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Overexpression of synthesized cephalosporin C acylase containing mutations in the substrate transport tunnel

Paper ID Volume ID Publish Year Pages File Format Full-Text
20784 43191 2012 6 PDF Available
Title
Overexpression of synthesized cephalosporin C acylase containing mutations in the substrate transport tunnel
Abstract

Cephalosporin C (CPC) acylase converts CPC into 7-aminocephalosporanic acid (7-ACA) by single-step enzymatic catalysis. An optimized CPC acylase gene with substituted codons and a reduced GC content was artificially designed, synthesized and overexpressed in recombinant Escherichia coli. The synthetic CPC acylase (sCPCAcy) exhibited 2.3 times more CPC specific deacylation activity with substrate CPC than with substrate glutaryl-7-ACA (GL-7-ACA). Site-directed mutagenesis of the residues around the active center showed that not only the residues that were adjacent to the CPC D-α-aminoadipyl moiety, but also the residues that were in the substrate transport tunnel (Leu666, Ala675, Leu677), played crucial roles in catalysis as the ones locating in active center. Mutant sCPCAcyLeu666Phe and sCPCAcyLeu677Ala exhibited significantly reduced specific enzymatic activity, while mutant sCPCAcyAla675Gly demonstrated enhanced activity. The specific activity of purified sCPCAcy and sCPCAcyAla675Gly was 10.0 U/mg and 11.3 U/mg, respectively. The optimal CPC acylase productivity of mutant sCPCAcyAla675Gly reached 5349 U/l after 24 h in culture, which was a 35% increase over the activity of sCPCAcy.

Keywords
Synthetic cephalosporin C acylase; 7-Aminocephalosporanic acid; Overexpression; Site-directed mutagenesis; Substrate transport tunnel
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Overexpression of synthesized cephalosporin C acylase containing mutations in the substrate transport tunnel
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Bioscience and Bioengineering - Volume 113, Issue 1, January 2012, Pages 36–41
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us