Peroxidase
Purpose:
The manganese peroxidase was needed to be tested in order to see whether it was
active.
Method:
A reaction mixture of 25mM lactate, 0.1 mM MnSO​4​, 1 mg BSA, 1 mg phenol red and
0.2 mL of culture filtrate from the yeast in 20 mM sodium acetate buffer(pH 4.5) into
a total volume of 2 mL. The reaction was started in the cuvette with addition of 0.1 M
H​2​O​2​ and stopped after one minute. The assay was measured at 610 nm, a blank
was also performed at the same wavelength but without enzyme mixture.
 4. Enzymatic assay for Manganese Peroxidase
Purpose:
The assay is designed to measure the oxidation of ABTS in order o get the laccase
and MnP activity.
Method:
The assay is performed with a spectrophotometry at 420 nm. A 0.5 mM ABTS
solution in 50 mM sodium acetate (pH 5.0). The MnP was mixed with 0.5 mM ABTS,
0.05 mM H​2​O​2​, 0.16 mM MnCl​2​, in 40 mM sodium citrate buffer (pH 4.5), the enzyme
sample is 100 μL in a 1 mL final volume. The cuvette was filled with the samples and
run in a spectrophotometry at 420 nm.

Lignin assays

5. Fractionation of Lignin

Purpose:
In order to use organic solvents to degrade or “melt” the lignin and because of the lignins
complex structure, a fractionation is needed. An organic media is used to make the lignin
groups that are similar in properties, stick together and therefore become a new fraction.
Kraft Lignin is the shape of the lignin as it is when it reaches the end of line at a paper-mill,
for example. Fractionation was in addition carried out in preparation for detection of
degradation using size exclusion chromatography, which was not used in the end.

Method:
Methanol sample: 2 grams of Kraft Lignin was mixed with 20 mL methanol. The solution was
stirred for 2 hours, at 500 rpm, and then centrifuged for 5 minutes in 4000 rpm. The
supernatant was saved for the next step.
The exact same setup was prepared but was run through a level 1 filter-paper. The
supernatant was saved for the next step.

Acetone fraction: The pellet from the earlier centrifuge step was mixed with 20 mL of
acetone and then stirred for 2 hour, at 500 rpm, and then centrifuged for 10 min at 4000 rpm.
Supernatant and pellet was saved separately.
The pellet from the filtered fraction was mixed with 20 mL of acetone and then stirred for 2
hours, at 500 rpm. The mix was then filtered through a level 3 filter-paper. The pellet and
supernatant was saved separately.

6. Native-PAGE
Purpose:
For the detection whether the lignin had been degraded or not several different methods
were used. The Native-PAGE was a first step in figuring out how to detect the lignin.
Because the lignin doesn’t resemble proteins, a gel without SDS was considered a good
start.
Method:
Sample buffer(2X)
49μL 1.5 M tris HCl, pH 8.8
50μL 25% glycerol
1μL 1% bromophenol Blue
All was mixed into eppendorf tube.
When the buffer was used, it was diluted 1:1 with ddH​2​O

Running buffer(10X):
275 mL ddH​2​O
125 mL Tris HCl pH8.8
100 mL glycerol (1.92M)
All was mixed a diluted to 1X when need to run the gel.
When the buffer was used, it was diluted 1:10 with ddH​2​O

The Native-PAGE
Stacking gel:
4.275 mL 0.375 M Tris-HCl pH 8.8
0.67 mL Acrylamide/Bis-acrylamide(30%)
0.05 mL 10% ammonium persulfate
5 μL TEMED

Separating gel 10%

3.4 mL Acrylamide/Bis-acrylamide(30%)
6.49 mL 0.375 Tris-HCl ph8.8
100 μL 10% ammonium persulfate
10 μL TEMED

First, the separating gel was added to the glass plates held by the casting frame. 200 μL
Isopropanol was on top of the gel in order to make the line straight. When the gel had
polymerized the stacking gel was added on top and the comb was added to the gel. The
glass was added into the chamber for the gel to run in and the samples was added. 1X
running buffer was added to the chamber before it was closed. A voltage of 80V was the
initial voltage for the stacking gel and when the samples had reached the separating gel the
volt was increased to 120 V. The samples ran until they reached the end of the glass.

7. SDS-PAGE
Purpose:
For the detection whether the lignin had been degraded or not several different methods
were used. The SDS-PAGE was the second step in the gel usage to find out whether the
lignin had been degraded or not. Several SDS-PAGE:s were done.
Method:
Separation gel:
Table 2: Separation gel with specifik percent
20 % 18 % 15 % 10 %

ddH​2​O 0.59 mL 1.334 mL 2.29 mL 4.1 mL

acrylamide/bis (30%) 6.7 mL 6.066 mL 5 mL 3.3 mL

Tris-HCl (1.5 M, pH 8.8) 2.5 mL 2.5 mL 2.5 mL 2.5 mL

SDS (10%) 100 μL 100 μL 100 μL 100 μL

TEMED 10 μL 10 μL 10 μL 10 μL

APS 100 μL 100 μL 100 μL 100 μL

The stacking gel that was used for all of the gels were a 4 % gel.

Stacking gel for 4%:

6.1 mL ddH​2​O
1.3 mL Acrylamide/bis (30%)
2.5 mL Tris-HCl (0.5M pH6.8)
100 μL SDS (10%)
100 μL APS (10%)
10 μL TEMED

First the separating gel was added to the glass plates held by the casting frame. 200 μL
Isopropanol was on top of the gel in order to make the line straight. When the gel had
polymerized the stacking gel was added on top and the comb was added to the gel. The
glass was added into the chamber for the gel to run in and the samples was added. 1X
running buffer was added to the chamber before it was closed. A voltage of 80V was the
initial voltage for the stacking gel and when the samples had reached the separating gel the
volt was increased to 120 V. The samples ran till they reached the end of the glass.
 8. Fractionating lignin in high pH
Purpose:
According to some articles, lignin is able to be degraded when exposed to a high pH.

Method:
A 0.5 M NaOH was prepared with 5.99 grams of solid NaOH in 300 mL ddH​2​O.
2 grams of Kraft Lignin was mixed with 0.5M NaOH and stirred overnight.
Some of the pellet from the acetone fraction was also mixed with 0.5M NaOH.

9. Degrading lignin with HRP extracted from horse-radish

Purpose:
In order to see whether we could degrade lignin with our enzyme, a number of experiments
were setup with different compounds. This experiment had a phosphate buffer to match the
buffer that the HRP extracted from the horse-radish from the local supermarket (ICA). The
samples were tested with a nanodrop and Platereader Tecan.
Method:
2 grams of sulfonated lignin was mixed with 20 mL of ddH​2​O.
The following setup was made.

Tabell 3: Sample setup for the degradation of lignin with HRP in phosphate buffer.
Sample Lignin H​2​O Phosphate H​2​O​2 Heat-inacti ICA HRP
buffer vated
enzyme

Sample 1 100 μL 9.9 mL - - - -

Sample 2 100 μL - 9.9 mL - - -

Sample 3 100 μL - 9.74 mL - 0.16 mL -

Sample 4 100 μL - 9.74 mL - - 0.16 mL

Sample 5 100 μL - 9.41 mL 0.33 mL - 0.16 mL

Sample 6 100 μL - 9.41 mL 0.33 mL 0.16 mL -

Sample 7 100 μL - 9.57 mL 0.33 mL - -

10. Sonicating lignin

Purpose:
 We wanted to see if a change in the lignin could be achieved by using sonication as a
method to change the lignin structure. Both the Kraft Lignin and the Sulfonated lignin was
sonicated.
Method:
1 mL of lignin solution was mixed with 4 mL the correct buffer depending on the original
solution. The fractionation with acetone had acetone and the fractionation with methanol had
methanol and the solutions with water was mixed with water. When the lignin was being
sonicated the amplitude was set to 50% and the pulse had a 5 second run and a 5 second
pause that it alternated between. The lignin was sonicated for 30 min.

11. Degrading lignin with enzyme and Tris-buffer

Purpose:
In order to see whether we could degrade lignin with our enzyme, a number of experiments
were setup with different compounds. This experiment had a Tris-HCl buffer to match the
buffer that the HRP extracted from the horse-radish from the local supermarket (ICA). The
samples was tested with nanodrop and Platereader Tecan.
Method:
2 grams of sulfonated lignin was added into 20 mL of ddH​2​O. Then the following setup was
made:

Tabel 4: Sample setup for degradation of lignin with HRP in Tris-HCl buffer
Sample Tris-HCl ICA HRP Sigma Heat-inactivated Heat-inactivated H​2​O​2 Lignin
HRP ICA Sigma

Sample 1 9.74 mL - 160 μL - - - 100 μL

Sample 2 9.74 mL 160 μL - - - - 100 μL

Sample 3 9.41 mL - 160 μL - - 330 100 μL

μL

Sample 4 9.41 mL 160 μL - - - 330 100 μL

μL

Sample 5 9.41 mL - - - 160 μL 330 100 μL

μL

Sample 6 9.41 mL - - 160 μL - 330 100 μL

μL

Sample 7 9.9 mL - - - - - 100 μL

 12. Degrading lignin with HRP with continuous addition of
hydrogen peroxide
Purpose:
According to an article, continuous addition of hydrogen peroxide to the solution with
enzyme could help the degradation of lignin. So this was done during a certain
period of time. The reaction were detected in a full spectrum nanodrop analysis.
Method:
1 gram of sulfonated lignin was added to 10 mL of ddH​2​O and stirred. HRP was
added to a concentration of 0.03 mg/mL and hydrogen peroxide with a concentration
of 90 ​μM was added every 120 minutes. The samples were stirred for 4 hours. To try the
samples in the nanodrop, 1.5 μL of the sample from different timepoints was added to the
nanodrop and measured in a full spectrum analysis.

13. Degrading lignin with HRP

Purpose:
To try different samples and to see whether the data could be affected by specific
components in the sample.
Method:
The following setup was done in order to test the different components in the sample.
Sample 2 would be the one with all the components in it.

Tabell 5: Sample setup of degradation of lignin with HRP with continuous addition of H​2​O​2
Sample Kraft Lignin HRP H​2​O​2 ddH​2​O

Sample 1 0.02 g - 33 μL 9.67 mL

Sample 2 0.02 g 250 μL 33 μL 9.717 mL

Sample 3 0.02 g - - 10 mL

Sample 4 0.02 g 250 μL - 9.75 mL

14. Lysation of yeast pellet with enzymes

Purpose:
One of the enzymes expressed by ​Pichia Pastoris​ was supposed to be secreted. When we
didn’t see any enzyme activity in the supernatant we suspected that the enzymes where still
in the yeast cells, therefore we wanted to lyse the cells.
Method:
The pellet from the Pichia Pastoris samples were sonicated with an amplitude of 50% and a
pulse of 5 seconds and paus of 5 seconds. The cells were sonicated for 30 minutes. The
sonicated cells were vortexed with glass-beads and then centrifuged down for 10 minutes in
4000 rpm. The supernatant was then tested in the FOX-assay(see protocol for FOX-assay).