Nitrilases

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Nitrilases

• Nitrile hydrolyzing enzymes

Megha Joshi 06530004
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Nitriles

• R-C N-
• 1. Present as cyanoglycosides produced by
  plants.
• 2. Nitrile compounds find extensive use in
  manufacturing of polymers and other chemicals.
• 3. Transformation of nitriles is a valuable
  industrial reaction. Eg- Hydrolysis

Appl Microbiol Biotechnol (2002) 603344
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• Chemical hydrolysis of nitriles is achieved in
  harsh conditions.
• Biocatalytic hydrolysis of nitriles is however,
  achieved in mild conditions.

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Nitrilases

Nitrilase, the first nitrile-metabolizing enzyme
to be discovered is known to convert indole-
3-acetonitrile to indole-3-acetic acid (an auxin)
in plants (Thimann and Mahadevan
1964). Microbial nitrilase was discovered much
later. Nitrilases are classified based on their
substrate specificity as aliphatic or aromatic
nitrilases.
Fig ref- http//www.nsf.gov/news/mmg/media/images/
Auxin_basic_h.jpg
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Enzyme mechanism
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Nitrilase producing microorganisms
Ref- Appl Microbiol Biotechnol (2002) 603344
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Novel Sensitive High-Throughput Screening
Strategy forNitrilase-Producing Strains

• Qing Zhu, Ao Fan, Yuanshan Wang, Xiaoqin Zhu,
  Zhao Wang, Minghuo Wu, and Yuguo Zheng
• APPLIED AND ENVIRONMENTAL MICROBIOLOGY (2007)
• 73(19), 6053-6057

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Introduction-

• Methods for efficient screening for nitrilase
  producing organisms are required.
• Conventional methods such as HPLC, liquid
  chromatography-mass spectrometry, or gas
  chromatography are tedious and time consuming.

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Previous screening methods

• Eg. 1-

Cell extract
NH3
1ml of 0.011 sodium nitroprusside and l ml of
20mM sodium hypochlorite.

• Limitation-
• Buffer was not stable, thus could not be used
  for a large no. of libraries
• Background caused by other proteins.

Heated for 10 min. on a boiling water-bath.
OD at 640 nm.
Int. J. Biochem. 17677683.
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• Eg. 2-

• Major Limitation-
• Unable to detect concentrations below 5 mM.

J. Mol. Catal. B Enzym. 39156159.
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Lanthanide ions as potential tools-

• Fluorescence based.
• The lanthanide ions, such as Tb(III), Eu(III),
  Sm(III), and Dy(III), exhibit typical
  fluorescence.
• Due to low molar absorptivity, evoking emission
  is hard.

• Ligands possess energy levels close to metal
  ions.
• Intramolecular energy transfer takes place.

Emission at ions characteristic wavelength
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Design principle of probes

• Salicylic acid serves as a photon antenna.
• Complex shows a large Stokes shift.

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Probe- Chemical variants
- Nitrilase activity was first measured using
non-fluorogenic probes, and product concentration
was monitored by HPLC.
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Enzyme assay by fluorescent probe
Probe
Pure enzyme

• Addition of EDTA-TbCl3 post incubation.
• Luminescence was measured at 545 nm after
  excitation at 328 nm.

• Ft and F0 represent the fluorescence at times t
  and 0.
• nst is the corresponding
• moles of the product standard.
• -Fst is the fluorescence resulting from nst of
  product.

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Determination of nitrilase activity from actual
cell culture
Probe
3 day cell culture pellet suspended in buffer
Samples removed after regular intervals
Incubation
EDTA TbCl3
Fluorescence readout
Control- Mixtures without cells. To exclude the
possibility of probe spontaneous hydrolysis.
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Results Discussion-a. Structure design of
fluorescent probes
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Subsequently-
Nitrilase from Rhodococcus 30ºC

• Probes 1-3 gave similar peaks.
• Probe 4 did not hydrolyze to corresponding acid.
• Similar results were obtained for Bacillus
  subtilis E9.

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1. Optimization of Buffer System
Probe 1 nitrilase from Rhodococcus
equi CCTCC.M.205114.
PBS / Tris / Borate pH- 6 / 7 / 8
30ºC
pH 8 50mM Tris buffer
2. Selectivity
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Kinetic Analysis
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Validating the High-Throughput screening strategy

• Effect of carbon source at different
  concentrations was observed.

By HPLC
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By Probe 2
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Summarizing

• This is a simple, rapid, and high throughput
  fluorescence nitrilase assay method with high
  specificity.
• The accuracy is higher than those of the other
  existing assay methods.

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Thank You!