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Screening and purification of potential Hydroxy
nitril lyase
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Introduction

• Hydroxynitrile lyases (Hnls) are biocatalysts for
  the synthesis of optically pure cyanohydrins
• It is a stereoselective C-C bond formation, forms
  chiral cyanohydrins
• Cyanohydrins synthesizes ß-adrenergic blocking
  precursors agents1, ACE inhibitors1, insecticides
  and biologically active compounds
• Cyanogenic glycosides (CGs) are found in higher
  plants and ferns for protection from herbivore

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NAME STRUCTURE
PRECURSOR APPLICATION
Applications of
cyanohydrins
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Objective of the experiment

• Screening for Discovery of New Potent Hydroxy
  nitrile lyase
• Finding out a new source possessing new catalytic
  or biochemical properties
• Convergent evolution shown by HNLs give high
  probability of natures own engineered HNL
• There are more than 3000 cyanogenic plant
  species, whereas the number of reported HNLs
  (characterized and non characterized) is about 40
  enzymes (less than 2 of cyanogenic organism)
• To expand the current substrate range

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Isolation and purification
Protein impurities
Fractional precipitation (NH4)2SO4
Column chromatography
Ion exchanged chromatography
Affinity chromatography
Isoelectric focusing
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Work Plan
Screening for discovery of new HNL
Selection of plant families , genus and species.
Collection of materials (leaves, root , etc)
Homogenization
Crude
Ammonium sulphate precipitation
Enzyme assay
Protein purification
Spectrometric activity
HPLC or GC
Chromatography
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Physiochemical characterization
pH
Temperature

• Optimum pH

• Optimum temperature

• pH stability

• Temperature stability

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