| PATENT ASSIGNEE'S COUNTRY | Denmark. |
| UPDATE | 11.99 |
| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | 02.11.99 |
| PATENT TITLE |
Method of preparing a variant of a lipolytic enzyme |
| PATENT 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. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | 22.08.96 |
| PATENT FOREIGN APPLICATION PRIORITY DATA | This data is not available for free |
| PATENT REFERENCES CITED | Abstract--Dialog; File 155, Medline, Dialog Accession No. 06804243. |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
We claim: 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. 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. 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. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
FIELD OF THE INVENTION The present invention relates to a method of preparing a variant of a parent lipolytic enzyme and to variants prepared by the method. Furthermore, the invention relates to a DNA construct encoding a variant of the invention, an expression vector and host cell comprising the DNA construct and a detergent additive or a detergent composition comprising a variant. BACKGROUND OF THE INVENTION For a number of years lipolytic enzymes have been used as detergent enzymes, i.e., to remove lipid or fatty stains from clothes and other textiles. For instance, various microbial lipases have been suggested as detergent enzymes. Examples of such lipases include a Humicola lanuginosa lipase, e.g., described in EP 258 068 and EP 305 216, a Rhizomucor miehei lipase, e.g., as described in EP 238 023, a Candida lipase, such as a C. antarctica lipase, e.g., the C. antarctica lipase A or B described in EP 214 761, a Pseudomonas lipase such as a P. alcaligenes and P. pseudoalcaligenes lipase, e.g., as described in EP 218 272, a P. cepacia lipase, e.g., as described in EP 331 376, a Bacillus lipase, e.g., a B. subtilis lipase (Dartois et al., 1993), a B. stearothermophilus lipase (JP 64/744992) and a B. pumilus lipase (EP 91 00664). Furthermore, a number of cloned lipases have been described, including the Penicillium camembertii lipase described by Yamaguchi, S. et al., 1991, the Geotricum candidum lipase (Schimada, Y. et al., 1989), and various Rhizopus lipases such as a R. delemar lipase (Hass, M. J et al., 1991), a R. niveus lipase (Kugimiya, W. 1992), and a R. oryzae lipase. Other types of lipolytic enzymes having been suggested as detergent enzymes include cutinases, e.g., derived from Pseudomonas mendocina as described in WO 88/09367, or a cutinase derived from Fusarium solani pisi (e.g., described in WO 90/09446). In recent years attempts have been made to prepare lipase variants having improved properties for detergent purposes. For instance, WO 92/05249 discloses lipase variants with improved properties, in which certain characteristics of wild-type lipase enzymes have been changed by specific, i.e., site-directed modifications of their amino acid sequences. More specifically, lipase variants are described, in which one or more amino acid residues of the so-called lipid contact zone of the parent lipase has been modified. PCT/DK93/00225 describes lipase variants with improved properties, in which an amino acid residue occupying a critical position of the lipase has been modified. EP 407 225 discloses lipase variants with improved resistance towards proteolytic enzymes, which have been prepared by specifically defined amino acid modifications. EP 260 105 describe hydrolases in which an amino acid residue within 15 .ANG. from the active site has been substituted. All of the above mentioned lipase variants have been constructed by use of site-directed mutagenesis resulting in a modification of specific amino acid residues which have been chosen either on the basis of their type or on the basis of their location in the secondary or tertiary structure of the parent lipase. An alternative approach for constructing mutants or variants of a given protein has been based on random mutagenesis. For instance, U.S. Pat. No. 4,898,331 and WO 93/01285 disclose such techniques. A need exists for novel lipolytic enzymes having improved washing and/or dishwashing properties, and the object of the present invention is to prepare such enzymes. BRIEF DISCLOSURE OF THE INVENTION The present inventors have now developed a novel method of preparing variants of lipolytic enzymes having improved washing and/or dishwashing performance as compared to their parent enzymes. The method is based on random or localized random mutagenesis of DNA sequences encoding a lipolytic enzyme. More specifically, in a first aspect the invention relates to a method of preparing a variant of a parent lipolytic enzyme, which method comprises (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 dependence to calcium and/or an improved tolerance towards a detergent or one or more detergent components as compared to the parent lipolytic enzyme. In the present context, the term "lipolytic enzyme" is intended to indicate an enzyme exhibiting a lipid degrading capability, such as a capability of degrading a triglycerid or a phospholipid. The lipolytic enzyme may, e.g., be a lipase, a phospholipase, an esterase or a cutinase. The term "random mutagenesis" is intended to be understood in a conventional manner, i.e., to indicate an introduction of one or more mutations at random positions of the parent enzyme (i.e., as opposed to site-specific mutagenesis). The random mutations are typically introduced by exposing a large number of copies of the DNA sequence to be modified to a mutagen and then screening for the presence of variants. Suitable techniques for introducing random mutations are discussed in detail below. The screening criteria of step c) are considered to be of particular use in identifying variants of parent lipolytic enzymes having improved washing and/or dishwashing performance as compared to their parent enzymes. In the present context, the term "decreased dependence to calcium" is intended to mean that the mutated lipolytic enzyme requires lower amounts of calcium for exhibiting the same degree of activity as the parent enzyme when tested under similar conditions. Preferably, the mutated lipolytic enzyme of the invention is substantially independant of the presence of calcium for exhibiting enzymatic activity. The term "improved tolerance towards a detergent or detergent component" is intended to mean that the mutated lipolytic enzyme is active at higher concentrations of the detergent or detergent component than the parent lipolytic enzyme. In the present context the term "detergent" is intended to indicate a mixture of detergent ingredients normally used for washing or dishwashing. Analogously, a "detergent component" is intended to indicate a component or ingredient normally found in detergent or dishwashing compositions, examples of which are given in the following description. It will be understood that the variant prepared by the method of the invention in addition to the decreased dependency to calcium and/or improved tolerance towards a detergent or one or more detergent components exhibits lipolytic activity preferably of a magnitude comparable to or exceeding that of the parent lipolytic enzyme, when tested under washing and/or dishwashing conditions. The screening criteria defined in step c) of the method of the invention may be determined by any suitable methods known in the art. A particular suitable assay developed for the present purpose is described in the Materials and Methods section below. In a further aspect the invention relates to a DNA construct comprising a mutated DNA sequence encoding a variant of a 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, which DNA sequence is isolated from the host cell selected in step (c) of the method of the invention. In a still further aspect the invention relates to a recombinant expression vector carrying the DNA construct, a cell which is transformed with the DNA construct or the vector as well as a method of producing the variant of the parent lipolytic enzyme by culturing said cell under conditions conducive to the production of the variant, after which the variant is recovered from the culture. In final aspects the invention relates to a variant of a lipolytic enzyme and the use of said variant as a detergent enzyme, in particular for washing or dishwashing, and to a detergent additive and a detergent composition comprising the variant. |
| PATENT PHOTOCOPY | Available on request |
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