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Protein Engineering for Long Term Space Exploration

Vanessa Gutierrez April 18, 2009 Mentor: Dr. Roberto Guzman NASA Space Grant Symposium. Protein Engineering for Long Term Space Exploration. Outline. Project Description Background Methods Results/Analysis Conclusion Acknowledgements. Project Description.

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Protein Engineering for Long Term Space Exploration

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  1. Vanessa Gutierrez April 18, 2009 Mentor: Dr. Roberto Guzman NASA Space Grant Symposium Protein Engineering for Long Term Space Exploration

  2. Outline • Project Description • Background • Methods • Results/Analysis • Conclusion • Acknowledgements

  3. Project Description • 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-NH2) were used to determine their effects on the activity of trypsin. PEG chemically bound to protease trypsin.

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

  5. Background • 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. Methods • Preparation of trypsin in aqueous solution of PEG (3500 Dalton) and substrate BAPNA. • Chemical modification of trypsin with MPEG-NH2 (2000 Dalton) via binding with glutaraldehyde. • Enzymatic kinetic assay measurements made with UV spectrophotometer. • Data analyzed using Michaelis-Menten analysis.

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

  8. Michaelis-Menten parameters:

  9. Results

  10. Results • PEG (3500 Dalton) in aqueous solution with trypsin yielded little effect on activity of enzyme. • Chemical modification of enzyme with MPEG-NH2 (2000 Dalton) showed decrease in the Michaelis-Menten constant Km as well as increase in Vmax compared to native trypsin.

  11. Conclusion • Addition of MPEG-NH2 (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. Acknowledgements 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

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