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Engineering Cytochrome P450BM3 for Oxidation and Silicon–Carbon Bond Cleavage of Volatile Methylsiloxanes


Sarai, Nicholas Singh (2023) Engineering Cytochrome P450BM3 for Oxidation and Silicon–Carbon Bond Cleavage of Volatile Methylsiloxanes. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/gn1j-fz77.


Directed evolution of enzymes can reveal activities that do not occur in the natural world. While most examples of directed evolution of new-to-nature chemistry have been applied in a synthetic direction, enzymatic biodegradation typically relies on wild-type enzymes. This thesis posits that directed evolution can generate enzymes capable of degrading non-biodegradable anthropogenic compounds, focusing on efforts to break silicon–carbon bonds, which are not known to be cleaved by enzymes in Nature. Chapter I establishes background on how enzymes evolve to catalyze degradation of compounds over long timescales in Nature, highlighting the enzymatic depolymerization of lignocellulosic biomass. This sets the stage for a case study of rapid enzyme evolution in response to anthropogenic molecules such as plastics and agrochemicals. With this background, directed evolution of new-to-nature synthetic activities is presented to demonstrate how new enzymatic activities can be evolved in the laboratory. In Chapter II, the state of the art for biocatalytic reactions involving organosilicon compounds is reviewed, starting with a description of how biology uses silicon and concluding with a perspective on future opportunities in this nascent field. Finally, Chapter III describes the engineering of a novel siloxane oxidase based on a cytochrome P450, which conducts two reaction steps in tandem to cleave silicon–carbon bonds. First, it hydroxylates the C–H bonds of siloxanes—the anthropogenic building blocks of silicone polymers—to yield a carbinol species, an activity reminiscent of the parent enzyme’s native hydroxylation of fatty acids. Via a function entirely different than its native activity, the enzyme converts this carbinol to a silanol species. In performing both of these steps, this is the first known enzyme that can cleave Si–C bonds, an activity which is the first step toward enzymatic degradation of these persistent, man-made compounds. In sum, this thesis demonstrates that directed evolution can reveal enzymatic degradation chemistries that are not known in Nature by establishing new-to-nature Si–C cleavage of siloxanes.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:siloxane; bioremediation; biocatalysis; directed evolution; enzyme engineering; brook rearrangement; silicon–carbon bond cleavage; cytochrome P450; P450BM3
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Biochemistry and Molecular Biophysics
Thesis Availability:Restricted to Caltech community only
Research Advisor(s):
  • Arnold, Frances Hamilton
Thesis Committee:
  • Newman, Dianne K. (chair)
  • Peters, Jonas C.
  • Gray, Harry B.
  • Arnold, Frances Hamilton
Defense Date:5 May 2023
Funding AgencyGrant Number
Dow Chemical Company227027AU
Dow Chemical Company227027AO
NSF Graduate Research FellowshipDGE-1745301
Record Number:CaltechTHESIS:05152023-034200704
Persistent URL:
Related URLs:
URLURL TypeDescription adapted for Chapter II work not included in thesis work not included in thesis
Sarai, Nicholas Singh0000-0002-4655-0038
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:15173
Deposited By: Nicholas Sarai
Deposited On:16 May 2023 16:08
Last Modified:10 May 2024 18:09

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