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EP-3768835-B1 - SUBTILASE VARIANTS AND COMPOSITIONS COMPRISING SAME

EP3768835B1EP 3768835 B1EP3768835 B1EP 3768835B1EP-3768835-B1

Inventors

  • FRIIS, ESBEN, PETER
  • LENHARD, Rolf, Thomas
  • CHRISTENSEN, LARS, LEHMANN, HYLLING
  • BAUER, Carl, Mikael

Dates

Publication Date
20260506
Application Date
20190321

Claims (16)

  1. A subtilase variant, comprising mutations at position 209 in combination with two or more mutations at positions selected from 63, 215 and 217, wherein the mutation in position 209 is L209W, the mutation in position 63 is S63G or S63A, the mutation in position 215 is G215A, and the mutation in position 217 is Y217L, Y217I, Y217V or Y217M, wherein positions are numbered according to SEQ ID NO: 1, wherein the variant has protease activity and a sequence identity to SEQ ID NO: 1 of at least 80% and less than 100%, and wherein the variant has an increased storage stability in a detergent composition compared to the subtilase of SEQ ID NO: 1.
  2. The subtilase variant of claim 1, comprising the mutations L209W in combination with S63G/A, G215A and Y217L/I/V/M.
  3. The subtilase variant of claim 2, comprising the substitutions S63G, L209W, G215A and Y217L.
  4. The subtilase variant of claim 3, further comprising at least one mutation selected from the group consisting of S9E/D, N76D/E, Y104V/I/L/M, G128S/A/T, G131*, G131P, S204D/E, Q206L/I/V/M, A216V/I/L/M, F261W/N/Y and Y262E/D.
  5. The subtilase variant of claim 4, comprising one of the following sets of mutations: • S9E S63G L209W G215A Y217L • S63G N76D L209W G215A Y217L • S63G S204D L209W G215A Y217L • S63G Q206L L209W G215A A216V Y217L • S63G Q206L L209W G215A Y217L • S63G L209W G215A Y217L F261W Y262E • S63G Y104V G128S G131* L209W G215A Y217L • S63G Y104V G128S G131P L209W G215A Y217L
  6. The subtilase variant of any of the preceding claims, wherein the variant has at least 85% sequence identity to SEQ ID NO: 1.
  7. The subtilase variant of claim 6, wherein the variant has at least 90% sequence identity to SEQ ID NO: 1.
  8. The subtilase variant of any of the preceding claims, wherein the variant has an increased storage stability in a detergent composition compared to the subtilase of SEQ ID NO: 1 when measured for 24 hours as described in storage stability assay I in Example 2 herein, and/or as measured as described in storage stability assay II in Example 3 herein.
  9. The subtilase variant of claim 8, wherein the variant has an increased storage stability in a detergent composition compared to the subtilase of SEQ ID NO: 1 of at least 25%, preferably at least 50%, more preferably at least 75%, such as at least 100%, when measured for 24 hours as described in storage stability assay I in Example 2 herein, and/or as measured as described in storage stability assay II in Example 3 herein.
  10. A method for stabilizing a subtilase variant, the method comprising introducing into a parent subtilase having protease activity and at least 80% sequence identity to SEQ ID NO: 1 the mutation L209W in combination with two or more substitutions selected from S63G/A, G215A and Y217L/I/V/M.
  11. The method of claim 10, wherein the stabilized subtilase variant has an increased storage stability in a detergent composition compared to the subtilase of SEQ ID NO: 1.
  12. The method of claim 11, wherein the variant has an increased storage stability in a detergent composition compared to the subtilase of SEQ ID NO: 1 when measured for 24 hours as described in storage stability assay I in Example 2 herein, and/or as measured as described in storage stability assay II in Example 3 herein.
  13. The method of claim 12, wherein the variant has an increased storage stability in a detergent composition compared to the subtilase of SEQ ID NO: 1 of at least 25%, preferably at least 50%, more preferably at least 75%, such as at least 100%, when measured for 24 hours as described in storage stability assay I in Example 2 herein, and/or as measured as described in storage stability assay II in Example 3 herein.
  14. A method for obtaining a subtilase variant, the method comprising: (a) providing a host cell comprising a polynucleotide encoding a variant of a parent protease having the mutations L209W and two or more mutations selected from S63G/A, G215A and Y217L/I/V/M compared to SEQ ID NO: 1, wherein the variant has protease activity and a sequence identity to SEQ ID NO: 1 of at least 80%, and wherein the variant has an increased storage stability in a detergent composition compared to the subtilase of SEQ ID NO: 1; (b) cultivating the host cell under conditions suitable for expression of the variant; and (c) recovering the variant.
  15. A detergent composition comprising a subtilase variant according to any of claims 1-9 and at least one detergent component, and optionally further comprising at least one additional enzyme.
  16. Use of the composition of claim 15 in a cleaning process, such as laundry or hard surface cleaning such as dish wash.

Description

Reference to a Sequence Listing This application contains a sequence listing in computer readable form, which is incorporated herein by reference. FIELD OF THE INVENTION The present invention relates to subtilase variants, compositions comprising the variants, polynucleotides encoding the variants, methods of producing the variants, and methods of using the variants. BACKGROUND OF THE INVENTION Subtilisins are serine proteases from the family S8, in particular from the subfamily S8A, as defined by the MEROPS database (https://www.ebi.ac.uk/merops/index.shtml). In subfamily S8A the key active site residues Asp, His and Ser are typically found in motifs that differ from those of the S8B subfamily. Subtilisin BPN' (also known under the acronym BASBPN) from Bacillus amyloliquefaciens has the MEROPS numbers S08.034 and is a member of the S8A subfamily. In the detergent industry, enzymes have for many decades been implemented in washing formulations. Enzymes used in such formulations comprise proteases, lipases, amylases, cellulases, mannosidases as well as other enzymes or mixtures thereof. Commercially, the most important enzymes are proteases. An increasing number of commercially used proteases for e.g. laundry and dishwashing detergents are protein engineered variants of naturally occurring wild type proteases, Further, other subtilase variants have been described in the art with alterations relative to a parent subtilase resulting in improvements such as better wash performance, thermal stability, storage stability or catalytic activity. However, various factors make further improvement of proteases advantageous. For example, washing conditions such as temperature and pH tend to change over time, and are also different in different countries or regions of the world, and many stains are still difficult to completely remove under conventional washing conditions. Another challenge in detergent compositions is enzyme stability, since the chemical components of these compositions as well as conditions of pH, temperature and humidity often tend to inactivate enzymes. Further, in-wash conditions can also result in inactivation of the enzymes (due to e.g. pH, temperature or chelation instability), resulting in loss of wash performance during the wash cycle. Thus, despite the intensive research in protease development there remains a need for new and improved proteases that have improved stability, for example improved storage stability, e.g. in a detergent composition, and which at the same time have similar or improved wash performance compared to the parent subtilase. The present invention addresses these challenges by providing subtilase variants with improved stability. EP1321513A1 and US2003/0157645A1 describe subtilisin variants with improved characteristics. Almog et al. reports the crystal structures of two thermally stabilized subtilisin BPN' variants (Journal of Biological Chemistry, vol. 277, no. 30, pp. 27553-27558, 2002). US2017/0305963A1 describes peptide fragment condensation and cyclisation using a subtilisin variant with improved synthesis over hydrolysis ratio. US6312936B1 describes multiple substituted protease variants. US6440717 B1 describes subtilisin variants with improved hydrolysis compared the wildtype enzyme. WO95/30010 A1 describes subtilisin variants with improved properties. WO89/09830 A1 describes mutated subtilisin variants having increased thermal stability. SUMMARY OF THE INVENTION The present invention relates to subtilase variants comprising mutations at position 209 in combination with two or more mutations at positions selected from 63, 215 and 217, wherein the mutation in position 209 is L209W, the mutation in position 63 is S63G or S63A, the mutation in position 215 is G215A, and the mutation in position 217 is Y217L, Y217I, Y217V or Y217M, wherein positions are numbered according to SEQ ID NO: 1, wherein the variant has protease activity and a sequence identity to SEQ ID NO: 1 of at least 80% and less than 100%, and wherein the variant has an increased storage stability in a detergent composition compared to the subtilase of SEQ ID NO: 1. The present invention also relates to compositions comprising the variants, in particular detergent compositions, polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of producing the variants. The present invention further relates to methods for stabilizing a subtilase variant and for producing a subtilase variant. DEFINITIONS Subtilase/protease: The terms "subtilase" and "protease" may be used interchangeably herein and refer to an enzyme that hydrolyses peptide bonds in proteins. This includes any enzyme belonging to the EC 3.4 enzyme group (including each of the thirteen subclasses thereof), and in particular endopeptidases (EC 3.4.21). The EC number refers to Enzyme Nomenclature 1992 from NC-IUBMB, Academic Press, San Diego, California, including supplements