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Methods of Enzyme Purification

This document outlines several common methods for purifying enzymes, including salt precipitation, ion exchange chromatography, gel permeation chromatography, hydrophobic interaction chromatography, affinity chromatography, and immunoaffinity chromatography. It also describes methods for assessing purity, such as SDS polyacrylamide gel electrophoresis, and defines units of enzyme activity like specific activity, enrichment, and percent purity and recovery.

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Dawlat Salama
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710 views2 pages

Methods of Enzyme Purification

This document outlines several common methods for purifying enzymes, including salt precipitation, ion exchange chromatography, gel permeation chromatography, hydrophobic interaction chromatography, affinity chromatography, and immunoaffinity chromatography. It also describes methods for assessing purity, such as SDS polyacrylamide gel electrophoresis, and defines units of enzyme activity like specific activity, enrichment, and percent purity and recovery.

Uploaded by

Dawlat Salama
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Methods of enzyme purification

1. salt precipitation

2. Chromatographic methods
i. ion exchange (cationic or anionic )
separation based on charge; the greater the net charge, the more strong
the interaction ; elute by increasing ionic strength of buffer or altering
pH

ii. gel permeation (size exclusion) chromatography:


separation based on size; larger proteins elute first

iii. Hydrophobic interaction chromatography:


separation based on hydrophobicity of protein surface; elute by
decreasing polarity of buffer ; more hydrophobic bind more strongly and
elute last.

iv. affinity chromatography:


separation based on specific biological interaction; eg enzyme –
substrate recognition; elute with substrate

v. immunoaffinity chromatography:
separation based on specificity of antibody recognition of peptide
sequence; elute by lowering pH to 2-3 to disrupt antibody – peptide
interaction. Neutralize quickly to avoid loss of activity.

Assessment of purity

 SDS polyacrylamide gel electrophoresis

 enrichment

 N-group analysis

Units of enzyme activity


 Activity: amount of product produced per unit time (eg. ?mols /
sec)

 Specific activity: amount of product produced per unit time per mg


protein

(eg. mmols / sec / mg)


 Enrichment: SA of fraction / SA of starting material

 Maximum possible enrichment: 100 / % of total protein


represented by protein of interest

 % purity : actual enrichment / maximum possible enrichment

 % recovery (yield) : total activity of fraction / total starting


activity

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