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Para-nitrobenzyl esterases with enhanced activity in aqueous and nonaqueous media

Patent ·
OSTI ID:871483
 [1];  [1]
  1. Pasadena, CA
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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.

Research Organization:
California Institute of Technology (Pasadena, CA)
DOE Contract Number:
FG02-93CH10578
Assignee:
California Institute of Technology (Pasadena, CA)
Patent Number(s):
US 5741691
OSTI ID:
871483
Country of Publication:
United States
Language:
English

References (3)

Directed evolution of a para-nitrobenzyl esterase for aqueous-organic solvents journal April 1996
Engineering proteins for nonnatural environments journal June 1993
Tuning the activity of an enzyme for unusual environments: sequential random mutagenesis of subtilisin E for catalysis in dimethylformamide. journal June 1993

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