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United States Patent (19) 11 Patent Number: 5,869,438 Svendsen Et Al

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United States Patent (19) 11 Patent Number: 5,869,438 Svendsen Et Al USOO5869438A United States Patent (19) 11 Patent Number: 5,869,438 Svendsen et al. (45) Date of Patent: Feb. 9, 1999 54) LIPASE WARIANTS 52 U.S. Cl. ......................... 510/226; 435/198; 435/69.1; 435/252.3; 435/320.1; 435/196; 536/23.2; 75 Inventors: Allan Svendsen, Birkerød; Shamkant 536/23.7; 530/350; 510/392; 510/305 Anant Patkar, Lyngby; Erik Gormsen, 58 Field of Search ..................................... 435/198, 196, Virum; Jens Sigurd Okkels; Marianne 435/187-188, 69.1, 252.3, 320.1, 71.1; Thellersen, both of Frederiksberg, all of 424/94.1; 536/22.2, 23.7; 510/305, 226, Denmark 392 73 Assignee: Novo Nordisk A/S, Bagsvaerd, 56) References Cited Denmark FOREIGN PATENT DOCUMENTS 21 Appl. No.: 479,275 O305 216 A1 3/1989 European Pat. Off.. O 407 225A1 1/1991 European Pat. Off.. 22 Filed: Jun. 7, 1995 WO95/09909 4/1995 WIPO. Related U.S. Application Data Primary Examiner Robert A. Wax ASSistant Examiner Tekchand Saidha 63 Continuation-in-part of PCT/DK94/00162, Apr. 22, 1994, which is a continuation-in-part of PCT/DK95/00079, Feb. Attorney, Agent, or Firm-Steve T. Belson; Elias J. 27, 1995, which is a continuation-in-part of Ser. No. 434, Lambiris 904, May 1, 1995, abandoned, which is a continuation of Ser. No. 977,429, which is a continuation of PCT/DK91/ 57 ABSTRACT 00271, Sep. 13, 1991, abandoned. The present invention relates to lipase variants which exhibit 30 Foreign Application Priority Data improved properties, detergent compositions comprising Said lipase variants, DNA constructs coding for Said lipase Sep. 13, 1990 DK Denmark ................................. 2194/90 Sep. 13, 1990 DK Denmark ...... ... 2195/90 variants, and methods of making Said lipase variants. Sep. 13, 1990 DK Denmark ...... ... 2196/90 Apr. 23, 1993 DK Denmark ...... ... O466/93 51 Claims, 10 Drawing Sheets Feb. 22, 1994 DKI Denmark ................................. O217/94 (51) Int. Cl. ............................................... C11D 3/386 (4 of 10 Drawing Sheet(s) Filed in Color) U.S. Patent Feb. 9, 1999 Sheet 1 of 10 5,869,438 A /O. U.S. Patent Feb. 9, 1999 Sheet 2 of 10 5,869,438 A/ 6. /b U.S. Patent Feb. 9, 1999 Sheet 3 of 10 5,869,438 A /O. 2 U.S. Patent Feb. 9, 1999 Sheet 4 of 10 5,869,438 U.S. Patent Feb. 9, 1999 Sheet 5 of 10 5,869,438 Lineorize plasmid with Unique restriction enzyme 3-step PCR incorporating mutotion EcoRI SOI --- Cleave with restriction enzymes, isolate frogment Reclone into expression plosmid Sequence verification of PCR frogment ACGT A/G 3 U.S. Patent Feb. 9, 1999 Sheet 6 of 10 5,869,438 5' STEPTEP 5' - T Hy-3' 3' — see -5 A H-H- --O U.S. Patent Feb. 9, 1999 Sheet 7 of 10 5,869,438 IIIDUH(999ÞÝ) U.S. Patent Feb. 9, 1999 Sheet 8 of 10 5,869,438 U.S. Patent Feb. 9, 1999 Sheet 9 of 10 5,869,438 Z9/-/ ~TREF></ 000ff 5,869,438 1 2 LPASE WARLANTS EP 260,105 describe hydrolases in which an amino acid residue within 15 A from the active site has been Substituted. CROSS-REFERENCE TO RELATED APPLICATIONS All of the above mentioned lipase variants have been constructed by use of Site-directed mutagenesis resulting in This application is a continuation-in-part of Ser. No. 5 a modification of Specific amino acid residues which have PCT/DK94/00162 filed Apr. 22, 1994, PCT/DK95/00079 been chosen either on the basis of their type or on the basis filed Feb. 27, 1995 and of Ser. No. 08/434,904, filed May 1, of their location in the Secondary or tertiary Structure of the 1995, now abandoned, which is a continuation of Ser. No. parent lipase. 07/977,429 filed Feb. 22, 1993, now abandoned, which is a continuation of PCT/DK91/00271 filed Sep. 13, 1991, An alternative approach for constructing mutants or vari which are incorporated herein by reference. ants of a given protein has been based on random mutagen esis. For instance, U.S. Pat. No. 4,898,331 and WO FIELD OF INVENTION 93/O1285 disclose such techniques. The present invention relates to lipase variants with It is an object of the present invention to prepare lipolytic improved properties, DNA constructs coding for the expres enzymes having improved washing and/or dishwashing Sion of Said variants, host cells capable of expressing the 15 properties. variants, and methods of producing the variants by cultivat ing Said host cells. SUMMARY OF THE INVENTION BACKGROUND OF THE INVENTION The present invention relates to variants of a parent For a number of years lipolytic enzymes have been used lipolytic enzyme which exhibit improved properties, deter in detergents to remove lipid or fatty Stains from clothes and gent compositions comprising Said lipase variants, DNA other textiles. constructs coding for Said lipase variants, and methods of For instance, various microbial lipases have been Sug making Said lipase variants. gested as detergent enzymes. Examples of Such lipases The present invention also relates to a method of prepar include a Humicola lanuginosa lipase, e.g., described in EP 25 ing variants of lipolytic enzymes having improved washing 258,068 and EP305,216, a Rhizomucor miehei lipase, e.g., and/or dishwashing performance as compared to their parent as described in EP 238,023, a Candida lipase, such as a C. enzymes. The method is based on random or localized antarctica lipase, e.g., the C. antarctica lipase A or B random mutagenesis of DNA sequences encoding a lipolytic described in EP 214,761, a Pseudomonas lipase such as a P enzyme. More specifically, this method comprises alcaligenes and P. pseudoalcaligenes lipase, e.g., as (a) Subjecting a DNA sequence encoding the parent described in EP 218,272, a P. cepacia lipase, e.g., as lipolytic enzyme to random mutagenesis, described in EP 331,376, a Bacillus lipase, e.g., a B. Subtilis (b) expressing the mutated DNA sequence obtained in lipase (Dartois et al., Biochemica et Biophysica Acta 1131, Step (a) in a host cell; and pp. 253-260 (1993)), a B. Stearothernophilus lipase (JP 35 (c) Screening for host cells expressing a mutated lipolytic 64/744992) and a B. pumilus lipase (EP 91 00664). enzyme which has a decreased dependence to calcium Furthermore, a number of cloned lipases have been and/or an improved tolerance towards a detergent or described, including the Penicillium camembertii lipase one or more detergent components as compared to the described by Yamaguchi et al., Gene 103, pp. 61-67 (1991), parent lipolytic enzyme. the Geotricum candidumn lipase (Shimada et al., J. Bio 40 chem. 106, 383-88 (1989)), and various Rhizopus lipases BRIEF DESCRIPTION OF THE DRAWINGS Such as a R. delemar lipase (HaSS et al., Gene 109, pp. 107-13 (1991)), a R. niveus lipase (Kugimiya, Biosci. Bio The file of this patent contains at least one drawing tech. Biochem. 56, pp. 716-19 (1992)), and a R. oryzae executed in color. Copies of this patent with color drawing lipase. (s) will be provided by the Patent and Trademark Office The primary Structure of a number of lipases has been 45 upon request and payment of necessary fee. determined and described in the literature (Boel et al., Lipids The present invention is described in the following with 23, pp. 701-06 (1988), de Caro et al., Biochim. BiophyS. reference to the appended drawings, in which: Acta 671, pp. 129-38 (1981), Winkler et al., Nature 343, pp. FIGS. 1A and B are computer models showing the three 771-74 (1990)). Furthermore, the tertiary structure of a dimensional Structure of the lipid contact Zone of the H. more limited number of lipases has been elucidated (Brady 50 lanuginosa lipase when the lipase is in inactive (A) and et al., Nature 343, 767-70 (1990) and Schrag et al., Nature active (B) form, respectively. “White” residues represent 351, pp. 761-64 (1991)). From these investigations it hydrophobic amino acids (Ala, Val, Leu, Ile, Pro, Phe, Trp, appears that lipases Seem to have certain Structural features Gly and Met), “yellow” residues represent hydrophilic in common, but that, on the other hand, major structural amino acids (Thr, Ser, Gln, ASn, Tyr and Cys), “blue” variations also exist among the lipases. 55 residues represent positively charged amino acids (LyS, Arg Other types of lipolytic enzymes include cutinases, e.g., a and His), and “red’ residues represent negatively charged cutinase derived from Pseudomonas mendocina (WO amino acids (Glu and Asp); 88/09367), or from Fusarium Solani pisi (WO 90/09446). FIGS. 2A and 2B are computer models showing the In recent years attempts have been made to prepare lipase 60 three-dimensional Structure of the lipid contact Zone of the variants having improved properties for detergent purposes. Rh. miehei lipase when the lipase is in inactive (A) and PCT/DK93/00225 describes lipase variants with active (B) form, respectively. improved properties, in which an amino acid residue occu FIG. 3 is a Schematic representation of the preparation of pying a critical position of the lipase has been modified. plasmids encoding lipase variants by polymerase chain EP 407.225 discloses lipase variants with improved resis 65 reaction (PCR); tance towards proteolytic enzymes, which have been pre FIG. 4 is a Schematic representation of the three-step pared by Specifically defined amino acid modifications. mutagenesis by PCR; 5,869,438 3 4 FIG. 5 shows a restriction map of plasmid paO1; oligonucleotides encoding multiple mutations by perform FIG. 6 shows a restriction map of plasmid paHL; ing minor alterations of the cassette, however, an even greater variety of mutations can be introduced at any one FIG.
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