Journal
JOURNAL OF BIOTECHNOLOGY
Volume 142, Issue 2, Pages 127-134Publisher
ELSEVIER
DOI: 10.1016/j.jbiotec.2009.03.005
Keywords
Directed evolution; Biocatalysis; Phenylalanine dehydrogenase; Error-prone PCR; High-throughput screening; Parallel over-expression
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Funding
- Science Foundation Ireland
- Ph.D. studentship
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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. (C) 2009 Elsevier B.V. All rights reserved.
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