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The development of an assay for nitroarene
reductase activity in Bakers yeast based on
RP-HPLC and investigations regarding purification
of this enzyme
Reporter Jing Xiaona Department of Surface
Biotechnology
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Where the origin idea come from?
Arylhydroxylamines are valuable key intermediates
in the synthesis of certain pharmaceutical
products such as paracetamol
NNA
HANA
ANA
valuable key intermediate but hard to get
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Formation of hydroxylamine and its reduction to
amine
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bakers yeast? a potential new way In 2004
the first example of the enzymatic reduction of
nitroarene compounds to arylhydroxylamines was
reported Chemoselective reduction of the
corresponding aromatic nitro compounds by bakers
yeast is highly efficient and occurs under mild,
environment friendly conditions some enzymes
with nitroreductase activity in bakers yeast
play a crucial role Therefore, the use of
whole-cell yeast, yeast homogenates or isolated
enzymes are regarded alternative strategies for
the development of new biotransformation
processes for the production of
arylhydroxylamines on an industrial scale.
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• Questions

• Strategy?
• 1 new method development
• 2 new enzyme purification
• System?
• ÄKTA Basic HPLC workstation for RP-HPLC
• ÄKTA FPLC workstation for DEAE HPLC

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Nitroreduction reaction systems

• 4-NNA(substrate) whole cell or enzyme fraction
  
• incubated at 26C
  
• Organic extraction
• Reduced pressure evaporation to
  concentrate the sample
• Product detection using RP-HPLC

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Products analysis using RP-HPLC

• ÄKTA Basic workstation
• a Kromasil C18 column
• Water and methanol gradient were used as mobile
  phase with a flow rate of 0.8 ml/min.
• Specific adsorption wavelength for 4-ANA
  detection is 424 nm in methanol.

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The system to detect the product
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. Calibration curve for 4-ANA production in the
reaction system
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Time-course of biotransformation by the crude
yeast homogenate
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Preliminary investigation for purification of the
novel enzyme

• A DEAE-Sepharose CL-6B exchange column was
  employed to separate the protein fractions in the
  yeast homogenates.
• The supernatant was transferred to the column
• Eluted with an increasing linear gradient 0100
  Buffer B (20 mM Tris-HCl, 0.5 M NaCl, pH 8.0).
• The eluted fractions were collected and incubated
  with the substrate 4-NNA at 26C
• The product was analyzed using the RP-HPLC method
  we developed.

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First tryDEAE anion exchange
chromatography
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fraction 4 from the DEAE-chromatography
separation contained most nitroreductase
activity

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Summary

• A simple method combined reverse-phase HPLC has
  been developed to assess 4-ANA nitroreductase
  activity from the bakers yeast homogenates by
  qualifying the reduction rate of the final
  product 4-ANA. Although the identification of the
  enzyme involving in the nitroarene reduction
  process in bakers yeast remains unknown, the
  method developed in our lab could contribute to
  investigation of nitroreduction mechanism.
• Further studies to purify the key enzyme in the
  nitroaromatic reduction are currently under way
  in our lab.

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My Harvest

• 1 cooperation !
• 2 A little protein purification strategy and
  common rules
• 3 Operation on ÄKTA HPLC workstation
• 4 Nice friends in the lab

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Acknowledgements

• Thanks to Prof. Jan-Christer Janson for being an
  helpful and kind supervisor. It has been great
  working in this lab.
• Thanks to Dr. Xu Jianqiang for his support in
  many ways during my time here and I learnt a lot
  form him.
• I also would like to thank Miss Moon and my
  classmate Nongyuan and other people at the lab
  for the friendly atmosphere and always making me
  feel pleasant at our lab.