Directed evolution of thermophilic enzymes
AssigneeDiversa Corporation
InventorJay M. Short
Life of this patent
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Abstract
Thermostable enzymes are subjected to mutagenesis to produce a thermophilic enzyme which is stable at thermophilic temperature and which has increased activities at least two-fold higher than the activity of the wild-type thermostable enzyme at lower temperatures, which are temperatures of 50° C. or lower.
Claims92
A method of producing a thermostable enzyme which is stable at temperatures of at least 60° C. and which exhibits higher activity at a desired lower temperature below 60° C., compared with a corresponding wild-type enzyme at its optimal temperature, comprising:
- (a) isolating nucleic acid from a thermophilic organism, said nucleic acid comprising at least one polynucleotide encoding a desired enzyme which is stable at a temperature of at least 60° C.;
- (b) preparing a library of clones generated from the nucleic acid of step (a);
- (c) mutating the nucleic acid sequence contained in a clone from the library comprising the at least one polynucleotide of step (a);
- and
- (d) screening mutants produced by the mutating step (c) to identify a mutated enzyme or a polynucleotide encoding a mutated enzyme, wherein the mutated enzyme is stable at a temperature of at least 60° C., and has increased enzyme activity at a lower temperature than the activity of the corresponding wild-type enzyme at its optimal temperature.
A method of producing a thermostable enzyme, said enzyme being stable at a temperature of at least 60° C. and having activity at a temperature of less than 60° C. that is higher than the corresponding activity of a wild-type enzyme, comprising:
- (a) inserting nucleic acid isolated from a thermostable organism into an expression vector, said nucleic acid encoding at least one enzyme which is stable at a temperature of at least 60° C.;
- (b) preparing an expression library containing the isolated nucleic acid of step (a) in a suitable host cell;
- (c) subjecting the library to random mutagenesis;
- and
- (d) screening mutants produced in (c) for a mutated enzyme or for a polynucleotide encoding a mutated enzyme, wherein the mutated enzyme is stable at a temperature of at least 60° C. and has increased enzyme activity at a lower temperature than the activity of a corresponding wild-type enzyme at its optimal temperature.
The method according to clam 36, wherein the mutated enzyme is a hydrolase or epoxide hydrolase.
A method of producing an enzyme that is stable at temperatures of at least 60° C., said enzyme having higher enzyme activity at a temperature lower than 60° C., compared with the activity of a corresponding wild-type enzyme at its optimal temperature, comprising:
- (a) isolating from a thermophilic organism at least one polynucleotide encoding a desired enzyme which is stable at a temperature of at least 60° C.;
- introducing the at least one polynucleotide of step (a) into a host organism having at least one deletion in its nucleic acid repair genes so as to mutate the at least one introduced polynucleotide encoding the desired enzyme so as to produce a mutated enzyme;
- and
- (c) screening nucleic acid or expression products from the host organism for a mutated enzyme, or for at least one polynucleotide encoding a mutated enzyme, said enzyme being stable at a temperature of at least 60° C. and having increased enzyme activity at a lower temperature than the activity of a corresponding wild-type enzyme at its optimal temperature.
Dependencies are inferred from the claim text.