Skip Navigation

This Article
Right arrow Full Text Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (6)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Sroga, G. E.
Right arrow Articles by Dordick, J. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sroga, G. E.
Right arrow Articles by Dordick, J. S.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Protein Engineering, Vol. 14, No. 11, 929-937, November 2001
© 2001 Oxford University Press

Generation of a broad esterolytic subtilisin using combined molecular evolution and periplasmic expression

Grazyna E. Sroga and Jonathan S. Dordick,1

Department of Chemical Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180-3590, USA

Concomitant activity improvement of an evolved enzyme toward two very different ester substrates was achieved when a unique combination of functional periplasmic enzyme expression in Escherichia coli, random mutagenesis, DNA shuffling and cell-based kinetic screenings was applied. Specifically, we focused on the conversion of subtilisin E into an enzyme with broader esterase activity as opposed to its native amidase activity. Cell-based microtiter assays were performed on N-acetyl-D,L-phenylalanine p-nitrophenyl ester (Phe-NPE) and sucrose 1'-adipate (S1'A), as well as on the tetrapeptide amide substrate N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide. After a single modified cycle of directed molecular evolution, we isolated a number of clones exhibiting increased activity toward Phe-NPE. In the following rounds of screenings, mutants with improved activity on Phe-NPE were also tested on S1'A. Three mutants were identified with increased esterolytic activity on Phe-NPE and S1'A, while having similar amidase activity to that of the parental enzymes. Because the two ester substrates are structurally distinct, we have evolved a more general esterolytic subtilisin and this may have important applications in synthesis.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Microbiol. Mol. Biol. Rev.Home page
L. Yuan, I. Kurek, J. English, and R. Keenan
Laboratory-Directed Protein Evolution
Microbiol. Mol. Biol. Rev., September 1, 2005; 69(3): 373 - 392.
[Abstract] [Full Text] [PDF]

Home page
 MicrobiologyHome page
V. Faraco, G. Palmieri, G. Festa, M. Monti, G. Sannia, and P. Giardina
A new subfamily of fungal subtilases: structural and functional analysis of a Pleurotus ostreatus member
Microbiology, February 1, 2005; 151(2): 457 - 466.
[Abstract] [Full Text] [PDF]


Disclaimer:
Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.