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