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Laboratory evolution of cytochrome P450 peroxygenase activity


Cirino, Patrick Carmen (2004) Laboratory evolution of cytochrome P450 peroxygenase activity. Dissertation (Ph.D.), California Institute of Technology.


The ability of the cytochrome P450 heme monooxygenases to catalyze difficult oxidation reactions, often with high specificity and selectivity, makes them attractive for numerous biotechnological applications. However they are generally limited by low turnover rates and low stability, and their minimum requirements for catalysis include a cofactor as source of electrons (NAD(P)H), partner proteins for electron transfer, and dioxygen. Some P450s are capable of supporting low levels of peroxygenase activity, in which a peroxide is utilized to drive catalysis via a ?shunt? pathway. This mechanism for substrate oxidation, although inefficient and not generally utilized in nature, simplifies P450 catalysis by eliminating the need for NAD(P)H. Our goal was to engineer an efficient P450 peroxygenase which utilizes hydrogen peroxide (H2O2). Directed evolution is a powerful enzyme engineering methodology which mimics nature's algorithm for evolution. Enzyme libraries are generated via DNA mutagenesis or recombination techniques, and variants with improved function are isolated using an appropriate screen or selection. Using this strategy, in combination with site-directed mutagenesis, we have created P450 BM-3 heme domain variants with more than 100-fold improved H2O2-driven hydroxylation activity compared to wild-type, showing both an improved kcat as well as a lower Km for H2O2. Thermostability was also improved by directed evolution. We have engineered a cell-free, biomimetic hydroxylase that requires only H2O2 to exploit the hydroxylating power of P450 BM-3. Peroxide-mediated inactivation as a result of heme destruction remains a major obstacle and presents an important enzyme engineering challenge. This research has broadened the potential applications of P450 biocatalysis by exploiting the versatility of heme-containing proteins.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:biocatalysis; cytochrome P450; directed evolution; oxidation; peroxygenase; protein engineering
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemical Engineering
Awards:Constantin G. Economou Memorial Prize, 1999
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Arnold, Frances Hamilton
Thesis Committee:
  • Arnold, Frances Hamilton (chair)
  • Tirrell, David A.
  • Gray, Harry B.
  • Davis, Mark E.
Defense Date:2 June 2003
Non-Caltech Author Email:cirino (AT)
Record Number:CaltechETD:etd-06062003-164310
Persistent URL:
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:2469
Deposited By: Imported from ETD-db
Deposited On:10 Jun 2003
Last Modified:11 Dec 2014 00:04

Thesis Files

PDF (cirinoCV.pdf) - Final Version
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PDF (cirinothesis.pdf) - Final Version
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