B12 -dependent dehydratases with improved reaction kinetics
AssigneeE.I. du Pont de Nemours and Company
InventorsKatharine J. Gibson, Der-Ing Liao, Xiao-Song Tang
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Abstract
Sequences of B12-dependent dehydratases with improved reaction kinetics are presented. Use of these B12-dependent dehydratases reduce the rate of the enzyme's suicide inactivation in the presence of glycerol and 1,3-propanediol. The enzymes were created using error-prone PCR and oligonucleotide-directed mutagenesis to target the DhaB1 gene, which encodes the α-subunit of glycerol dehydratase. Mutants with improved reaction kinetics were rapidly identified using high throughput assays.
Claims3
A method of screening for B12-dependent dehydratases having improved reaction kinetics, comprising:
- (a) contacting a B12-dependent dehydratase holoenzyme with a mixture comprising glycerol and 1,3-propanediol, wherein the 1,3-propanediol is at least 25 mM;
- and
- (b) screening the B12-dependent dehydratase holoenzyme to estimate the turnover ratio of the B12-dependent dehydratase.
A method for identifying B12-dependent dehydratase with improved reaction kinetics relative to a reference dehydratase comprising
- (a) providing a population of mutant dehydratase holoenzymes;
- (b) separately contacting each mutant dehydratase holoenzyme with 5-10 mM glycerol 10-300 mM 1,3-propanediol;
- (c) measuring at least two time points in a high throughput assay sufficiently separated to estimate kcat and total enzyme turnover for each mutant dehydratase holoenzyme of (b);
- (d) separately contacting the reference holoenzyme with 5-10 mM glycerol 10-300 mM 1,3-propanediol;
- (e) measuring at least two time points in a high throughput assay sufficiently separated to estimate kcat and total enzyme turnover for the reference holoenzyme of (d);
- and
- (f) selecting a B12-dependent mutant having improved reaction kinetics relative to the reference dehydratase, said kinetics determined by comparing the measurements of step (c) with the measurements of step (e).
A method for identifying B12-dependent dehydratase with improved reaction kinetics relative to a reference dehydratase comprising
- (a) providing a population of mutant dehydratase holoenzymes;
- (b) separately contacting each mutant dehydratase holoenzyme with 5-50 mM glycerol and >300 mM 1,3-propanediol;
- (c) measuring one time point in a high throughput assay sufficiently separated from T0 to estimate the total enzyme turnover number for each mutant dehydratase holoenzyme of (b);
- (d) separately contacting the reference holoenzyme with 5-50 mM glycerol and >300 mM 1,3-propanediol;
- (e) measuring one time point in a high throughput assay sufficiently separated from T0 to estimate the total enzyme turnover number for the reference holoenzyme of (d);
- and
- (f) selecting a B12-dependent mutant having improved reaction kinetics relative to the reference dehydratase, said kinetics determined by comparing the measurements of step (c) with the measurements of step (e).
Dependencies are inferred from the claim text.