1. Vanessa Gutierrez April 18, 2009 Mentor: Dr. Roberto Guzman NASA Space Grant Symposium

2.  Project Description  Background  Methods  Results/Analysis  Conclusion  Acknowledgements

3.  Protein engineering (chemical modification) was explored to enhance the activity and use of the protease trypsin.  Polyethylene glycol (PEG) and mono amino polyethylene glycol (MPEG-NH 2 ) were used to determine their effects on the activity of trypsin. PEG chemically bound to protease trypsin.

4.  Enzymes catalyze specific reactions and substrates. Unreacted substrate and enzyme Substrate-enzyme complex Converted substrate (product) and regenerated enzyme

5.  Enzymes may lose activity at high temperatures and high pH in aqueous solutions.  Enzyme activity represented by Michaelis- Menten kinetics. [E] + [S] [E-S] [E] + [P]

6.  Preparation of trypsin in aqueous solution of PEG (3500 Dalton) and substrate BAPNA.  Chemical modification of trypsin with MPEG- NH 2 (2000 Dalton) via binding with glutaraldehyde.  Enzymatic kinetic assay measurements made with UV spectrophotometer.  Data analyzed using Michaelis-Menten analysis.

7. Comparison of peak wavelengths for different species: Native trypsin, PEG, trypsin + PEG, trypsin-PEG

8. Michaelis-Menten parameters:

9. Results K m (mM) V max (mol/s) Trypsin (native) 1.280 ± 0.222.754 ± 0.17 Trypsin + PEG 1.305 ± 0.143.054 ± 0.28 Trypsin–MPEG- NH2 1.096 ± 0.174.001 ± 0.37

10.  PEG (3500 Dalton) in aqueous solution with trypsin yielded little effect on activity of enzyme.  Chemical modification of enzyme with MPEG-NH 2 (2000 Dalton) showed decrease in the Michaelis-Menten constant K m as well as increase in V max compared to native trypsin.

11. Conclusion  Addition of MPEG-NH 2 (2000 Dalton) onto trypsin yielded higher activity in aqueous solution using Michaelis-Menten kinetics.  Future directions :  Purify and characterize derivatives of PEG.  Analysis of activity and kinetic effects of PEGs onto enzymes of different moieties.  Analysis of chemical activity with other proteins.

12. Special thank you to: NASA Space Grant Consortium Biomolecular Engineering and Separation Sciences Laboratory: Professor Roberto Guzman Lian Wang – Post doctorate Shellie Knights - Undergraduate Mariano Garcia Soto - Graduate Omar Gonzalez - Graduate Brenda Verdugo - Graduate Pedro Ayala - Graduate Phillip Zinsli - Undergraduate