Electrochemical characterisation of cellobiose dehydrogenases from different microorganisms based on

1
Electrochemical characterisation of cellobiose
dehydrogenases from different microorganisms
based on SAM on Au electrodes

• Leonard Stoica, Tautgirdas Ruzgas, Lo Gorton,
  Lund University
• and Nina Dimcheva, Plovdiv University

FINAL PuBLIER meeting, 29 August 2003
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Introduction

• CDH sources
• Trametes villosa Phanerochaete sordida
• Myriococcum thermophilum (bacterial)
  Phanerochaete chrysosporium
• Thiols used for SAM on Au electrodes

• Techniques used for studies
• CV and QSWV

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Model of enzyme characterisation
1. Thiol type influneces the enzyme orientation.
2. Redox communication (DET)
4. Information about Linker domain (IET)
3-2gt
3. Study the enzyme activity (kcat, IET, DET)
pH is modifying the enzyme activity and the net
charge of the enzyme, so it will affect the
enzyme orientation (1) gt (2) gt (3) gt (4)
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pH influenceCDH (T.v.) Aldrithiol-SAM
Au-electrode
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QSWV shows the shifting of formal potential with
pH
3.5
4.0
4.5
5.0
5.5
6
Cysteine-SAM CDH (T.v.)
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Cysteine-SAM CDH (T.v.)
4.5
4.0
3.5
6.0
5.5
5.0
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QSWV CDH (T.v.)cysteine
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Trametes villosa-thiol comparison
10 mV/sec, 20 mM acetate buffer, pH 3.5, Trametes
villosa
10 mM cellobiose
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Aldrithiol-MUDAL (955)
10 mV/sec, 20 mM acetate buffer, pH 3.5, Trametes
villosa
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Cystamine-MUDAL (13)
10 mV/sec, 20 mM acetate buffer, pH 3.5, Trametes
villosa
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CVCDH (Panerochaete sordida) thioglycerol
3.5
4.5
5.5
4.5
6.5
7.5
3.5
5.5
6.5
7.0
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QSWVCDH (Panerochaete sordida)1 thioglycerol
4.5 -5 mV
5.5 -35 mV
3.5 35 mV
6.5 -50 mV
7.0 -55 mV
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pH influence on DET, SS and METat CDH from (P.c.)
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Comparison of DET/MET ratios for CDH from T.v.
and P.c.
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Conclusions

• CDHs from different microorganisms show
  distinctive properties one to each other

Trametes villosa Highest catalytic activity at
pH 4.5 DET much more higher than MET even at pH
6.
Phanerochaete chrysosporium Highest activity at
pH 6 with high efficiency of MET and a strong
self-limitation of DET
The high efficiency of DET suggests the lack of
the linker region and the existance of an
interdomains docking. The low MET can be an
indication that MET is realised from the linker
region for P.c.
The role of 26 amino acids from the linker region
must play a very important role in order to the
enzyme to modulate its functions
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Acknowledgements

• Swedish National Board of Industrial and
  Technical Development
• Studies and Applications of Biosensors and
  Bioanalysis
• INCO project PuBLIER (ICA2-CT-2000-10050)

Thank you for your attention!