Directed evolution of thermophilic enzymes

• US 5,830,696 A
• Filed: 12/05/1996
• Issued: 11/03/1998
• Est. Priority Date: 12/05/1996
• Status: Expired due to Term

• Current Assignee: BP Corporation North America Incorporated (BP PLC)
• Original Assignee: Diversa Corporation (BASF SE)
• Inventors: Short, Jay M.
• Primary Examiner(s): Achutamurthy, Ponnathapura
• Assistant Examiner(s): PARK, HANKYEL
• Application Number:
  US08/760,489
• Time in Patent Office: 698 Days
• Field of Search: 435/69.1, 530/350
• US Class Current: 435/69.1
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/1034 Isolating an individual clone by screening libraries
• CPC Class Codes: C12N 15/1058 Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms
• CPC Class Codes: C12N 9/00 Enzymes; Proenzymes; Compositions thereof preparations containing enzymes for cleaning teeth A61K8/66, A61Q11/00; medicinal preparations containing enzymes or proenzymes A61K38/43; enzyme containing detergent compositions C11D; enzymes with nucleic acid structure, e.g. ribozymes, C12N15/113; Processes for preparing, activating, inhibiting, separating or purifying enzymes preparation of malt C12C1/00
• CPC Class Codes: C12N 9/16 acting on ester bonds (3.1)
• CPC Class Codes: C12N 9/2405 Glucanases
• CPC Class Codes: C12N 9/2468 acting on beta-galactose-glycoside bonds, e.g. carrageenases (3.2.1.83; 3.2.1.157); beta-agarase (3.2.1.81)
• CPC Class Codes: C12N 9/2471 Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
• CPC Class Codes: C12Y 302/01021 Beta-glucosidase (3.2.1.21)
• CPC Class Codes: C12Y 302/01023 Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase

Abstract

Thermostable enzyme 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.

Claims

1. A method for providing a thermostable enzyme having improved enzyme activities as compared to a corresponding wild-type enzyme at lower temperatures, comprising:

(a) subjecting at least one polynucleotide encoding an enzyme which is stable at a temperature of at least 60°

C. to a random mutagenesis; and

(b) screening mutant enzymes produced in (a) 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.;

has an enzyme activity at a temperature at least 10°

C. below its optimal temperature range; and

has activity greater than the enzyme encoded by the polynucleotide of step (a) prior to mutagenesis.

2. The method of claim 1, wherein the enzyme is selected from the group consisting of lipase, esterase, protease, glycosidase, glycosyl transferase, phosphatase, kinase, monoxygenase, dioxygenase, haloperoxidase, lignin peroxidase, diarylpropane peroxidase, epoxide hydrolase, nitrite hydratase, nitrile nitrilase, transaminase, amidase and acylase.