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Subtilase Variants (19) & (11) EP 2 333 055 A1 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.: 15.06.2011 Bulletin 2011/24 C12N 9/54 (2006.01) C11D 3/386 (2006.01) C12N 1/15 (2006.01) C12N 1/19 (2006.01) (2006.01) (21) Application number: 10180093.6 C12N 1/21 (22) Date of filing: 12.10.2001 (84) Designated Contracting States: (72) Inventors: AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU • Nørregaard-Madsen, Mads MC NL PT SE TR DK-3560, Birkerød (DK) • Larsen, Line, Bloch (30) Priority: 13.10.2000 DK 200001528 DK-2880, Bagsværd (DK) • Hansen, Peter Kamp (62) Document number(s) of the earlier application(s) in DK-4320, Lejre (DK) accordance with Art. 76 EPC: 01978206.9 / 1 326 966 Remarks: This application was filed on 27-09-2010 as a (71) Applicant: Novozymes A/S divisional application to the application mentioned 2880 Bagsvaerd (DK) under INID code 62. (54) Subtilase variants (57) Novel subtilase variants having a reduced ten- dry detergent compositions and dishwash composition, dency towards inhibition by substances present in eggs, including automatic dishwash compositions. Also, isolat- such as trypsin inhibitor type IV- 0. In particular, variants ed DNA sequences encoding the variants, expression comprising at least one additional amino acid residue vectors, host cells, and methods for producing and using between positions 42-43, 51-56, 155-161, 187-190, the variants of the invention. Further, cleaning and de- 216-217, 217-218 or 218-219 (in BASBPN numbering). tergent compositions comprising the variants are dis- These subtilase variants are useful exhibiting excellent closed. or improved wash performance on egg stains when used in e.g. cleaning or detergent compositions, such as laun- EP 2 333 055 A1 Printed by Jouve, 75001 PARIS (FR) EP 2 333 055 A1 Description TECHNICAL FIELD 5 [0001] The present invention relates to novel subtilase variants having a reduced tendency towards inhibition by substances present in eggs, such as trypsin inhibitor type IV-0. In particular, the present invention relates to novel subtilase variants where the variants comprise at least one additional amino acid residue between positions 42-43, 51-55, 155-160, 187-189, 217-218 or 218-219 (in subtilisin BPN’ (BASBPN) numbering,vide infra). These subtilase variants are useful exhibiting excellent or improved wash performance on egg stains when used in e.g. cleaning or 10 detergent compositions, such as laundry detergent compositions and dishwash composition, including automatic dish- wash compositions. The present invention also relates to isolated DNA sequences encoding the variants, expression vectors, host cells, and methods for producing and using the variants of the invention. Further, the present invention relates to cleaning and detergent compositions comprising the variants of the invention. 15 BACKGROUND OF THE INVENTION [0002] In the detergent industry enzymes have for more than 30 years been implemented in washing formulations. Enzymes used in such formulations comprise proteases, lipases, amylases, cellulases, as well as other enzymes, or mixtures thereof. Commercially most important enzymes are proteases. 20 [0003] An increasing number of commercially used proteases are protein engineered variants of naturally occurring wild type proteases, e.g. DURAZYM® (Novozymes A/S), RELASE ® (Novozymes A/S), MAXAPEM ® PURAFECT® (Ge- nencor International, Inc.). [0004] Further, a number of protease variants are described in the art. A thorough list of prior art protease variants is given in WO 99/27082. 25 [0005] However, even though a number of useful proteases and protease variants have been described, there is still a need for new improved proteases or protease variants for a number of industrial uses. [0006] In particular, the problem of removing egg stains from e.g. laundry or hard surfaces has been pronounced due to the fact that substances present in the egg white inhibit many serine proteases. Examples of such substances include trypsin inhibitor type IV-0 (Ovo-inhibitor) and trypsin inhibitor type III-0 (Ovomucoid). 30 [0007] Therefore, an object of the present invention is to provide improved subtilase enzymes, which are not, or which are only to a limited extent, inhibited by such substances. A further object of the present invention is to provide improved subtilase enzymes that are suitable for removal of egg stains from, for example, laundry and/or hard surfaces. SUMMARY OF THE INVENTION 35 [0008] Thus, in a first aspect the present invention relates to a subtilase variant selected from the group consisting of a subtilase variant comprising an insertion of at least one additional amino acid residue between positions 42 and 43 (BASBPN numbering); a subtilase variant comprising an insertion of at least one additional amino acid residue between positions 51 and 56 40 (BASBPN numbering); a subtilase variant comprising an insertion of at least one additional amino acid residue between positions 155 and 161 (BASBPN numbering); a subtilase variant comprising an insertion of at least one additional amino acid residue between positions 187 and 190 (BASBPN numbering); 45 a subtilase variant comprising an insertion of at least one additional amino acid residue between positions 216 and 217 (BASBPN numbering); and a subtilase variant comprising an insertion of at least one additional amino acid residue between positions 217 and 218 (BASBPN numbering); and a subtilase variant comprising an insertion of at least one additional amino acid residue between positions 218 and 219 50 (BASBPN numbering). [0009] In a second aspect the present invention relates to an isolated nucleic acid sequence comprising a nucleic acid sequence that encodes for a subtilase variant according to the invention. [0010] In a third aspect the present invention relates to a nucleic acid construct comprising the nucleic acid sequence according to the invention operably linked to one or more control sequences capable of directing the expression of the 55 subtilase in a suitable host. [0011] In a fourth aspect the present invention relates to a recombinant expression vector comprising the nucleic acid construct according to the invention, a promoter, and transcriptional and translational stop signals. [0012] In a fifth aspect the present invention relates to a recombinant host cell comprising the nucleic acid construct 2 EP 2 333 055 A1 of the invention. [0013] In a sixth aspect the present invention relates to a method for producing a subtilase variant according to the invention, the method comprising: 5 (a) cultivating a recombinant host cell according to the invention under conditions conducive to the production of the subtilase; and (b) recovering the subtilase variant. 10 [0014] In a seventh aspect the present invention relates to a method for producing a subtilase variant according to the invention, the method comprising: (a) cultivating a strain from the genus Bacillus, preferably from the species Bacillus clausii, such as Bacillus clausii DSM 13585, to produce a supernatant comprising the subtilase variant; and 15 (b) recovering the subtilase variant. [0015] In an eight aspect the present invention relates to a cleaning or detergent composition, preferably a laundry or dishwash composition, comprising a subtilase variant according to the invention. 20 [0016] Further aspects of the present invention relate to use of the subtilases according to the invention in a cleaning or detergent composition; use of the subtilases or the compositions according to the invention for removal of egg stains; a method for cleaning or washing, including a method for removal of egg stains from, a hard surface or laundry comprising contacting the hard surface or the laundry with the composition of the invention. [0017] In a seventh aspect the present invention relates to a method for removal of egg stains from a hard surface or 25 from laundry, the method comprising contacting the egg stain- containing hard surface or the egg stain- containing laundry with a cleaning or detergent composition, preferably a laundry or dishwash composition, which contains a subtilase variant according to the invention. [0018] Concerning alignment and numbering reference is made to Fig. 1 which shows an alignments between subtilisin BPN’(a) (BASBPN)(SEQ ID NO:7) and subtilisin 309 (b) (BLSAVI)(SEQ ID NO:8). 30 [0019] These alignments are in this patent application used as a reference for numbering the residues. DEFINITONS [0020] Prior to discussing this invention in further detail, the following terms and conventions will first be defined. 35 NOMENCLATURE OF AMINO ACIDS AND NUCLEIC ACIDS [0021] Throughout the specification, figures and claims the recognized IUPAC nomenclature for amino acid residues will be used, mostly in the one letter code form, but also in the three letter code form. Similarly recognized IUPAC 40 nomenclature for nucleic acids will be used throughout the specification, figures and claims. NOMENCLATURE AND CONVENTIONS FOR DESIGNATION OF VARIANTS [0022] In describing the various subtilase enzyme variants produced or contemplated according to the invention, the 45 following nomenclatures and conventions have been adapted for ease of reference: A frame of reference is first defined by aligning the isolated or parent enzyme with subtilisin BPN’ (BASBPN). [0023] The alignment can be obtained by the GAP routine of the GCG package version 9.1 to number the variants 50 using the following parameters: gap creation penalty = 8 and gap extension penalty = 8 and all other parameters kept at their default values. Another method is to use known recognized alignments between subtilases, such as the alignment indicated in WO 91/00345. In most cases the differences will not be of any importance. [0024] Thereby a number of deletions and insertions will be defined in relation to BASBPN. In Fig. 1, subtilisin 309 has 6 deletions in positions 36, 58, 158, 162, 163, and 164 in comparison to BASBPN. These deletions are in Fig. 1 55 indicated by asterixes (*).
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