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Andreas Bommarius
Professor
Contact Information
Building: Petit IBB
Office:
3310
Phone: 404.385.1334
Fax: 404.894.2291
email
Mailing Address
Georgia Institute of Technology
School of Chemical &
Biomolecular Engineering
311 Ferst Drive, N.W.
Atlanta, GA 30332-0100
Links
Research Areas
1. Investigation and Enhancement of Protein Stability
Determination of long-term process stability of protein biocatalysts through short-term experiments
The deactivation of protein biocatalysts even at relatively low temperatures is one of the principal drawbacks to their use. We have derived an equation for both time- and temperature-dependent activity of the biocatalyst based on known concepts such as transition state theory and the Lumry-Eyring model. We then derived an analytical solution for the total turnover number (ttn), under isothermal operation, as a function of the catalytic constant kcat, the unfolding equilibrium constant K, and the intrinsic first-order deactivation rate constant(s) kd,i. Employing an immobilized glucose isomerase biocatalyst in a CSTR and utilizing a linear temperature ramp beyond the Tm of the enzyme, we demonstrate an accelerated method for extracting the thermodynamic and kinetic constants describing the biocatalyst system. In addition, we demonstrate that the predicted biocatalyst behavior at different temperatures and reaction times is consistent with the experimental observations.
2. Modeling Biocatalyst Development
Improving directed evolution through pooling
Pooling in directed evolution experiments will greatly increase the throughput of screening systems, but important parameters such as the number of good mutants created and the activity level increase of the good mutants will depend highly on the protein being engineered. We developed and validated a Monte-Carlo simulation model of pooling that allows the testing of various scenarios in silico before starting experimentation. Using our test enzymes, -galactosidase (supermutant, or greatly improved enzyme) and -glucuronidase (dud, or enzyme with ancestral level of activity), the model accurately predicted the number of supermutants detected in experiments within a factor of 2. Pooling is most suited to cases such as the directed evolution of new function in a protein, where a large increase in activity over the background level will occur. Pooling can be used when new mutants have a lower level of activity increase, if a very sensitive assay is employed. Using our model will increase the throughput of screening procedures for directed evolution experiments and thus, lead to speedier engineering of proteins.
3. Development of Novel Biocatalysts
Development and characterization of NAD(P)H oxidase from Lactobacillis sanfranciscensis
A possible solution for the regeneration of NAD+ from NADH is the oxidation of NADH with concomitant reduction of oxygen catalyzed by NADH oxidase (E.C. 1.6.-.-). We employ NADH oxidase from Lactobacillus sanfranciscensis, which reduces O2 to innocuous H2O, and (R)-alcohol dehydrogenase ((R)-ADH) from Lactobacillus brevis to perform enantioselective oxidation of racemic phenylethanol to acetophenone and (S)-phenylethanol with regeneration of either NADH or NADPH to their respective oxidized precursors. NADH oxidase from L. sanfranciscensis accepts both NADH and NADPH; in contrast, the wildtype (R)-ADH only accepts NADP(+)(H) whereas its G37D mutant strongly prefers NAD(+)(H). Highly pure NADH oxidase (221 U/mg, two-step protocol) was coupled with wildtype-ADH from L. brevis on NADP(H) and mutant ADH from L. brevis on NAD(H) to achieve 50% conversion of racemic phenylethanol to (S)-phenylethanol and acetophenone. Depending on the relative concentration of alcohol to cofactor, up to more than 100 turnovers were observed. We believe that this is the first demonstration of a regeneration scheme for both NAD+ from NADH and NADP+ from NADPH with the same enzyme.
Solving the crystal structure of NAD(P)H oxidase
In collaboration with Allen Orville's lab, we recently published the first crystal structure of an NADH oxidase.
Coupled reaction from MSG to alpha-ketoglutarate with regeneration of NADH to NAD+ with NADH oxidase
Alpha-ketoglutarate, employed to treat mild chronic renal insufficiency, was obtained through enzymatic oxidation of monosodium glutamate (MSG) catalyzed by L-glutamate dehydrogenase (L-gluDH) coupled with NADH oxidase for the regeneration of NADH back to NAD+. The irreversible reduction of molecular oxygen to water by NADH oxidase is demonstrated to drive oxidation of MSG to -ketoglutarate to completion. L-gluDH was found to be inhibited by all three oxidative deamination products, -ketoglutarate, NADH, and ammonia. As the pH in the current system was balanced by sodium and NADH was recycled to NAD+, inhibition of L-gluDH by -ketoglutarate is believed to present the biggest challenge to an efficient process. In a batch experiment, we achieved a volumetric productivity of 1 g/(L·d).
