Para-nitrobenzyl esterases with enhanced activity in aqueous and

Details Para-nitrobenzyl esterases with enhanced activity in aqueous and Para-nitrobenzyl esterases with enhanced activity in aqueous and

1996-01-23

1998-04-21

Wax, Robert A.

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

435 691, 435 712, 4352523, 43525233, 4353201, 4351721, 4351723, 536 232, 935 14, 935 29, 935 43, 935 56, 935 73, C12N 1555, C12N 918, C12N 121, C12N 1563

Patent

active

057416915

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.

REFERENCES:
patent: 5316935 (1994-05-01), Arnold et al.
patent: 5468632 (1995-11-01), Cantwell et al.
Arnold, F.H., "Engineering proteins for nonnatural environments," The FASEB Journal, vol. 7, pp. 744-749, 1993.
Chen, K., et al., "Tuning the activity of an enzyme for unusual environments: Sequential random mutagenesis of subtilisin E for catalysis in dimethylformamide," Proc. Natl. Acad. Sci. USA, vol. 90, pp. 5618-5622, 1993.
Moore et al. (1996). Nature Biotechnology 14: 458-467, Apr. 1996.

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