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Efficient screening for new amino acid dehydrogenase activity: Directed evolution of Bacillus sphaericus phenylalanine dehydrogenase towards activity with an unsaturated non-natural amino acid

Paper ID Volume ID Publish Year Pages File Format Full-Text
24584 43528 2009 8 PDF Available
Title
Efficient screening for new amino acid dehydrogenase activity: Directed evolution of Bacillus sphaericus phenylalanine dehydrogenase towards activity with an unsaturated non-natural amino acid
Abstract

Mutants of phenylalanine dehydrogenase (PheDH) from Bacillus sphaericus with improved activity and specificity towards an unsaturated non-natural amino acid of commercial interest, propargylglycine, have been obtained by directed evolution after screening 10,000 colonies. An effective high-throughput screening assay for the detection of amino acid dehydrogenase activity using spectrophotometric measurement of formazan colour has been developed and explored. Parallel over-expression of the target protein library in 96-well plates was optimized by using auto-induction medium. Using these methodologies and medium/high mutation frequency error-prone polymerase chain reaction, combinations of mutations and multi-site saturation mutagenesis targeting two “hot-spots” close to the active site of the enzyme, one mutant was obtained with 7.4-fold improved catalytic efficiency with the target substrate together with 612-fold improvement of selectivity between the target substrate and the natural one. In the high mutation frequency epPCR and saturation mutagenesis, a robotic colony picker and automation workstation were employed for the high-throughput screening.

Keywords
Directed evolution; Biocatalysis; Phenylalanine dehydrogenase; Error-prone PCR; High-throughput screening; Parallel over-expression
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Efficient screening for new amino acid dehydrogenase activity: Directed evolution of Bacillus sphaericus phenylalanine dehydrogenase towards activity with an unsaturated non-natural amino acid
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 142, Issue 2, 15 June 2009, Pages 127–134
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