Praktikum Chemische Biotechnologie Versuch BPT1 und BPT2

WS 2016/2017 Chemische Biotechnologie BPT1/BPT2

1

Praktikum Chemische Biotechnologie

Versuch BPT1 und BPT2

Wintersemester 2016/2017

Betreuer: BPT

Labor: G1 3.19 / G1 1.OG


2

ContentEnrichment and characterization of 2‐hydroxybiphenyl 3‐monooxygenase (HbpA) .............................. 3

Introduction: ........................................................................................................................................ 3

BPT1: Enrichment of 2‐hydroxybiphenyl 3‐monooxygenase (HbpA) out of recombinant E. coli ........... 4

Cell disruption using French press: ..................................................................................................... 4

Ultracentrifugation: ............................................................................................................................. 4

Proteinenrichment using anion exchange chromatography: .............................................................. 5

Preparing a SDS‐polyacrylamide‐gel according to Lämmli: ................................................................. 6

BPT2: Characterization of enriched 2‐hydroxybiphenyl 3‐monooxygenase (HbpA) ............................... 8

Determination of protein concentration by Bradford method: .......................................................... 8

Determination of protein size and purity using SDS‐PAGE: ................................................................ 9

Measurements of HbpA activity by spectrophotometric assay: ....................................................... 10

Reporting: .............................................................................................................................................. 12

Questions: ............................................................................................................................................. 13

Bibliography: ......................................................................................................................................... 13


3

Enrichment and characterization of 2‐hydroxybiphenyl 3‐monooxygenase

(HbpA)

Introduction:

The enzyme used in this study is 2‐hydroxybiphenyl 3‐monooxygenase (HbpA) (EC 1.14.13.44). It is a

tetrameric enzyme (composed of four identical units) that catalyzes regioselective ortho‐

hydroxylation of 2‐substituted phenols (Figure 1). This enzyme exhibits a broad substrate specify [1,

2] and thus is an interesting biocatalyst for the production of 3‐substituted catechols that can be

later used for synthesis of blood pressure lowering agents and anti‐tumor drugs.

HbpA belongs to the group of FAD dependent Flavin monooxygenases [3, 4] and requires NADH,

molecular oxygen and the respective substrate for catalytic activity.

Figure 1: Schematic representation of HbpA catalyzed hydroxylation

E.coli can be used to produce the enzyme HbpA. Therefor E.coli JM101 harboring the plasmid

pHBP461 was grown in a bioreactor. After the batch phase using glucose as carbon source, the cells

were fed with glycerol. During the fed‐batch the gene expression was induced with IPTG. After

harvesting the cells were stored at ‐20 °C.

In this exercise you will first purify and then characterize this catalyst. First the cells have to be

disrupted and the cell debris have to be separated from the soluble fractions. For the purification

(enrichment) anion exchange chromatography is used and the characterization will consist of purity

assessment (SDS‐PAGE gel technique [5]) and activity measurements in a spectrophotometric

method. Last but not least you will measure the protein concentration of the samples and you will be

able to comment on the enrichment factor and the purity of the fractions.

WS 2016/2017 Chemische Biotechnologie BPT1

4

BPT1: Enrichment of 2‐hydroxybiphenyl 3‐monooxygenase (HbpA) out of

recombinant E. coli

Cell disruption using French press:

Materials:

Cell pellet E. coli JM 101 HbpA461 (~ 5 g)

100 mL buffer A (20 mM TrisHCl pH 7.5)

Crushed ice

3 x 50 mL falcon‐tubes

Method:

French press is used for cell disruption. The equipment is stored in the fridge and need to be

assembled.

1) Add 16 mL buffer A to cell pellet (~ 4 g)

2) Suspend the cells using vortex and/or pipette. There should be no pellet left at the bottom of

the falcon tube or in the solution.

3) The falcon tube should always be on ice.

4) Take a sample (1 mL) for BPT2.

5) Assemble the French press cell, fill in the cell suspension and assemble the French press.

6) The disruption will take place twice at 1000 psi. The cell suspension should come out of the

French press cell drop wise to ensure an efficient disruption. Keep the sample on ice as often

as possible. The high pressure and the shear force during French press result in heating up

the cell suspension.

At the end you will have the crude extract in hand. Take a sample (1 mL) for BPT2.

Ultracentrifugation:

Materials:

Crude extract

2 x centrifugation‐tubes

Pipette

Balance

Seralpure‐Water


5

Method:

The water insoluble fractions (membrane fractions, not disrupted cells) will be separated using

ultracentrifuge. These molecules would inhibit the column run and plug the column material

irreversible.

1) Transfer the crude extract in the centrifuge tube. The second tube should be filled with

water.

2) Use the accuracy balance to tare the centrifuge tubes to +/‐ 10 mg. Because of the high g

force it is necessary to load the centrifuge exactly to ensure any unbalance.

3) Take the rotor out of the cold room to the centrifuge.

4) Load the centrifuge crosswise.

5) Switch on the centrifuge and program according to the assistant. Parameter: 4°C, 30

Minutes, 20000 rpm.

6) Take out the centrifuge tubes after centrifugation and keep on ice.

7) Turn off centrifuge, keep lid open, transfer rotor back to cold room.

Now you will have the cell free extract in hand. Take a sample (1 mL) for BPT2. This can be used to

load the column.

Proteinenrichment using anion exchange chromatography:

Materials:

Cell free extract

DEAE – Sepharose column (XK 16) CV=22.12 mL

1 L buffer A (20 mM TrisHCl, pH 7.5)

1 L buffer B (buffer A + 1M NaCl)

1 L Seralpure water

Tubes for the fractions

60 mL syringe

Sterile filter (0,22 µM, Millipore)

Method:

In this practicum a preparative chromatography (Bio‐Rad) is used. The cell free extract has to be

filtered before loading onto the column to enrich the protein HbpA.

The eluents A and B are pumped through the column and detector into fraction tubes. The column is

filled with DEAE sepharose which is an anion exchange medium. The stationary phase is positively

charged and the protein is negatively charged at a pH of 7.5. HbpA has an isoelectric point of 6.3. The

protein binds to the stationary phase. During the method, the concentration of sodium chloride


6

increases and the negatively charged protein will be exchanged by the chloride ions. The protein will

elute. During the method absorbance at 280 nm and conductivity is measured.

1) Dilute the supernatant to 40 mL with Buffer A.


3) Filter through a sterile filter.


5) Load the diluted cell free extract on the column with using a pump.

6) Run the method.

7) Program (CV = column volume = 22, 12 mL):

a. Wash 1 (2 CV; 1 mL/min; 95% Buffer A; 5% Buffer B)

b. Gradient (2 CV; 1 mL/min; 5% to 30% Buffer B)

c. Wash 2 (1,5 CV; 1 mL/min; 30% Buffer B)

d. Cleaning (3 CV; 1 mL/min; 100% Buffer B)

e. Eqilibrating (2 CV; 1 ml/min; 95% Buffer A)

8) Take samples from the interesting yellow fractions. Why yellow?

Figure 1. protein fractions of the HbpA purification

Preparing a SDS‐polyacrylamide‐gel according to Lämmli:

Materials:

Clean SDS PAGE plates and accessories

2 beakers and magnetic stirrer

Isopropanol

Solutions according to scheme

Method:

ATTENTION: You are working with toxic substances. Take care of your protective clothing and wear

gloves. Do not discard solutions in sink.


7

1) Mount two plates.

2) Prepare fresh: 1 mL 10% (w/v) ammonium persulfate in water

3) Pipette the components of the separating gel (12 % acrylamide) in a small beaker with a

magnetic stirrer inside. Stir the solution.

H2O (mL) 6,6

Separating gel buffer (mL) 3,75

Acrylamidesol. (mL) 4,5

Starter:

APS‐Lsg. (µL) 75

TEMED (µL) 7,5

Endvolume (mL) 15

4) NOTE: Pour solution between the glass plates straight after adding the starter to avoid

polymerization in the beaker.

5) Add 2 mL isopropanol on top of the gel and let it polymerize for about 30 min.

6) Decant isopropanol.

7) Put clean combs out.

8) Pipette the components of the stacking gel (3.6 % acrylamide) in a small beaker with a

magnetic stirrer inside. Stir the solution.

H2O (mL) 3,2

Stacking gel buffer (mL) 1,25

Acrylamidesol. (mL) 0,45

Starter:

APS‐Lsg. (µL) 50

TEMED (µL) 5

Endvolume (mL) 5

9) NOTE: Pour solution between the glass plates straight after adding the starter to avoid

polymerization in the beaker.

10) Insert the comb.

11) Take of clamps after polymerization of the gel.

12) Put the gel in wet paper towel and plastic bag. Sign the bag and put it in the fridge.


8

BPT2: Characterization of enriched 2‐hydroxybiphenyl 3‐monooxygenase

(HbpA)

Determination of protein concentration by Bradford method:

Materials:

Samples cell extract (CE), ultracentrifugation (UC), filtration (FIL), subsequent fractions (F1,

F2,…)

BSA protein standard 2 mg/mL

Bradford Reagent for Macro assay for protein concentration from 0 to 40 µg/mL

Seralpure water

1.5 mL PMMA cuvettes

Method:

1) Prepare the samples for the standard curve first. Make the dilution of BSA protein standard

(2 mg/mL) according to the table below.

Concentration [mg/mL] Water [µL] Volume sample [µL] Taken from

2 200 2

1,5 50 150 2

1 100 100 2

0,75 100 100 1.5

0,5 100 100 1

0,25 100 100 0.5

0,125 100 100 0.25

0 200 0 water

2) Distribute 1000 µL of Bradford Reagent into separate disposable PMMA cuvettes. You will do

the standard curve in duplicates. Do not forget the blank!

3) Add 20 µL of prepared protein solution directly into the cuvette (mix the content thoroughly

by vortexing 5 s) – check if the samples are homogenous, if not mix again.

4) Incubate for 12 min at room temperature and measure absorbance of the sample at 595 nm.

5) To determine the protein concentration in the unknown samples collected on the first day,

first dilute the samples ______ and ______ times (keep the final volume at 100 µL)

6) Distribute 1000 µL of Bradford Reagent into separate disposable PMMA cuvettes.

7) Add 20 µL of the diluted protein sample directly into the cuvette (mix the content thoroughly

by vortexing 5 s) – make sure it is homogenous. Make each sample twice.


9

8) Incubate for 12 min at room temperature and measure absorbance of the sample at 595 nm.

If absorbance values measured are too high and extend the range covered by the standards make an

additional dilution of the sample and repeat steps 6 – 8.

9) For preparing the standard curve, calculate the average value of the blank. Subtract the blank

from measured absorbance values. Calculate the average values and measurement error as a

standard deviation. Generate the graph in Excel where X’s are your expected standard

protein concentration and Y’s are the measured absorption values.

10) Fit in the trend line and display the R² and equation on the graph. Use the equation to

calculate protein concentration in your samples.

Determination of protein size and purity using SDS‐PAGE:

Materials:

SDS‐PAGE chamber

Precast gels

1x running buffer

Samples from cell lysis and chromatography

2x concentrated SDS sample loading buffer

Protein Size Marker – Page Ruler Unstained Protein Ladder (Fermentas #26614)

Coomassie Brilliant Blue staining solution

Destaining solution (acetic acid and methanol in water)

Method:

1) Assemble the SDS‐PAGE chamber

2) Dilute the protein samples to 0.8 mg/mL if possible. Keep the volume of the sample at least

50 µL. If concentration of the protein is lower than that use it as it is.

3) Mix 50 µL of the sample with 50 µL of loading buffer. You should do it under the hood

because the sample loading buffer contains DTT that you should not inhale.

4) Mix by vortexing and spin down for 10 s on a bench‐top centrifuge.

5) Incubate in the thermoshaker for 7 min at 95°C with 300 rpm mixing – this step will denature

the proteins and led the dye bind to the peptide back bone of the protein. Therefore we say

that we run the gel with denaturing conditions.

6) Centrifuge the samples down for 2 min at 13200 rpm on the bench‐top centrifuge.

7) Load 10 µL of the sample’s supernatant into separate well. Keep the record of the loading

order of your samples! The gel should be loaded asymmetrically to let you distinguish the

samples’ order after staining and destaining.


10

8) Load 5 µL of the protein marker into first and the last well after your sample. Marker lets you

tell the size of proteins in a band.

9) Run the gel at 80 V through stacking gel and increase to 140 V in resolving gel.

10) You will know that the run is finished when the blue dye starts eluting from the gel into the

buffer chamber at the bottom.

11) Remove the gel carefully, wash it with VE water and stain it for 5 min with a fresh Coomassie

Brilliant Blue staining solution.

12) Transfer the staining solution back into the bottle. Wash the gel with VE water once and put

it into the destaining solution for at least 1 h. Roll the paper towel and put it into the

destaining solution next to the gel. It will accelerate destaining process by adsorbing the

staining dye.

13) When clear protein bands are visible you can stop the process. Discard destaining solution

and flush the gel with water. If you still have time you may take the picture of the gel,

otherwise we will send the picture after the practical course.

Measurements of HbpA activity by spectrophotometric assay:

Materials:

Samples from cell lysis and chromatography

0.5 mL 25 mM NADH solution in water

0.5 mL 2 mM FAD in water

0.5 mL 25 mM 2‐hydroxybiphenyl in methanol

50 mL 50 mM potassium phosphate buffer (KPi, pH 7.5)

Method:

1) Switch the spectrophotometer ON. Load the method file and switch the thermostat ON.

2) Since NADH is consumed during the HbpA catalyzed reaction, activity can be measured by

following the decrease of the absorbance at 340 nm (± 4 nm).

3) Dispense 950 µL of 50 mM potassium phosphate buffer (KPi) to the plastic PMMA cuvette

4) Add 12 µL of 25 mM NADH (final concentration of NADH is 0.3 mM)

5) Add 10 µL of 20 mM FAD (final concentration of FAD 20 µM)

6) Add 20 µL of enzyme solution (taken from one of your samples either CE, UC, FIl or F1…)

7) Mix solution well with a P200 pipette by pipetting up and down severel times.

8) Incubate sample for at least one minute before starting the measurement for the sample to

reach 30°C.


11

9) Start the measurement and follow background activity for 1.5 min (the slope will be

calculated automatically)

10) Add 8 µL of 25 mM 2‐hydroxybiphenyl solution (final concentration 0.2 mM).

11) Quickly start the measurement and follow substrate specific activity for 1.5 min (the slope

will again be displayed automatically)

12) Subtract background activity (slope from time point 0.5 till 1.5 min) from substrate specific

activity (slope from time point 0 till 1 min) to calculate ∆A per minute.

13) Measure the samples in duplicate.

If ∆A per minute exceeds 0.25 dilute the enzyme to reach appropriate slope. Enzyme volume used for

the reaction can also be reduced to 10 µL if only 2 times dilution is needed.

14) Calculate enzyme’s volumetric and specific activity by applying Beer‐Lambert Law and results

from Bradford assay. Molar absorption coefficient for NADH is 6220 M‐1cm‐1.

∆6.22

∗1000

∗ μ

∗ . 1

. 2

15) Fill in the purification table. Calculate the purification factor by dividing the specific activity of

the sample from the Anion Exchange Chromatography (AEX) by specific activity of the cell

extract (CE) and total amount of the activity in the pooled fractions by multiplying fractions

volume by volumetric activity.

Nr Purification

Step

Protein

Concentration

[mg/mL]

Volumetric

activity

[U/mL]

Specific

activity

[U/mgprot]

Purification

factor

Total Activity

[U]

1 CE

2 UC


12

Reporting:Report should be prepared according the scheme IMRaD, meaning Introduction, Methods, Results

and Discussion. Keep the writing short and write only the essential information e.g. do not write how

the chromatography or SDS‐PAGE works, give a relevant reference only. The same applies to every

technique used – a reference is enough. Introduction part should therefore be kept short and consist

of 2 – 3 sentences summarizing aim of the experiments. This IMRaD structure is the most commonly

used layout in scientific writing. Reports should be submitted via email within one week after the

second exercise is finished. You only have to prepare one report from those two exercises.

Let me just shortly comment on figures and their captions. Figure is a stand‐alone entity, meaning if

one sees it and reads the caption it is clear what each line or a bar represents, therefore be specific

and precise. Do not let the reader ‘deduce or guess’– tell the reader directly what every line means!

Below is an example of a well presented SDS PAGE figure (Figure 3).

Figure 3. Typical SDS‐PAGE gel of proteins (10μg). Protein Page Ruler (SM#0661,

Fermentas) (lanes M), E coli JM101 pBTac2 C23S crude cell extract (lane 1),

supernatant after centrifugation (lane 2), flow through after sample loading onto the

DEAE Streamline 50 column (lane 3), concentrated sample of most active fractions

after AEX and filtration through 30kDa cut‐off filter paper (lane 4), concentrated

sample of most active fractions of HbpA originating from E coli JM101 pHBP461 after

AEX and filtration through 30kDa cut‐off filter paper (lane 5)

Caption contains name of the technique used, origin of sample (strain and preparation method) and

amount of the protein loaded on the gel. Moreover two of the lanes show the molecular marker and

respective lines show relative protein sizes. Caption contains some abbreviations e.g. AEX (Anion

Exchange Chromatography) or DEAE which were previously used in the materials and method section

of the paper so there is no need to repeat them here. Please try to present your figures as clearly as


13

possible. If you want your report to be accepted as it is write it clear so that someone who did not do

this practical course know what was done!

Questions:1) What did you learn?

2) What do you not completely understand?

3) How have your ideas changed as a result of this lab?

4) What new questions do you have?

5) How would you improve what you did?

6)

Bibliography:1) Held, M., et al., Preparative scale production of 3‐substituted catechols using a novel monooxygenase

from Pseudomonas azelaica HBP 1. Journal of Molecular Catalysis B: Enzymatic, 1998. 5(1‐4): p. 87‐93.

2) Kohler, H.P., D. Kohler‐Staub, and D.D. Focht, Degradation of 2‐hydroxybiphenyl and 2,2'‐

dihydroxybiphenyl by Pseudomonas sp. strain HBP1. Applied and environmental microbiology, 1988.

54(11): p. 2683‐2688.

3) Suske, W.A., et al., Purification and characterization of 2‐hydroxybiphenyl 3‐monooxygenase, a novel

NADH‐dependent, FAD‐containing aromatic hydroxylase from Pseudomonas azelaica HBP1. J Biol

Chem, 1997. 272(39): p. 24257‐65.

4) van Berkel, W.J.H., N.M. Kamerbeek, and M.W. Fraaije, Flavoprotein monooxygenases, a diverse class

of oxidative biocatalysts. Journal of Biotechnology, 2006. 124(4): p. 670‐689.

5) Sambrook, J. and D.W. Russell, Molecular Cloning: A Laboratory Manual. 2001: Cold Spring Harbor

Laboratory Press.

Documents

Praktikum Chemische Biotechnologie Versuch BPT1 und BPT2