B12 dependent dehydratases with improved reaction kinetics

Details B12 dependent dehydratases with improved reaction kinetics B12 dependent dehydratases with improved reaction kinetics

C - Chemistry, Metallurgy

12

N

C12N 15/60 (2006.01) C12N 9/88 (2006.01) C12Q 1/527 (2006.01) G01N 33/573 (2006.01)

Patent

CA 2508662

Sequences of B12-dependent dehydratases with improved reaction kinetics are presented, thus reducing the rate of the enzyme's suicide inactivation in the presence of glycerol and 1,3-propanediol. These enzymes were created using error-prone PCR and oligonucleotide-directed mutagenesis to target the DhaB1 gene, encoding the a-subunit of glycerol dehydratase. Mutants with improved reaction kinetics were rapidly identified using high throughput assays, also disclosed herein.

L'invention concerne des séquences de déshydratases dépendantes de la co-enzyme B¿12 ?à cinétique de réaction améliorée, ce qui permet de réduire le taux d'inactivation de type suicide de l'enzyme en présence de glycérol et de 1,3-propanediol. Lesdites enzymes ont été créées par PCR sujette à l'erreur et mutagenèse dirigée à l'aide d'un oligonucléotide pour cibler le gène DhaB1, lequel code la sous-unité .alpha. de la glycérol déshydratase. Des mutants à cinétique de réaction améliorée ont été rapidement identifiés à l'aide de dosages à haut rendement.

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