Vanessa Gutierrez April 18, 2009 Mentor: Dr. Roberto Guzman NASA Space Grant Symposium.
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Vanessa Gutierrez April 18, 2009 Mentor: Dr. Roberto Guzman NASA Space Grant Symposium
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Transcript of Vanessa Gutierrez April 18, 2009 Mentor: Dr. Roberto Guzman NASA Space Grant Symposium.
Vanessa GutierrezApril 18, 2009Mentor: Dr. Roberto GuzmanNASA Space Grant Symposium
Project DescriptionBackgroundMethodsResults/AnalysisConclusionAcknowledgements
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.
Enzymes catalyze specific reactions and substrates.
Unreacted substrate and enzyme
Substrate-enzyme complex
Converted substrate
(product) and regenerated
enzyme
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]
rp Vmax[S]
Km [S]
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.
Comparison of peak wavelengths for different species: Native trypsin, PEG, trypsin + PEG, trypsin-PEG
max
1
Vb
maxV
Km m
Michaelis-Menten parameters:
][
1
S
pr
1
Results
Km (mM) Vmax(mol/s)
Trypsin (native)
1.280 ± 0.22 2.754 ± 0.17
Trypsin + PEG 1.305 ± 0.14 3.054 ± 0.28
Trypsin–MPEG-NH2
1.096 ± 0.17 4.001 ± 0.37
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.
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.
Special thank you to:NASA Space Grant Consortium
Biomolecular Engineering and Separation Sciences Laboratory:
Professor Roberto GuzmanLian Wang – Post doctorate
Shellie Knights - UndergraduateMariano Garcia Soto - Graduate
Omar Gonzalez - GraduateBrenda Verdugo - Graduate
Pedro Ayala - GraduatePhillip Zinsli - Undergraduate
