Para-nitrobenzyl esterases with enhanced activity in aqueous and nonaqueous media
Abstract
A method for isolating and identifying modified para-nitrobenzyl esterases which exhibit improved stability and/or esterase hydrolysis activity toward selected substrates and under selected reaction conditions relative to the unmodified para-nitrobenzyl esterase. The method involves preparing a library of modified para-nitrobenzyl esterase nucleic acid segments (genes) which have nucleotide sequences that differ from the nucleic acid segment which encodes for unmodified para-nitrobenzyl esterase. The library of modified para-nitrobenzyl nucleic acid segments is expressed to provide a plurality of modified enzymes. The clones expressing modified enzymes are then screened to identify which enzymes have improved esterase activity by measuring the ability of the enzymes to hydrolyze the selected substrate under the selected reaction conditions. Specific modified para-nitrobenzyl esterases are disclosed which have improved stability and/or ester hydrolysis activity in aqueous or aqueous-organic media relative to the stability and/or ester hydrolysis activity of unmodified naturally occurring para-nitrobenzyl esterase.
- Inventors:
-
- Pasadena, CA
- Issue Date:
- Research Org.:
- California Institute of Technology (CalTech), Pasadena, CA (United States)
- OSTI Identifier:
- 872300
- Patent Number(s):
- 5906930
- Assignee:
- California Institute of Technology (Pasadena, CA)
- Patent Classifications (CPCs):
-
C - CHEMISTRY C12 - BIOCHEMISTRY C12N - MICROORGANISMS OR ENZYMES
- DOE Contract Number:
- FG02-93CH10578
- Resource Type:
- Patent
- Country of Publication:
- United States
- Language:
- English
- Subject:
- para-nitrobenzyl; esterases; enhanced; activity; aqueous; nonaqueous; media; method; isolating; identifying; modified; exhibit; improved; stability; esterase; hydrolysis; selected; substrates; reaction; conditions; relative; unmodified; involves; preparing; library; nucleic; acid; segments; genes; nucleotide; sequences; segment; encodes; expressed; provide; plurality; enzymes; clones; expressing; screened; identify; measuring; ability; hydrolyze; substrate; specific; disclosed; ester; aqueous-organic; naturally; occurring; selected reaction; modified enzymes; para-nitrobenzyl esterases; acid segments; hydrolysis activity; involves preparing; exhibit improved; reaction conditions; naturally occurring; nucleotide sequences; nucleotide sequence; method involves; nucleic acid; aqueous media; para-nitrobenzyl esterase; modified para-nitrobenzyl; improved stability; selected substrate; organic media; selected substrates; nonaqueous media; method involve; identifying modified; esterase hydrolysis; enhanced activity; modified enzyme; acid segment; /435/999/
Citation Formats
Arnold, Frances H, and Moore, Jeffrey C. Para-nitrobenzyl esterases with enhanced activity in aqueous and nonaqueous media. United States: N. p., 1999.
Web.
Arnold, Frances H, & Moore, Jeffrey C. Para-nitrobenzyl esterases with enhanced activity in aqueous and nonaqueous media. United States.
Arnold, Frances H, and Moore, Jeffrey C. Fri .
"Para-nitrobenzyl esterases with enhanced activity in aqueous and nonaqueous media". United States. https://www.osti.gov/servlets/purl/872300.
@article{osti_872300,
title = {Para-nitrobenzyl esterases with enhanced activity in aqueous and nonaqueous media},
author = {Arnold, Frances H and Moore, Jeffrey C},
abstractNote = {A method for isolating and identifying modified para-nitrobenzyl esterases which exhibit improved stability and/or esterase hydrolysis activity toward selected substrates and under selected reaction conditions relative to the unmodified para-nitrobenzyl esterase. The method involves preparing a library of modified para-nitrobenzyl esterase nucleic acid segments (genes) which have nucleotide sequences that differ from the nucleic acid segment which encodes for unmodified para-nitrobenzyl esterase. The library of modified para-nitrobenzyl nucleic acid segments is expressed to provide a plurality of modified enzymes. The clones expressing modified enzymes are then screened to identify which enzymes have improved esterase activity by measuring the ability of the enzymes to hydrolyze the selected substrate under the selected reaction conditions. Specific modified para-nitrobenzyl esterases are disclosed which have improved stability and/or ester hydrolysis activity in aqueous or aqueous-organic media relative to the stability and/or ester hydrolysis activity of unmodified naturally occurring para-nitrobenzyl esterase.},
doi = {},
journal = {},
number = ,
volume = ,
place = {United States},
year = {Fri Jan 01 00:00:00 EST 1999},
month = {Fri Jan 01 00:00:00 EST 1999}
}
Works referenced in this record:
Directed evolution of a para-nitrobenzyl esterase for aqueous-organic solvents
journal, April 1996
- Moore, Jeffrey C.; Arnold, Frances H.
- Nature Biotechnology, Vol. 14, Issue 4
Engineering proteins for nonnatural environments
journal, June 1993
- Arnold, Frances H.
- The FASEB Journal, Vol. 7, Issue 9
Tuning the activity of an enzyme for unusual environments: sequential random mutagenesis of subtilisin E for catalysis in dimethylformamide.
journal, June 1993
- Chen, K.; Arnold, F. H.
- Proceedings of the National Academy of Sciences, Vol. 90, Issue 12