Document Detail


Evolution of an acylase active on cephalosporin C.
MedLine Citation:
PMID:  16260759     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Semisynthetic cephalosporins are synthesized from 7-amino cephalosporanic acid, which is produced by chemical deacylation or by a two-step enzymatic process of the natural antibiotic cephalosporin C. The known acylases take glutaryl-7-amino cephalosporanic acid as a primary substrate, and their specificity and activity are too low for cephalosporin C. Starting from a known glutaryl-7-amino cephalosporanic acid acylase as the protein scaffold, an acylase gene optimized for expression in Escherichia coli and for molecular biology manipulations was designed. Subsequently we used error-prone PCR mutagenesis, a molecular modeling approach combined with site-saturation mutagenesis, and site-directed mutagenesis to produce enzymes with a cephalosporin C/glutaryl-7-amino cephalosporanic acid catalytic efficiency that was increased up to 100-fold, and with a significant and higher maximal activity on cephalosporin C as compared to glutaryl-7-amino cephalosporanic acid (e.g., 3.8 vs. 2.7 U/mg protein, respectively, for the A215Y-H296S-H309S mutant). Our data in a bioreactor indicate an ~90% conversion of cephalosporin C to 7-amino-cephalosporanic acid in a single deacylation step. The evolved acylase variants we produced are enzymes with a new substrate specificity, not found in nature, and represent a hallmark for industrial production of 7-amino cephalosporanic acid.
Authors:
Loredano Pollegioni; Simona Lorenzi; Elena Rosini; Giorgia Letizia Marcone; Gianluca Molla; Roberto Verga; Walter Cabri; Mirella S Pilone
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Publication Detail:
Type:  Journal Article     Date:  2005-10-31
Journal Detail:
Title:  Protein science : a publication of the Protein Society     Volume:  14     ISSN:  0961-8368     ISO Abbreviation:  Protein Sci.     Publication Date:  2005 Dec 
Date Detail:
Created Date:  2005-12-02     Completed Date:  2006-02-03     Revised Date:  2013-06-07    
Medline Journal Info:
Nlm Unique ID:  9211750     Medline TA:  Protein Sci     Country:  United States    
Other Details:
Languages:  eng     Pagination:  3064-76     Citation Subset:  IM    
Affiliation:
Dipartimento di Biotecnologie e Scienze Molecolari, Università degli Studi dell'Insubria, Via J.H. Dunant, 3, 21100 Varese, Italy. loredano.pollegioni@uninsubria.it
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MeSH Terms
Descriptor/Qualifier:
Amidohydrolases / chemistry*,  genetics,  metabolism*
Amino Acid Sequence
Amino Acid Substitution
Base Sequence
Cephalosporins / metabolism*
Directed Molecular Evolution*
Gene Library
Kinetics
Models, Molecular
Molecular Sequence Data
Molecular Structure
Mutation / genetics
Protein Structure, Quaternary
Substrate Specificity
Chemical
Reg. No./Substance:
0/Cephalosporins; 3XIY7HJT5L/cephalosporin C; EC 3.5.-/Amidohydrolases; EC 3.5.1.4/amidase
Comments/Corrections

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