Production of enzymes having desired activities by mutagenesis

• US 5,939,250 A
• Filed: 05/22/1996
• Issued: 08/17/1999
• Est. Priority Date: 12/07/1995
• 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): Wax, Robert A.
• Assistant Examiner(s): Nashed, Nashaat T.
• Application Number:
  US08/651,568
• Time in Patent Office: 1,182 Days
• Field of Search: 435/183, 435/189, 435/195, 435/196, 435/197, 435/198, 435/199, 435/207, 435/212, 435/219, 435/4, 435/69.1, 536/23.1, 536/23.2
• US Class Current: 435/4
• 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 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 9/2434 acting on beta-1,4-glucosidic bonds
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Y 301/03001 Alkaline phosphatase (3.1.3.1)
• CPC Class Codes: C12Y 302/01021 Beta-glucosidase (3.2.1.21)

Abstract

Disclosed is a process for obtaining an enzyme having a specified enzyme activity derived from a heterogeneous DNA population by screening, for the specified enzyme activity, a library of clones containing DNA from the heterogeneous DNA population which have been exposed to directed mutagenesis towards production of the specified enzyme activity. Also disclosed is a process for obtaining an enzyme having a specified enzyme activity by screening, for the specified enzyme activity, a library of clones containing DNA from a pool of DNA populations which have been exposed to directed mutagenesis in an attempt to produce in the library of clones DNA encoding an enzyme having one or more desired characteristics which can be the same or different from the specified enzyme activity.

Claims

1. A process for obtaining a specified protein or enzymatic activity of interest encoded by DNA contained in a heterogeneous population of microorganisms comprising:

(a) screening a library containing a plurality of clones containing DNA isolated from a heterogeneous population of microorganisms for a specified protein or enzymatic activity;

(b) isolating a clone which is positive for the specified activity;

(c) introducing at least one mutation into the DNA contained in the clone of (b);

and

(d) comparing the activity of a DNA expression product from (c) with the activity encoded by a non-mutagenized form of the DNA present in (b), wherein a difference in activity is indicative of an effect of introducing at least one mutation, thereby providing the specified protein or enzymatic activity of interest.

2. The process of claim 1, further comprising, prior to (c), obtaining DNA from a clone which is positive for the specified activity.

3. The process of claim 2, wherein obtaining the DNA contained in the clone positive for the specified activity comprises contacting the clone with a complementary nucleic acid, or fragment thereof, thereby allowing hybridization of the clone DNA with the complementary nucleic acid and isolation thereof.

4. The process of claim 3, wherein the complementary nucleic acid or fragment thereof comprises a solid phase bound hybridization probe.

5. The process of claim 1, wherein the mutation is introduced by a method selected from the group consisting of error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis and site-specific mutagenesis.

6. The process of claim 1, comprising screening the heterogeneous population of DNA for a further specified protein or enzymatic activity, prior to exposing the DNA to mutagenesis.

7. A process for obtaining an enzyme having a specified enzyme activity, comprising:

screening a heterogenous population of DNA obtained from a pool of DNA populations containing a plurality of activities and which have been modified by mutagenesis, for an enzyme having one or more desired characteristics which can be the same or different from the specified enzyme activity.

8. The process of claim 7, further comprising, prior to said mutagenesis, obtaining from the heterogeneous population, DNA which comprises DNA sequences coding for at least one common enzyme characteristic, which can be the same or different from the specified enzyme activity.

9. The process of claim 8, wherein obtaining the DNA comprises contacting the population with a complementary nucleic acid, or fragment thereof, thereby allowing hybridization of the clone DNA with the complementary nucleic acid and isolation thereof.

10. The process of claim 9, wherein the complementary nucleic acid, or fragment thereof comprises a solid phase bound hybridization probe.

11. The process of claim 10, comprising obtaining DNA from clones containing DNA from the heterogeneous population which exhibit the class of enzyme activity.

12. The process of claim 7, wherein the mutagenesis is introduced by a method selected from the group consisting of error-prone PCR shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis and site-specific mutagenesis.