NITRILASE ASSAYS

Ashlesha Bhondwe

INTRODUCTION

Nitrilases are enzymes that catalyse the hydrolysis of organontiriles to the corresponding carboxylic acids and ammonia.

NITRILASES

Play an important role in disposing off toxic nitriles.

They are regioselective enzymes. Play an important role in the synthesis of pharmaceutical intermediates.

These enzymes are classified based on their substrate specificity as aromatic or aliphatic nitrilases.

MECHANISM OF THE ENZYME

METHODS FOR SCREENING OF NITRILASE ACTIVITY

High throughput microbial nitrilase screening are necessary .

Techniques such as HPLC, mass spectroscopy are time consuming.

A high throughput amenable colorimetric assay for enantioselective screening of nitrilase producing micro organisms using pH sensitive Indicators.(Banerjee et.al, 2003)

pH drop determined due to formation of acids

Material used for the assay: •Bromothymol blue 0.01%•Phosphate Buffer (10mM, pH- 7.2)•Mandelonitrile (500mM stock)

R- CN R-COO- + H+

pKa of indicator – 7.2 (similar to the pKa of the buffer)

Proportionality between the rate of change of absorbance of indicator and rate of the reaction, given by buffer factor, Q

Yellow

Bromothym

ol

Blue

CALCULATION

Q= (Cb / Cin )* (1/Δ ε*l)

Q= Buffer FactorCb = concentration of buffer

Cin = concentration of indicator

Δ ε = molar extinction coefficient

Rate (µmol/min) = (dA/dt) * Q

ASSAY

Assay carried out in a 96 well microplate for rapid screening of microorganisms. Absorbance at 616nm

The decrease in absorbance is proprtional to the concentration of proton released.

A Metal ion based method for screening of Nitrilases.

(Daniel R et.al, 2006)

Metal ion cobalt was used to determine the amount of ammonia released by the action of nitrilase.

ASSAY

Material used for assay : BufferedCobalt chloride (10mM) – made in

10mM Tris pH- 7. Substrate – hydrocinnamonitrile and

isobutylsuccinonitrile Reaction quenched by increasing the

temperature from 30 ° C to 95° C Assay validated by HPLC. As soon as the cobalt chloride solution is

added to the reaction mixture a yellow colored complex is formed. Absorbance at 375nm.

LIMITATION

This technique has a high background interference due to the initial colour of cobalt chloride.

For this reason higher concentrations of ammonia can be detected (above 5mM).