CaltechAUTHORS
  A Caltech Library Service

Cost-Effective Whole-Cell Assay for Laboratory Evolution of Hydroxylases in Escherichia coli

Schwaneberg, Ulrich and Otey, Christopher and Cirino, Patrick C. and Farinas, Edgardo and Arnold, Frances H. (2001) Cost-Effective Whole-Cell Assay for Laboratory Evolution of Hydroxylases in Escherichia coli. Journal of Biomolecular Screening, 6 (2). pp. 111-117. ISSN 1087-0571. http://resolver.caltech.edu/CaltechAUTHORS:20180824-101913620

Full text is not posted in this repository. Consult Related URLs below.

Use this Persistent URL to link to this item: http://resolver.caltech.edu/CaltechAUTHORS:20180824-101913620

Abstract

Cytochrome P450 BM-3 from Bacillus megaterium catalyzes the subterminal hydroxylation of medium- and long-chain fatty acids at the to-1, w-2, and c-3 positions. A continuous spectrophotometric assay for P450 BM-3 based on the conversion of p-nitrophenoxycarboxylic acids (pNCA) to co-oxycarboxylic acids and the chromophore p-nitrophenolate was reported recently. However, this pNCA assay procedure contained steps that limited its application in high throughput screening, including expression of P450 BM-3 variant F87A in 4-ml cultures, centrifugation, resuspension of the cell pellet, and cell lysis. We have shown that permeabilization of the outer membrane of Escherichia coli DH5a with polymyxin B sulfate, EDTA, polyethylenimine, or sodium hexametaphosphate results in rapid conversion of 12-pNCA. A NADPH-generating system consisting of NADP+, D/L-isocitric acid, and the D/L-isocitrate dehydrogenase of E. coli DH5a reduced the cofactor expense more than 10-fold. By avoiding cell lysis, re-suspension, and centrifugation, the high throughput protocol allows screening of thousands of samples per day.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1177/108705710100600207DOIArticle
ORCID:
AuthorORCID
Cirino, Patrick C.0000-0002-7245-6205
Arnold, Frances H.0000-0002-4027-364X
Additional Information:© 2001 The Society for Biomolecular Screening.
Record Number:CaltechAUTHORS:20180824-101913620
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20180824-101913620
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:89148
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:24 Aug 2018 17:25
Last Modified:24 Aug 2018 17:25

Repository Staff Only: item control page