Isolation of various Industrially important enzymes. Enzyme kinetics of alpha-amylase, urease, acidphosphatase etc. Strategies for purification and characterization of enzymes. Immobilization techniques.

This is a lab, which will be including in the 6th semester of biotechnology course. This lab is very important to gain complete knowledge about enzymes. This lab mainly includes the extraction of enzymes from various sources, determining the activity of various enzymes and it also helps us to know the optimum pH, temperature requirements of enzymes and along with this we can know the kinetics of enzymes such as Km and the effect of inhibitors on enzymes. The other part includes different strategies of purification and characterization of enzymes using various techniques like ammonium sulphate fractionation, column chromatography, and Molecular weight determination of proteins using molecular sieving and by gel electrophoresis. Another important part of this lab is immobilization of enzymes and determining the kinetics of immobilized enzymes. Demo experiments in this laboratory include PCR and restriction analysis of a plasmid by agarose gel electrophoresis.

So the thorough knowledge of this lab will help the students to enter into various industries based on purification and characterization of enzyme on a large scale.

1. Isolation of alpha-amylase from sweet potato or saliva.
2. Isolation of urease from horse gram or kidney gram.
3. Isolation of acid phosphatases from sweet potato.
4. Determination of lysozyme activity.

 5. Determination of Km of an enzyme.
6. Effect of pH and temperature on enzyme activity.
7. Determination of specific activity of an enzyme.
8. Effect of inhibitors on enzyme activity.

9. Ammonium sulphate fractionation and column chromatography.
10. Molecular weight determination of a protein by molecular sieving.
11. Molecular weight determination of a protein by gel electrophoresis.

12. Immobilization of glucose oxidase.
13. Kinetics of immobilized enzymes.

14. Restriction analysis of plasmid by agarose gel electrophoresis.
15. PCR