University of Groningen HMF oxidase Dijkman, …129 Curriculum vitae Willem Pieter Dijkman was born on the seventh of March 1988, in Bergum. He grew up in Noardburgum and moved to

University of Groningen

HMF oxidaseDijkman, Willem Pieter

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AAppendices

129

Curriculum vitae

Willem Pieter Dijkman was born on the seventh of March 1988, in Bergum. He grew up in Noardburgum and moved to Groningen in 2007, during the first year of his studies Biology at the University of Groningen. After obtaining the bachelor degree in Molecular Life Sciences, he continued with the master program Molecular Biology and Biotechnology in 2009. In addition to writing a thesis in the Membrane Enzymology group of prof. Bert Poolman, he conducted two research projects during his master. The first project was performed in the Microbial Physiology group of prof. Lubbert Dijkhuizen at the University of Groningen. A second research project was conducted in the group of dr. Florian Hollfelder at the University of Cambridge (United Kingdom). Back in Groningen, Willem started as a PhD student in the Molecular Enzymology group of prof. Marco Fraaije in November 2011. This research was part of a BE-Basic project focusing on an integrated process for the biocatalytic production of the bioplastic precursor 2,5-furandicarboxylic acid. The results of the enzymatic oxidation of a key intermediate to 2,5-furandicarboxylic acid are described in this thesis.

List of publications

Dijkman W.P., Binda C., Fraaije M.W., Mattevi A., Structure-based enzyme tailoring of 5-hydroxymethylfurfural oxidase. ACS Catalysis, 2015, 5, p. 1833–1839.

Ewing T.A., Dijkman W.P., Vervoort J.M., Fraaije M.W., van Berkel W.J.H., The Oxidation of thiols by flavoprotein oxidases: a biocatalytic route to reactive thiocarbonyls. Angewandte Chemie International Edition in English, 2014, 53, p. 13206–13209.

Dijkman W.P., Groothuis D.E., Fraaije M.W., Enzyme-catalyzed oxidation of 5-hydroxymethylfurfural to furan-2,5-dicarboxylic Acid. Angewandte Chemie International Edition in English, 2014, 53, p.6515–6518.

Dijkman W.P., Fraaije M.W., Discovery and characterisation of a 5-(hydroxymethyl)furfural oxidase from Methylovorus sp. Strain MP688. Applied Environmental Microbiology, 2013, 80, p. 1082–1090.

Dijkman W.P., Gonzalo G., Mattevi A., Fraaije M.W., Flavoprotein oxidases: classification and applications. Applied Microbiology and Biotechnology, 2013, 97, p. 5177–5188.

Gatti-Lafranconi P., Dijkman W.P., Devenish S.R., Hollfelder F., A single mutation in the core domain of the lac repressor reduces leakiness. Microbial Cell Factories, 2013, 12(67).

Leemhuis H., Dijkman W.P., Dobruchowska J.M., Pijning T., 4,6-α-Glucanotransferase activity occurs more widespread in Lactobacillus strains and constitutes a separate GH70 subfamily. Applied Microbiology and Biotechnology, 2013, 97, p. 181–193.

Appendices

130 characterization, application and engineering of 5-(hydroxymethyl)furfural oxidase

Acknowledgements

After several chapters of scientific content, it is time to write down a short personal note. And although the English language might give the following chapter a bit of an impersonal feel for some, I am sincerely grateful for all the support and advice I received during my research. This chapter is therefore dedicated to everyone who was directly or indirectly involved in this thesis.

First and foremost I would like to thank Marco. In the past four years I did not only learn about HMFO, but more importantly also about how to perform research. Your critical view and knowledge were essential for this process. Your interest in the project and the very rapid and constructive corrections on the reporting (even outside working hours), were definitely fundamental to the quick finishing of my PhD research.

The different collaborations which came with the project is something that I did not, and likely could not have, foreseen back in November 2011. Very pleasant was the work done on the structure of HMFO, together with Andrea, Claudia and Stefano. I am very happy with the resulting paper. In addition I would like the thank the past and present members of the Mattevi group for hospitality during the visits to Italy.

Less pleasant was the work on thiol oxidation, in collaboration with Tom and Willem (van Berkel). Nevertheless I would like to thank you both for finishing this with a good paper! How two labs can discover the same thing at the same time is still puzzling me, especially since this finding could have been done 30 years earlier. On this topic I would also like to thank Patrícia, for the numerous organic chemistry approaches you taught me, in search for the product of the reaction.

Being a PhD-student does not only involve research and learning, but also teaching. This teaching was in many cases closely intertwined with my research. Therefore I would like to thank all those students who contributed to my research during several practical courses.

From all students, especially the students who performed their master or bachelor project were of great value. Not only were many experiments performed by students, supervision also made me think about what I was doing, and sometimes about who I am.

From these students I supervised I’d first like to thank Piotr. You came when I just started and at that time the research was heading in no clear direction. All the experiments you performed clearly showed where to go with the project.

Daphne, thanks for the great work, just have a look at chapter 3 and 5. If we would have skipped all the small talk and performed more work, your research might have resulted in a second publication, but I am glad we didn’t. It was great having you around.

Remke, although ten weeks is way too short for research, you managed to work on two projects. The mutants made were less promising than I hoped, but inspired a whole new set of experiments.

Lisa, from all students you had the hardest job. And even though your enzyme would not have fitted in this thesis, it was, and still is an intriguing oxidase. In addition, thanks a lot for making the best mutant ever (chapter 4)! I hope you learned a lot and I am sure you will remember the time in Groningen.

This whole thesis is based on HMFO, and the reaction it catalyzes. The industrial relevance of this topic has been an important reason for me to embark on the project. Therefore I would also like thank the BE-Basic partners involved, Harald and Marc from Bird Engineering and the groups from DSM and the universities of Wageningen and Delft. I enjoyed

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the project meetings in the beginning of the project. I did not foresee these meetings would become so scarce later on, but this is probably due to my naivety.

Even though the research was tough in the beginning of the project, many people were of great help. Therefore I would like to thank the members of lab 109: Hanna, Maarten, Edwin and especially Remko, for starting up the HMF oxidase project and being a unofficial supervisor in the first months of the project. But also later on I received help. I am especially grateful to Gosia and Elvira for their assistance with the mechanistical studies.

I would have been nowhere without the tricks I learned during my research projects as a student. For this, I would like to thank Anette, Hans and Pietro. And for all the tricks I never learned: Philip, you did a great job with the layout!

The many small steps and breakthroughs on the lab were not the only thing which made the past years so enjoyable. I would like to thank all past and present members of the two groups for the amazing time I had: I am looking back at yet another best time of my life. Coffee breaks, conferences, drinks and dinners would not have been the same without you. One highlight was being paranymph, Hugo, thanks for choosing me.

And then there is the King’s Office. Thanks to Alex, Geoffrey and Hemant the long days of writing and data analysis were very pleasant, with a good balance between work and distraction.

Dear Marjolein, thanks for being there all along. You made it easy to forget the lab and the days without results. The five minutes bike ride to you was a pleasant and quick transition to the other, real, world. The world without research. I hope we can combine those two in our future.

Willem

Appendices


Supporting data

Table S1. Names and structures of commonly used compounds.

compound name number structure CAS numbercommonly used

abbriviation

[5-(hydroxymethyl)furan-2-yl]methanol 5 1883-75-6 HMF-OH

5-(hydroxymethyl)furfural 1 67-47-0 HMF

furan-2,5-dicarbaldehyde 2 823-82-5 DFF

5-(hydroxymethyl)furan-2-carboxylic acid

6 6338-41-6 HMF-Ac

5-formylfuran-2-carboxylic acid 3 13529-17-4 FFCA or FFA

2,5-furandicarboxylic acid 4 3238-40-2 FDCA

[4-(hydroxymethyl)phenyl]methanol 7 589-29-7 -

4-(hydroxymethyl)benzaldehyde 8 52010-97-6 -

terephthalaldehyde 9 623-27-8 -

4-(hydroxymethyl)benzoic acid 10 3006-96-0 -

4-formylbenzoic acid 11 619-66-9 -

terephthalic acid 12 100-21-0 -

OO OH

OO O

OO OH

O

OO O

O

O

O

O

O

O

OHO OH

OHO

OO

O

O

O

O

O

O

OHHO

OHO

OO

133

Table S2. Percentage of products formed during the oxidation of 2 mm [5-(hydroxymethyl)furan-2-yl]methanol (5) by 1 µm HMFO in time at 25 °C, pH 7.0. Average values of two experiments.

reaction time (hr)

precentage of 5-(hydroxymethyl)furfural (1) derivatives present

5 1 2 6 3 4

0.25 n.d.[a] n.d. 80 0.05[b] 20 0.8

0.50 n.d. n.d. 55 0.05 44 0.9

1.0 n.d. n.d. 26 n.d 73 1.1

4.0 n.d. n.d. n.d. n.d. 98 1.9

[a] n.d. is not detected [b] the substrate preparation ([5-(hydroxymethyl)furan-2-yl]methanol (5)) contains 0.01 % 5-(hydroxymethyl)furan-2-carboxylic acid (6) as impurity.

Table S3. Percentage of products formed during the oxidation of 2 mm 5-(hydroxymethyl)furfural (1) by 1 µm HMFO in time at 25 °C, pH 7.0. Average values of two experiments.

reaction time (hr)


5 1 2 6 3 4

0.25 n.d.[a] n.d. 65 0.1[b] 35 0.5

0.50 n.d. n.d. 39 0.09 60 0.6

1.0 n.d. n.d. 12 0.08 87 0.6

4.0 n.d. n.d. n.d. n.d. 98 1.8

[a] n.d. is not detected [b] the substrate preparation (5-(hydroxymethyl)furfural (1)) contains 0.5 % 5-(hydroxymethyl)furan-2-carboxylic acid (6) as impurity.

Table S4. Percentage of products formed during the oxidation of 2 mm furan-2,5-dicarbaldehyde (2) by 1 µm HMFO in time at 25 °C, pH 7.0. Average values of two experiments.

reaction time (hr)


5 1 2 6 3 4

0.25 n.d.[a] n.d. 55 0.05[b] 45 0.2

0.50 n.d. n.d. 32 n.d. 68 0.3

1.0 n.d. n.d. 6.3 n.d. 93 0.3

4.0 n.d. n.d. n.d. n.d. 98 1.7

[a] n.d. is not detected [b] the substrate preparation (furan-2,5-dicarbaldehyde (2)) contains 0.04 % 5-(hydroxymethyl)furan-2-carboxylic acid (6) and 0.5% 5-formylfuran-2-carboxylic acid (3) as impurity.

Appendices


Table S5. Percentage of products formed during the oxidation of 2 mm 5-(hydroxymethyl)furan-2-carboxylic acid (6) by 1 µm HMFO in time at 25 °C, pH 7.0. Average values of two experiments. In control samples with only substrate, the only compound detected was the starting material.

reaction time (hr)


5 1 2 6 3 4

0.25 n.d.[a] n.d. n.d. 40 32 0.6

0.50 n.d. n.d. n.d. 32. 40 0.6

1.0 n.d. n.d. n.d. 24 48 0.7

4.0 n.d. n.d. n.d. 17 54 0.7

[a] n.d. is not detected [b] 5-(hydroxymethyl)furan-2-carboxylic acid (6) was found highly esterified when purchased. After de-esterification (Wierckx et al., Microbial Biotechnology 2010 3(3) p336-430), 0.5 mm of the esterified 5-(hydroxymethyl)furan-2-carboxylic acid (6) remained present in the 2 mm substrate solution, this 25% is not converted.

Table S6. Percentage of products formed during the oxidation of 2 mm 5-formylfuran-2-carboxylic acid (3) by 1 µm HMFO in time at 25 °C, pH 7.0. Average values of two experiments. In control samples with only substrate, the only compound detected was the starting material.

reaction time (hr)


5 1 2 6 3 4

0.25 n.d.[a] n.d. n.d. n.d. 99 0.9

0.50 n.d. n.d. n.d. n.d. 99 1.0

1.0 n.d. n.d. n.d. n.d. 99 1.3

4.0 n.d. n.d. n.d. n.d. 97 2.9

[a] n.d. is not detected

Table S7. Percentage of products formed during the oxidation of 2 mm [4-(hydroxymethyl)phenyl]-methanol (7) by 1 µm HMFO in time at 25 °C, pH 8.0. Average values of two experiments. In control samples with only substrate, the only compound detected was the starting material.

reaction time (hr)

precentage of [4-(hydroxymethyl)phenyl]methanol (7) derivatives present

7 8 9 10 11 12

0.25 82 15 2.0 n.d.[a] n.d. n.d.

0.50 70 20 10 n.d. n.d. n.d.

1.0 50 31 21 n.d. n.d. n.d.

4.0 23 24 53 n.d. n.d. n.d.


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Table S8. Percentage of products formed during the oxidation of 2 mm 4-(hydroxymethyl)benzaldehyde (8) by 1 µm HMFO in time at 25 °C, pH 8.0. Average values of two experiments. In control samples with only substrate, the only compound detected was the starting material.

reaction time (hr)


7 8 9 10 11 12

0.25 n.d.[a] 72 28 n.d. n.d. n.d.

0.50 n.d. 50 50 n.d. n.d. n.d.

1.0 n.d. 18 81 n.d. 1.5 n.d.

4.0 n.d. 8.9 84 n.d. 6.6 n.d.


Table S9. Percentage of products formed during the oxidation of 2 mm terephthalaldehyde (9) by 1 µm HMFO in time at 25 °C, pH 8.0. Average values of two experiments. In control samples with only substrate, the only compound detected was the starting material.

reaction time (hr)


7 8 9 10 11 12

0.25 n.d.[a] n.d. 93 n.d. 7 n.d.

0.50 n.d. n.d. 79 n.d. 21 n.d.

1.0 n.d. n.d. 67 n.d. 33 n.d.

4.0 n.d. n.d. n.d. n.d. 86 14


Table S10. Percentage of products formed during the oxidation of 2 mm 4-(hydroxymethyl)benzoic acid (10) by 1 µm HMFO in time at 25 °C, pH 8.0. Average values of two experiments. In control samples with only substrate, the only compound detected was the starting material.

reaction time (hr)


7 8 9 10 11 12

0.25 n.d.[a] n.d. n.d. 83 17 n.d.

0.50 n.d. n.d. n.d. 54 46 n.d.

1.0 n.d. n.d. n.d. 3 92 5

4.0 n.d. n.d. n.d. n.d. 60 40


Appendices


Table S11. Percentage of products formed during the oxidation of 2 mm 4-formylbenzoic acid (11)by 1 µm HMFO in time at 25 °C, pH 8.0. Average values of two experiments. In control samples with only substrate, the only compound detected was the starting material.

reaction time (hr)


7 8 9 10 11 12

0.25 n.d.[a] n.d. n.d. n.d. 100 n.d.

0.50 n.d. n.d. n.d. n.d. 87 13

1.0 n.d. n.d. n.d. n.d. 80 20

4.0 n.d. n.d. n.d. n.d. 50 50


Table S12. Percentage of products formed during the oxidation of 4 mm [5-(hydroxymethyl)furan-2-yl]methanol (5) and 5-(hydroxymethyl)furfural (1) by 20 µm HMFO in the presence of 20 µm FAD, at 25 °C, pH 7.0. 24 hr reaction time. Average values of three experiments.

substrate[HMFO] µm

percentage of 5-(hydroxymethyl)furfural (1) derivatives present

5 1 2 6 3 4

[5-(hydroxymethyl)furan-2-yl]methanol (5) 20 n.d.[a] n.d. n.d. n.d. 8.0 92

[5-(hydroxymethyl)furan-2-yl]methanol (5) - 100 n.d. n.d. <0.01 <0.01 <0.01

5-(hydroxymethyl)furfural (1) 20 n.d. n.d. n.d. n.d. 4.8 95

5-(hydroxymethyl)furfural (1) - n.d. 99.6 n.d. 0.4 n.d. n.d.


Table S13. Products formed during the oxidation of 4 mm [4-(hydroxymethyl)phenyl]methanol (7) and 4-(hydroxymethyl)benzaldehyde (8) by 20 µm HMFO in the presence of 20 µm FAD, at 25 °C, pH 7.0. 24 hr reaction time. Average values of three experiments.

substrate[HMFO] µm

percentage of [4-(hydroxmethyl)phenyl]methanol (7) derivatives present

7 8 9 10 11 12

[4-(hydroxymethyl)phenyl]methanol (7) 20 n.d.[a] n.d. n.d. n.d. n.d. >99

[4-(hydroxymethyl)phenyl]methanol (7) - 100 0.02 n.d. n.d. n.d. n.d.

4-(hydroxymethyl)benzaldehyde (8) 20 n.d. n.d. n.d. n.d. n.d. >99

4-(hydroxymethyl)benzaldehyde (8) - n.d. 100 n.d. n.d. n.d. n.d.


137

Table S14. Steady state kinetics of HMFO, HMFO V101H and HMFO F67V-V101H

enzyme kcat (s-1) KM (mm) kcat/KM (s-1 mm-1)

wild type 21 ± 0.42 0.72 ± 0.066 29

V101H not active not active not active

F67V-V101H 0.0033 ± 0.0003 0.61 ± 0.26 0.0054

Enzyme concentrations were 200 nm, 6.0 µm and 5.0 µm for HMFO, HMFO V101H and HMFO F67V-V101H respectively. Experiments were performed at 25 °C in a 50 mm potassium phosphate pH 8.0 at atmospheric oxygen concentration. All three enzymes were heated at 80 °C in the presence of 1% w/v sodium dodecyl sulfate. Samples were run on an SDS-PAGE gel and the gel was subsequently soaked in 5% v/v acetic acid. No fluorescence of the FAD cofactor was monitored on a UVdoc HD2/20 MX imager (Uvitec) indicating FAD is not covalently bound in the wild type or the mutants.

Table S15. Spectral properties and apparent melting temperatures of HMFO and mutants

enzyme chapter Amax (nm) εmax (mM-1 cm-1) Tm’ (°C)

wild type 2 456 10.7 48

V101H 4 445 8.7 45

F67V-V101H 4 445 12.3 44

M103A 4 449 12.6 46

V367K 4 458 10.8 47

V367R 4 456 11.4 45

W369A 4 458 10.8 44

F434A 4 458 10.9 47

V465A 4 459 10.4 48

W466F 4 449 13.4 43

W466A 4 445 13.7 42

V367R-W466F 4 449 11.3 41

H333M-F352Y-V367R-W466F 5 449 12.4 45

H467A 2 464 9.9 55

N511A 4 456 10.0 49

The Tm’ was determined using the Thermo FAD method (Forneris et al., FEBS journal 2010 276, p2833-2840)

Appendices


Table S16. Activity of HMFO, HMFO W466A and HMFO W466F on secondary alcohols

enzyme enantiomer kobs (s-1) enantiomer kobs (s-1)

wild type S < 0.00001 ± 0.00003 R < 0.0001 ± 0.00005

W466A S 0.0029 ± 0.00006 R < 0.0001 ± 0.00008

W466F S 0.00085 ± 0.00004 R < 0.0001 ± 0.00002

The activity of the HMFO variants towards 5 mm S-1-phenylethanol and R-1-phenylethanol was followed by measuring formation of the product, acetophenone, at 247 nm (ε247 = 10.8 mm-1 cm-1) . Experiments were performed at pH 8.0, 25 °C.

Table S17. Effect on catalysis for HMFO V367R mutant towards several substrates

enzyme substrate kcat (s-1) KM (mm) kcat/KM (s-1 mm-1)

wild type 5-(hydroxymethyl)furfural (1) 9.9 1.4 7.1

V367R 5-(hydroxymethyl)furfural (1) 4.2 0.68 6.2

wild type 4-(hydroxymethyl)benzoic acid (10) 11.7 1.0 11.7

V367R 4-(hydroxymethyl)benzoic acid (10) 19.5 2.1 9.3

wild type 5-formylfuran-2-carboxylic acid (3) - > 4.0 0.0005

V367R 5-formylfuran-2-carboxylic acid (3 0.056 1.38 0.041

The introduction of a positive charge in the active site to stabilize the negative carboxylic acid group hardly influences the oxidation of 4-(hydroxymethyl)benzoic acid (10), but has a large effect on the oxidation of 5-formylfuran-2-carboxylic acid (3).

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Documents

University of Groningen HMF oxidase Dijkman, …129 Curriculum vitae Willem Pieter Dijkman was born on the seventh of March 1988, in Bergum. He grew up in Noardburgum and moved to