Method of preparing a variant of a lipolytic enzyme

• US 5,976,855 A
• Filed: 08/22/1996
• Issued: 11/02/1999
• Est. Priority Date: 02/22/1994
• Status: Expired due to Term

• Current Assignee: Novozymes A/S (Novozymes)
• Original Assignee: Novo Nordisk A/S
• Inventors: Okkels, Jens Sigurd, Svendsen, Allan, Clausen, Ib Groth, Thellersen, Marianne
• Primary Examiner(s): Wax, Robert A.
• Assistant Examiner(s): Saidha, Tekchand
• Application Number:
  US08/701,339
• Time in Patent Office: 1,167 Days
• Field of Search: 435/198, 435/832, 435/849, 435/874, 435/886, 435/913, 435/471, 435/69.1, 435/252.3, 435/320.1, 536/23.2
• US Class Current: 435/198
• CPC Class Codes: C11D 3/38627 containing lipase
• CPC Class Codes: C11D 3/38663 Stabilised liquid enzyme compositions
• CPC Class Codes: C12N 9/20 Triglyceride splitting, e.g. by means of lipase
• CPC Class Codes: Y10S 435/832 Bacillus
• CPC Class Codes: Y10S 435/849 Escherichia coli
• CPC Class Codes: Y10S 435/874 Pseudomonas
• CPC Class Codes: Y10S 435/886 Streptomyces
• CPC Class Codes: Y10S 435/911 using fungi
• CPC Class Codes: Y10S 435/913 Aspergillus

Abstract

The present invention relates to a method of preparing a variant of a parent lipolytic enzyme, comprising (a) subjecting a DNA sequence encoding the parent lipolytic enzyme to random mutagenesis, (b) expressing the mutated DNA sequence obtained in step (a) in a host cell, and (c) screening for host cells expressing a mutated lipolytic enzyme which has a decreased dependance to calcium and/or an improved tolerance towards a detergent or a detergent component as compared to the parent lipolytic enzyme.

Claims

1. A method of preparing a variant of a final lipolytic enzyme, which comprises

(a) subjecting a DNA sequence encoding the fungal lipolytic enzyme to random mutagenesis,

(b) expressing the mutated DNA sequence obtained in step (a) in a host cell, and

(c) screening for host cells expressing a mutated lipolytic enzyme which requires lower amounts of calcium for exhibiting the same degree of activity.

2. The method according to claim 1, in which the random mutagenesis is performed by use of a physical or a chemical mutagenizing agent, by use of an oligonucleotide or by use of PCR generated mutagenesis.

3. The method according to claim 1, in which the mutagenizing agent is selected from formic acid, UV irradiation, hydroxylamine, N-methyl-N'"'"'-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine, nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, and nucleotide analogues.

4. The method according to claim 1, in which the mutated DNA sequence is expressed by transforming a suitable host cell with the mutated DNA sequence and culturing the host cell obtained in step (b) under suitable conditions for expressing the mutated DNA sequence.

5. The method according to claim 1, in which the host cell is a microbial cell.

6. The method according to claim 5, in which the host cell is a fungal or bacterial cell.

7. The method according to claim 6, in which the host cell is an Aspergillus cell.

8. The method according to claim 6, in which the host cell is a Bacillus cell.

9. The method according to claim 1, in which the variant has an improved tolerance towards a non-ionic, anionic, cationic, zwitterionic or amphoteric surfactant.

10. The method according to claim 9, in which the non-ionic surfactant is an alcohol ethoxylate and the anionic surfactant is a linear alkylbenzenesulfonate or an alkyl sulphate.

11. The method according to claim 1, wherein host cells screened in step (c) are subjected to a second mutagenesis treatment, to rescreening, to reisolation and to recloning.

12. The method according to claim 1, in which the random mutagenesis is localized to a part of the DNA sequence encoding the fungal lipolytic enzyme.

13. The method according to claim 1, in which the fungal lipolytic enzyme is a lipase, esterase, cutinase or phospholipase.

14. The method according to claim 13, in which the parent lipolytic enzyme is a lipase and the localized random mutagenesis is performed on a part of the DNA sequence encoding a lipid contact zone of the lipase or a part thereof.

15. The method according to claim 13, in which the localized random mutagenesis is performed on a part of the DNA sequence encoding a lid region and a hydrophobic cleft of the lipase or a part thereof.

16. The method according to claim 1, wherein the parent lipolytic enzyme is a Humicola sp., Rhizomucor sp., Rhizopus sp., Candida sp. lipolytic enzyme.

17. The method according to claim 16, wherein the parent lipolytic enzyme is a H. lanuginosa, Rh. mucor, or C. antarctica lipase.

18. The method according to claim 17, in which the lipase is obtained from DSM 4109 and the DNA sequence is mutated in at least one of the regions defined by the amino acid residues 21-27, 56-64, 81-99, 108-116, 145-147, 174, 202-213, 226-227, 246-259 or 263-269.

19. The method according to claim 18, in which the localized random mutagenesis is performed in at least two of the regions.

21. The method of claim 1, futher comprising screening for host cells expressing a mutated lipolytic enzyme which has an improved tolerance towards a detergent or a detergent component as compared to the parent lipolytic enzyme.

20. In a method of subjecting a DNA sequence encoding a fungal lipolytic enzyme to random mutagenesis and expressing the mutated DNA sequence in a host cell, the improvement comprising screening for host cells expressing a mutated lipolytic enzyme which requires lower amounts of calcium for exhibiting the same degree of activity than the parent lipolytic enzyme.