LIPASE VARIANTS AND COMPOSITIONS COMPRISING SURFACTANT AND LIPASE VARIANT

Abstract

The present invention relates to lipase variants and compositions comprising (i) at least one surfactant and (ii) at least one lipase variant of the invention. Furthermore, the present invention relates to methods of using the compositions.

Claims

1. A lipase variant of a parent lipase, wherein the parent lipase is SEQ ID NO: 1, wherein said variant: (a) comprises a modification in at least one position corresponding to positions E1, V2, N33, F51, E56, L69, K98, V176, H198, E210, Y220, L227, and K237 of SEQ ID NO: 1; and optionally further comprises a modification in at least one position corresponding to positions D27, G38, D96, D111, G163, T231, N233, D254, and P256 of SEQ ID NO: 1; (b) has a sequence identity of at least 75% but less than 100% to SEQ ID NO: 1; and (c) has lipase activity.

2. The lipase variant according to claim 1, comprising modification in at least one of the following positions: E1, V2, D27, N33, G38, F51, E56, L69, D96, K98, D111, G163, V176, H198, E210, Y220, L227, T231, N233, K237, D254, and P256 of SEQ ID NO: 1.

3. The lipase variant according to claim 1, comprising at least one of the following substitutions: E1C, V2Y, D27R, N33K, N33Q, G38A, F51V, E56K, L69R, D96E, D96L, K98I, K98Q, D111A, G163K, V176L, H198S, E210K, Y220F, L227G, T231R, N233R, N233C, K237C, D254S, and P256T of SEQ ID NO: 1.

4. The lipase variant according to claim 1 comprising one of the following substitutions: S54T, S83T, G91A, A150G, I255A, and E239C of SEQ ID NO: 1.

5-6. (canceled)

7. The lipase variant according to claim 1, comprising substitutions E1C+N233C, and one or more additional substitutions.

8. The lipase variant according to claim 1, wherein the variant comprises or consists of one of the following sets of substitutions of SEQ ID NO: 1: E1C+H198L+N233C, E1C+H198G+N233C, E1C+L69V+N233C, E1C+L69T+N233C, E1C+L69S+N233C, E1C+L69H+N233C, E1C+L69F+N233C, E1C+L69C+N233C, E1C+H198Y+N233C, E1C+H198T+N233C, E1C+H198G+N233C, E1C+L227F+N233C, E1C+L227R+N233C, E1C+E210T+N233C, E1C+E210N+N233C, E1C+V176M+N233C, E1C+K98T+N233C, E1C+K98E+N233C, E1C+E56S+N233C, E1C+E56Q+N233C, E1C+E56R+N233C, E1C+F51M+N233C, E1C+D27R+F51Y+N233C, E1C+V2I+N233C, E1C+V2N+N233C, E1C+V2K+N233C, E1C+V2A+N233C, E1C+D96L+N233C, E1C+L69R+N233C, E1C+V2Y+N233C, E1C+N233C+P256T, E1C+N233C+D254S, E1C+T231R+N233C, E1C+H198S+N233C, E1C+D111A+N233C, E1C+D96E+N233C, E1C+G38A+N233C, E1C+N33Q+N233C, E1C+N33K+N233C, E1C+E210A+N233C, E1C+E210Q+N233C, E1C+E210R+N233C, E1C+H198D+N233C, E1C+K98R+N233C, E1C+K98V+N233C, E1C+F51L+N233C, E1C+F51I+N233C, E1C+K237C, E1C+L227G+N233C, E1C+E210K+N233C, E1C+V176L+N233C, E1C+K98Q+N233C, E1C+E56K+N233C, E1C+L147S+N233C+D254S, E1C+Y220F+N233C, E1C+K98I+N233C, E1C+N233C, E1C+D27R+F51I+E56R+K98E+T231R+N233C, E1C+D27R+F51I+E56R+K98E+T231R+N233C+D254S, E1C+D27R+G38A+F51L+K98I+D111A+G163S+H198S+Y220F+T231R+N233C+P256T, E1C+D27R+G38A+F51L+D96E+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+D254S+P256T, E1C+D27R+G38R+F51L+D96E+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+D254S+P256T, E1C+D27R+F51L+D96I+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+P256T, E1C+D27R+F51L+D96E+K98I+D111A+G163S+H198S+Y220F+T231R+N233C+P256T, E1C+D27R+F51L+D96E+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+P256T, E1C+D27R+G38A+F51I+D96E+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+D254S+P256T, E1C+D27R+G38A+F51V+D96E+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+P256T, E1C+D27R+F51V+D96E+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+P256T, E1C+D27R+F51V+D96E+K98I+D111A+G163S+H198S+Y220F+T231R+N233C+D254S+P256T, E1C+D27R+F51V+D96I+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+D254S+P256T, E1C+D27R+F51V+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+D254S+P256T, E1C+D27R+F51V+D96E+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+D254S+P256T, E1C+D27R+G38A+F51V+D96E+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+D254S+P256T, E1C+F51V+D96E+K98I+D111A+G163S+H198S+Y220F+T231R+N233C+P256T, E1C+F51L+D96E+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+D254S+P256T, E1C+G38A+F51V+D96E+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+P256T, E1C+F51V+D96E+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+P256T, E1C+F51V+D96I+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+D254S+P256T, E1C+F51V+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+D254S+P256T, E1C+F51I+D96E+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+D254S+P256T, E1C+D27R+F51L+D96E+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+D254S+P256T, E1C+D27R+N33K+G38A+F51V+D96E+K98I+D111A+G163K+H198S+Y220F+T231R+N233C, E1C+G38R+F51V+D96E+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+D254S+P256T, E1C+F51V+D96E+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+D254S+P256T, E1C+G38A+F51V+D96E+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+D254S+P256T, and E1C+D27R+G38R+F51V+D96E+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+D254S+P256T.

9. (canceled)

10. A composition comprising (i) at least one surfactant and (ii) a lipase variant of a parent lipase, wherein the parent lipase is SEQ ID NO: 1, wherein said variant: (a) comprises a modification in at least one position corresponding to positions E1, V2, N33, F51, E56, L69, K98, V176, H198, E210, Y220, L227, and K237 of SEQ ID NO: 1; and optionally further comprises a modification in at least one position corresponding to positions D27, G38, D96, D111, G163, T231, N233, D254, and P256 of SEQ ID NO: 1; (b) has a sequence identity of at least 75% but less than 100% to SEQ ID NO: 1; (c) has lipase activity; and (d) has an improved activity as compared to the parent lipase.

11. (canceled)

12. The composition according to claim 10, wherein said at least one surfactant comprises an anionic surfactant, including alkylbenzene sulfonate (LAS) or alcohol ether sulfate (AEOS).

13. The composition according to claim 10, wherein a first surfactant is an anionic surfactant and a second surfactant is a non-ionic surfactant.

14. The composition according to claim 10, comprising linear alkylbenzene sulfonate (LAS) surfactant and a lipase variant comprising one of the following sets of substitutions of SEQ ID NO: 1: E1C+H198T+N233C, E1C+H198G+N233C, E1C+L227F+N233C, E1C+E210T+N233C, E1C+E210N+N233C, E1C+V176M+N233C, E1C+K98T+N233C, E1C+K98E+N233C, E1C+E56S+N233C, E1C+E56Q+N233C, E1C+E56R+N233C, E1C+F51M+N233C, E1C+D27R+F51Y+N233C, E1C+V2I+N233C, E1C+V2N+N233C, E1C+V2K+N233C, E1C+V2A+N233C, E1C+D96L+N233C, E1C+L69R+N233C, E1C+V2Y+N233C, E1C+N233C+P256T, E1C+N233C+D254S, E1C+H198S+N233C, E1C+D111A+N233C, E1C+D96E+N233C, E1C+G38A+N233C, E1C+N33Q+N233C, E1C+N33K+N233C, E1C+V176L+N233C, E1C+E56K+N233C, E1C+E210A+N233C, E1C+E210Q+N233C, E1C+E210R+N233C, E1C+K98R+N233C, E1C+K98V+N233C, E1C+F51L+N233C, E1C+F51I+N233C, E1C+L227G+N233C, E1C+E210K+N233C, E1C+K98Q+N233C, E1C+Y220F+N233C, E1C+H198G+N233C, E1C+L69V+N233C, E1C+L69T+N233C, E1C+L69S+N233C, E1C+L69H+N233C, E1C+L69F+N233C, E1C+L69C+N233C, E1C+K237C, E1C+K98I+N233C, E1C+D27R+F51 I+E56R+K98E+T231R+N233C, E1C+D27R+F51 I+E56R+K98E+T231R+N233C+D254S, E1C+D27R+G38A+F51L+K98I+D111A+G163S+H198S+Y220F+T231R+N233C+P256T, and E1C+D27R+G38A+F51L+D96E+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+D254S+P256T.

15. The composition according to claim 10, comprising alcohol ether sulfate (AEOS) surfactant and a lipase variant comprising one of the following sets of substitutions of SEQ ID NO: 1: E1C+H198Y+N233C, E1C+H198T+N233C, E1C+H198G+N233C, E1C+H198S+N233C, E1C+H198L+N233C, and E1C+H198G+N233C.

16. The composition according to claim 10, comprising linear alkylbenzene sulfonate (LAS) surfactant and alcohol ethoxylate (AEO) surfactant, and a lipase variant comprising one of the following sets of substitutions of SEQ ID NO: 1: E1C+H198Y+N233C, E1C+H198T+N233C, E1C+H198G+N233C, E1C+L227F+N233C, E1C+L227R+N233C, E1C+E210T+N233C, E1C+E210N+N233C, E1C+V176M+N233C, E1C+K98T+N233C, E1C+K98E+N233C, E1C+E56S+N233C, E1C+E56Q+N233C, E1C+E56R+N233C, E1C+F51M+N233C, E1C+D27R+F51Y+N233C, E1C+V2I+N233C, E1C+V2N+N233C, E1C+V2K+N233C, E1C+V2A+N233C, E1C+D96L+N233C, E1C+L69R+N233C, E1C+V2Y+N233C, E1C+N233C+P256T, E1C+N233C+D254S, E1C+T231R+N233C, E1C+H198S+N233C, E1C+D111A+N233C, E1C+D96E+N233C, E1C+G38A+N233C, E1C+N33Q+N233C, E1C+N33K+N233C, E1C+V176L+N233C, E1C+E56K+N233C, E1C+E210A+N233C, E1C+E210Q+N233C, E1C+E210R+N233C, E1C+H198D+N233C, E1C+K98R+N233C, E1C+K98V+N233C, E1C+F51L+N233C, E1C+F51I+N233C, E1C+L227G+N233C, E1C+E210K+N233C, E1C+K98Q+N233C, E1C+Y220F+N233C, E1C+H198L+N233C, E1C+H198G+N233C, E1C+L69V+N233C, E1C+L69T+N233C, E1C+L69S+N233C, E1C+L69H+N233C, E1C+L69F+N233C, E1C+L69C+N233C, E1C+K237C, E1C+K98I+N233C, E1C+D27R+F51 I+E56R+K98E+T231R+N233C, E1C+D27R+F51 I+E56R+K98E+T231R+N233C+D254S, E1C+D27R+G38A+F51L+K98I+D111A+G163S+H198S+Y220F+T231R+N233C+P256T, E1C+D27R+G38A+F51L+D96E+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+D254S+P256T, E1C+D27R+G38R+F51L+D96E+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+D254S+P256T, E1C+D27R+F51L+D96I+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+P256T, E1C+D27R+F51L+D96E+K98I+D111A+G163S+H198S+Y220F+T231R+N233C+P256T, E1C+D27R+F51L+D96E+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+P256T, E1C+D27R+G38A+F51I+D96E+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+D254S+P256T, E1C+D27R+G38A+F51V+D96E+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+P256T, E1C+D27R+F51V+D96E+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+P256T, E1C+D27R+F51V+D96E+K98I+D111A+G163S+H198S+Y220F+T231R+N233C+D254S+P256T, E1C+D27R+F51V+D96I+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+D254S+P256T, E1C+D27R+F51V+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+D254S+P256T, E1C+D27R+F51V+D96E+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+D254S+P256T, E1C+D27R+G38A+F51V+D96E+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+D254S+P256T, E1C+F51V+D96E+K98I+D111A+G163S+H198S+Y220F+T231R+N233C+P256T, E1C+G38A+F51V+D96E+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+P256T, E1C+F51V+D96E+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+P256T, E1C+F51V+D96I+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+D254S+P256T, E1C+F51V+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+D254S+P256T, E1C+F51I+D96E+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+D254S+P256T, E1C+D27R+F51L+D96E+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+D254S+P256T, E1C+D27R+N33K+G38A+F51V+D96E+K98I+D111A+G163K+H198S+Y220F+T231R+N233C, E1C+G38R+F51V+D96E+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+D254S+P256T, E1C+F51V+D96E+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+D254S+P256T, E1C+G38A+F51V+D96E+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+D254S+P256T, and E1C+D27R+G38R+F51V+D96E+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+D254S+P256T.

17-18. (canceled)

19. The composition according to claim 10, further comprising a chelating agent, including EDTA, at a concentration between 0.001 and 100 mM.

20. The composition according to claim 10, comprising linear alkylbenzene sulfonate (LAS) surfactant, alcohol ethoxylate (AEO) surfactant, and a chelating agent, including EDTA, and a lipase variant comprising one of the following sets of substitutions of SEQ ID NO: 1: E1C+E210T+N233C, E1C+E210N+N233C, E1C+E56R+N233C, E1C+D96L+N233C, E1C+N233C+D254S, E1C+E56K+N233C, E1C+E210A+N233C, E1C+E210Q+N233C, E1C+E210R+N233C, E1C+E210K+N233C, E1C+D27R+F51 I+E56R+K98E+T231R+N233C, E1C+D27R+F51 I+E56R+K98E+T231R+N233C+D254S, E1C+D27R+G38A+F51L+D96E+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+D254S+P256T, E1C+D27R+G38R+F51L+D96E+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+D254S+P256T, E1C+D27R+F51L+D96I+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+P256T, E1C+D27R+G38A+F51I+D96E+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+D254S+P256T, E1C+D27R+F51V+D96E+K98I+D111A+G163S+H198S+Y220F+T231R+N233C+D254S+P256T, E1C+D27R+F51V+D96I+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+D254S+P256T, E1C+D27R+F51V+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+D254S+P256T, E1C+D27R+F51V+D96E+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+D254S+P256T, E1C+D27R+G38A+F51V+D96E+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+D254S+P256T, E1C+F51V+D96I+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+D254S+P256T, E1C+F51V+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+D254S+P256T, E1C+F51I+D96E+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+D254S+P256T, E1C+D27R+F51L+D96E+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+D254S+P256T, E1C+G38R+F51V+D96E+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+D254S+P256T, E1C+F51V+D96E+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+D254S+P256T, E1C+G38A+F51V+D96E+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+D254S+P256T, and E1C+D27R+G38R+F51V+D96E+K98I+D111A+G163K+H198S+Y220F+T231R+N233C+D254S+P256T.

21. The composition according to claim 10, comprising linear alkylbenzene sulfonate (LAS) surfactant, alcohol ethoxylate (AEO) surfactant, and a lipase variant comprising one of the following sets of substitutions of SEQ ID NO: 1: E1C+H198Y+N233C, E1C+H198T+N233C, E1C+H198G+N233C, E1C+H198S+N233C, E1C+H198L+N233C, and E1C+H198G+N233C.

22. The composition according to claim 10, further comprising at least one additional enzyme, including an amylase, protease, cellulase, another lipase, beta-glucanase, or mannanase.

23. The composition according to claim 10, further comprising at least one chelating agent; at least one polymer; at least one hydrotope; at least one builder or co-builder; at least one perfume; or at least one bleaching system.

24-26. (canceled)

27. A method of laundering, comprising laundering a fabric with a composition according to claim 10 at a temperature of 50° C. or less.

28. A method of pre-treating a fabric with a composition according to claim 10, comprising the steps of adding said composition to said fabric, and leaving the composition on the fabric for a period of time, and rinsing off said composition from said fabric.

Description

EXAMPLES

Example 1—Assays

[0307] p-Nitrophenyl (PNP) Assay (General Lipase Activity Assay):

[0308] The hydrolytic activity of a lipase may be determined by a kinetic assay using p-nitrophenyl acyl esters as substrate.

[0309] A 100 mM stock solution in DMSO of the substrates: p-Nitrophenyl butyrate (C4), p-Nitrophenyl caproate (C6), p-Nitrophenyl caprate (C10), p-Nitrophenyl laurate (C12) and p-Nitrophenyl palmitate (C16) (all from Sigma-Aldrich Danmark A/S, Kirkebjerg Allé 84, 2605 Brøndby; Cat. no.: C4:N-9876, C6: N-0502, C10: N-0252, C12: N-2002, C16: N-2752) may be diluted to a final concentration of 1 mM 25 into assay buffer (50 mM Tris; pH 7.7; 0.4% TritonX-100).

The lipase of the invention, the parent lipase and appropriate controls e.g. Buffer (negative), Lipolase™ & Lipex™ (positive) in 50 mM Hepes; pH 8.0; 10 ppm TritonX-100; +/−20 mM CaCl.sub.2 may be added to the substrate solution in the following final concentrations: 0.01 mg/ml; 5×10−3 mg/ml; 2.5×10−4 mg/ml; and 1.25×10−4 mg/ml in 96-well NUNC plates (Cat. No:260836, Kamstrupvej 90, DK-4000, Roskilde). Release of p-nitrophenol by hydrolysis of p-nitrophenyl acyl may be monitored at 405 nm for 5 minutes in 10 second intervals on a Spectra max 190 (Molecular Devices GmbH, Bismarckring 39, 88400 Biberach an der Riss, GERMANY). The hydrolytic activity towards one or more substrates of a variant may be compared to that of the parent lipase.

Relative Wash Performance (RP(Wash))

[0310] Washing experiments may be performed using Automatic Mechanical Stress Assay (AMSA) in order to assess the wash performance in laundry. The AMSA plate has a number of slots for test solutions and a lid firmly squeezing the laundry sample, the textile to be washed against all the slot openings. During the washing time, the plate, test solutions, textile and lid are vigorously shaken to bring the test solution in contact with the textile and apply mechanical stress in a regular, periodic oscillating manner. For further description see WO02/42740 especially the paragraph Special method embodiments (at page 23-24.

[0311] The laundry experiments are conducted under the experimental conditions specified below:

TABLE-US-00019 TABLE 1 Experimental conditions Detergent: 3.3 g/L Detergent B Test solution volume: 160 uL Wash time: 20 minutes Temperature: 30° C. Lipase dosage: 0 ppm or 0.35 ppm Test material: Cream Annatto stained EMPA221 cotton textile prepared as described in WO06/125437 except to exchanging turmeric with annatto (Annatto: A-320-WS, Chr. Hansen A/S, Boege Alle′, 10-12 DK-2970, Hoersholm, Denmark & EMPA221: EMPA, Lerchenfeldstrasse 5, CH-9014, St. Gallen, Switzerland)

[0312] Water hardness is adjusted to 15° dH by addition of CaCl.sub.2, MgCl.sub.2 and NaHCO.sub.3 (Ca.sup.2+:Mg.sup.2+:HCO.sub.3.sup.−=4:1:7.5).

TABLE-US-00020 TABLE 2 Model detergent B Composition Detergent B wt % NaOH, pellets (>99%) 1.05 Linear alkylbenzene sulfonic acid (LAS) (97%) 7.20 Sodium laureth sulfate (SLES) (28%) 10.58 Soy fatty acid (>90%) 2.75 Coco fatty acid (>99%) 2.75 Alcohol ethoxylate (AEO) with 8 mol EO; Lutensol TO 8 6.60 (~100%) Triethanol amine (100%) 3.33 Na-citrate, dihydrate (100%) 2.00 DTMPA; 0.48 diethylenetriaminepentakis(methylene)pentakis(phosphonic acid), heptasodium salt (Dequest 2066 C) (~42% as Na7 salt) MPG (>98%) 6.00 EtOH, propan-2-ol (90/10%) 3.00 Glycerol (>99.5) 1.71 Sodium formate (>95%) 1.00 PCA (40% as sodium salt) 0.46 Water up to 100

TABLE-US-00021 TABLE 3 Model detergent A Content of active compound Compound (% w/w) LAS 12.0 AEOS, SLES 6.6 Soy fatty acid 2.6 Coco fatty acid 2.6 AEO 10.3 Sodium hydroxide 1.9 Ethanol/Propan-2-ol 2.5/0.3 MPG 11.9 Glycerol 1.9 TEA 2.8 Sodium formate 0.9 Sodium citrate 1.9 DTMPA (as Na.sub.7-salt) 0.06 PCA (as Na-salt) 0.17 Ion exchanged water 38.1
Water hardness may be adjusted to 15° dH by addition of CaCl2, MgCl2, and NaHCO.sub.3 (Ca.sup.2+:Mg.sup.2+:HCO3.sup.−=4:1:7.5) to the test system. After washing the textiles are flushed in tap water and dried.

TABLE-US-00022 TABLE 4 Model detergent X Content of Active compound component Compound (% w/w) (% w/w) LAS 16.5 91 AEO* 2 99.5 Sodium carbonate 20 100 Sodium (di)silicate 12 82.5 Zeolite A 15 80 Sodium sulfate 33.5 100 PCA 1 100 *Model detergent X is mixed without AEO. AEO is added separately before wash. Water hardness may be adjusted to 12° dH by addition of CaCl2, MgCl2, and NaHCO3 (Ca.sup.2+:Mg.sup.2+:HCO3.sup.− = 2:1:4.5) to the test system. After washing the textiles are flushed in tap water and dried.

TABLE-US-00023 TABLE 5 Model detergent Z Content of % active compound component Compound (% w/w) (% w/w) LAS 7.0 85.3 Soap 1.1 93 AEO* 1.5 99.5 Soda ash 20.1 99.5 Hydrous sodium silicate 10.0 80.1 Zeolite A 5.0 80 Sodium citrate 2.0 100 HEDP-Na.sub.4 0.2 84 Polyacrylate 1.1 92 Sodium sulfate 52.0 100 *Model detergent Z is mixed without AEO. AEO is added separately before wash. Water hardness may be adjusted to 15° dH by addition of CaCl2, MgCl2, and NaHCO3 (Ca.sup.2+:Mg.sup.2+:HCO3.sup.− = 4:1:7.5) to the test system. After washing the textiles are flushed in tap water and dried. pH was adjusted with 4M NaOH.

TABLE-US-00024 TABLE 6 Model detergent Z with bleach Content of % active compound component Compound (% w/w) (% w/w) LAS 7.0 85.3 Soap 1.1 93 AEO* 1.5 99.5 Soda ash 20.1 99.5 Hydrous sodium silicate 10.0 80.1 Zeolite A 5.0 80 Sodium citrate 2.0 100 HEDP-Na.sub.4 0.2 84 Polyacrylate 1.1 92 Sodium percarbonate 9.3 86 TEAD 1.1 91.8 Sodium sulfate 41.6 100 *Model detergent Z is mixed without AEO. AEO is added separately before wash.

TABLE-US-00025 TABLE 7 Model detergent A′ Content of active Compound compound (% w/w) LAS 11.6 AEOS, SLES 4.9 Soy fatty acid 2.7 Coco fatty acid 2.8 AEO 11 Sodium hydroxide 1.8 Ethanol/Propan-2-ol 2.7/0.3 MPG 6 Glycerol 1.7 TEA 3.3 Sodium formate 1 Sodium citrate 2 DTMPA (as Na.sub.7-salt) 0.2 PCA (as Na-salt) 0.18 Ion exchanged water 33.6

[0313] Water hardness may be adjusted to 15° dH by addition of CaCl2, MgCl2, and NaHCO3 (Ca.sup.2+:Mg.sup.2+:HCO3.sup.−=4:1:7.5) to the test system. After washing the textiles are flushed in tap water and dried.

[0314] After washing the textiles are flushed in tap water and excess water was removed from the textiles using filter paper and immediately thereafter the textiles are dried at 100° C. for 15 minutes.

[0315] The wash performance is measured as the color change of the washed soiled textile. The soil may be cream mixed with annatto. Annatto contains the colorant norbixin, which function as a pH indicator by having pH dependent color change. Lipase activity leads to release of free fatty acids from the cream acylglycerols and this leads to pH decrease and thereby color change of the norbixin pH indicator. Lipase wash performance can therefore be expressed as the extent of color change of light reflected-emitted from the washed soiled textile when illuminated with white light.

[0316] Color measurements are made with a professional flatbed scanner (EPSON EXPRESSION 10000XL, Atea A/S, Lautrupvang 6, 2750 Ballerup, Denmark), which is used to capture an image of the washed soiled textile. To extract a value for the light intensity from the scanned images, 24-bit pixel values from the image were converted into values for red, green and blue (RGB).

[0317] Color change due to lipase activity was measured as the change in the reflection-emitting of green light (G) relative to the light intensity value (Int) calculated as:

Int=R.sup.2+G.sup.2+B.sup.2

The relative wash performance (RP(Wash)) of a lipase relative to a reference lipase was calculated as:

RP(Wash)=(G/Int(variant)−G/Int(no enzyme))/(G/Int(lipase ref.)−G/Int(no enzyme)).

[0318] A lipase is considered to exhibit improved wash performance, if it performs better than the reference (RP(Wash)>1). In the context of the present invention the reference enzyme is a lipase having a single amino acid substitution relative to the tested lipase.

Odor Detection by Solid Phase Micro Extraction Gas Chromatograph Measurements.

[0319] The butyric acid release (odor) from the lipase washed swatches are measured by Solid Phase Micro Extraction Gas Chromatography (SPME-GC) using the following method.

[0320] Cream Annatto stained EMPA221 cotton textile is washed as specified above and after wash, excess water is removed from the textile using filter paper and the textile is thereafter dried at 25° C. for 2 h. Each SPME-GC measurement was performed with four pieces of the washed and dried textile (5 mm in diameter), which are transferred to a Gas Chromatograph (GC) vial and the vial was closed. The samples are incubated at 30° C. for 24 hours and subsequently heated to 140° C. for 30 minutes and stored at 20° C.-25° C. for at least 4 hours before analysis. The analyses are performed on a Varian 3800 GC equipped with a Stabilwax-DA w/Integra-Guard column (30m, 0.32 mm ID and 0.25 um df) and a Carboxen PDMS SPME fiber (85 micro-m). Sampling from each GC vial is done at 50° C. for 8 minutes with the SPME fiber in the head-space over the textile pieces and the sampled compounds are subsequently injected onto the column (injector temperature=250° C.). Column flow=2 ml helium/minute. Column oven temperature gradient: 0 minute=50° C., 2 minutes=50° C., 6 minutes 45 seconds=240° C., 11 minutes 45 seconds=240° C. Detection is done using a Flame Ionization Detector (FID) and the retention time for butyric acid is identified using an authentic standard.

[0321] The relative odor release (RP(Odor)) of a lipase is the ratio between the amount butyric acid released (peak area) from a lipase washed swatch and the amount butyric acid released (peak area) from a reference lipase washed swatch, after both values will be corrected for the amount of butyric acid released (peak area) from a non-lipase washed swatch (blank). The reference lipase is a polypeptide having a single amino acid substitution relative to the tested lipase. The relative odor performance (RP(Odor)) of the polypeptide is calculated in accordance with the below formula:

RP(Odor)=(odor(variant)−odor(no enzyme))/(odor(lipase ref.)−odor(no enzyme))

[0322] Where odor is the measured butyric acid (peak area) released from the textile surface.

Alpha-Amylase Activity Assay—pNP-G7 Assay

[0323] The alpha-amylase activity may be determined by a method employing the G7-pNP substrate. G7-pNP which is an abbreviation for 4,6-ethylidene(G.sub.7)-p-nitrophenyl(G.sub.1)-α,D-maltoheptaoside, a blocked oligosaccharide which can be cleaved by an endo-amylase, such as an alpha-amylase. Following the cleavage, the alpha-Glucosidase included in the kit digest the hydrolysed substrate further to liberate a free PNP molecule which has a yellow color and thus can be measured by visible spectophometry at λ=405 nm (400-420 nm.). Kits containing G7-pNP substrate and alpha-Glucosidase is manufactured by Roche/Hitachi (cat. No. 11876473).

Reagents:

[0324] The G7-pNP substrate from this kit contains 22 mM 4,6-ethylidene-G7-pNP and 52.4 mM HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid), pH 7.0).

The alpha-Glucosidase reagent contains 52.4 mM HEPES, 87 mM NaCl, 12.6 mM MgCl.sub.2, 0.075 mM CaCl.sub.2, ≥4 kU/L alpha-glucosidase).

[0325] The substrate working solution is made by mixing 1 mL of the alpha-Glucosidase reagent with 0.2 mL of the G7-pNP substrate. This substrate working solution is made immediately before use. Dilution buffer: 50 mM MOPS, 0.05% (w/v) Triton X100 (polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether (C.sub.14H.sub.22O(C.sub.2H4O).sub.n (n=9-10))), 1 mM CaCl.sub.2, pH8.0.

Procedure:

[0326] The amylase sample to be analyzed is diluted in dilution buffer to ensure the pH in the diluted sample is 7. The assay is performed by transferring 20 μl diluted enzyme samples to 96 well microtiter plate and adding 80 μl substrate working solution. The solution is mixed and pre-incubated 1 minute at room temperature and absorption is measured every 20 sec. over 5 minutes at OD 405 nm.

[0327] The slope (absorbance per minute) of the time dependent absorption-curve is directly proportional to the specific activity (activity per mg enzyme) of the alpha-amylase in question under the given set of conditions. The amylase sample should be diluted to a level where the slope is below 0.4 absorbance units per minute.

Alpha-Amylase Activity Assay—Phadebas Activity Assay

[0328] The alpha-amylase activity may also be determined by a method using the Phadebas substrate (from for example Magle Life Sciences, Lund, Sweden). A Phadebas tablet includes interlinked starch polymers that are in the form of globular microspheres that are insoluble in water. A blue dye is covalently bound to these microspheres. The interlinked starch polymers in the microsphere are degraded at a speed that is proportional to the alpha-amylase activity. When the alpha-amylase degrades the starch polymers, the released blue dye is water soluble and concentration of dye can be determined by measuring absorbance at 620 nm. The concentration of blue is proportional to the alpha-amylase activity in the sample.

[0329] The alpha-amylase sample to be analyzed is diluted in activity buffer with the desired pH. Two substrate tablets are suspended in 5 mL activity buffer and mixed on magnetic stirrer. During mixing of substrate transfer 150 μl to microtiter plate (MTP) or PCR-MTP. Add 30 μl diluted amylase sample to 150 μl substrate and mix. Incubate for 15 minutes at 37° C. The reaction is stopped by adding 30 μl 1M NaOH and mix. Centrifuge MTP for 5 minutes at 4000×g. Transfer 100 μl to new MTP and measure absorbance at 620 nm.

[0330] The alpha-amylase sample should be diluted so that the absorbance at 620 nm is between 0 and 2.2, and is within the linear range of the activity assay.

Alpha-Amylase Activity Assay—Amylazyme Activity Assay

[0331] The alpha-amylase activity may also be determined by a method using the Amylazyme substrate (Megazyme® Amylazyme Test, supplied by Megazyme for the assay of cereal and bacterial amylases) comprising AZCL-amylose, which has been mixed with lactose and magnesium stearate and tabletted. A blue dye is covalently bound to these microspheres. The interlinked amylose polymers in the microsphere are degraded at a speed that is proportional to the alpha-amylase activity. When the alpha-amylase degrades the starch polymers, the released blue dye is water soluble and concentration of dye may be determined by measuring absorbance at 590 nm. The concentration of blue is proportional to the alpha-amylase activity in the sample.

[0332] The alpha-amylase sample to be analyzed is diluted in activity buffer with the desired pH. Two substrate tablets are suspended in 5 mL activity buffer and mixed on magnetic stirrer. During mixing of substrate 150 μl is transferred to a microtiter plate (MTP) or PCR-MTP. Next, 25 μl diluted amylase sample is added to 150 μl substrate and mixed. The mixture is incubated for 10 minutes at 37° C. The reaction is stopped by adding 25 μl 1M NaOH and mixed. MTP is centrifuged for 5 minutes at 4000×g, followed by transferring 100 μl to a new MTP and absorbance is measured at 590 nm.

Protease Activity Assays:

1) Suc-AAPF-pNA Activity Assay:

[0333] The proteolytic activity can be determined by a method employing the Suc-AAPF-PNA substrate. Suc-AAPF-PNA is an abbreviation for N-Succinyl-Alanine-Alanine-Proline-Phenylalanine-p-Nitroanilide, and it is a blocked peptide which can be cleaved by endo-proteases. Following cleavage a free PNA molecule is liberated and it has a yellow colour and thus can be measured by visible spectrophotometry at wavelength 405 nm. The Suc-AAPF-PNA substrate is manufactured by Bachem (cat. no. L1400, dissolved in DMSO).

[0334] The protease sample to be analyzed was diluted in residual activity buffer (100 mM Tris pH8.6). The assay was performed by transferring 60 μl of diluted enzyme samples to 96 well microtiter plate and adding 140 μl substrate working solution (0.72 mg/ml in 100 mM Tris pH8.6). The solution was mixed at room temperature and absorption is measured every 20 sec. over 5 minutes at OD 405 nm.

[0335] The slope (absorbance per minute) of the time dependent absorption-curve is directly proportional to the specific activity (activity per mg enzyme) of the protease in question under the given set of conditions. The protease sample should be diluted to a level where the slope is linear.

Assay for Measurement of Free Calcium Ions

[0336] The following assay may be used for the measurement of free calcium ions in solution, and thus for the determination of chelating agents (chelants) ability to reduce the concentration of free calcium ions (Ca.sup.2+) from e.g. 2.0 mM to 0.10 mM at pH 8.

Assay Principle:

[0337] Various amounts of chelants are added to a solution of 2.0 mM Ca.sup.2+ and the free Ca.sup.2+ concentration is determined by using a Calcium Ion Selective Electrode at fixed pH and temperature. The concentration of chelant necessary to reduce the concentration of free calcium from 2.0 mM to 0.10 mM can be determined from a plot of the free calcium concentration measured versus the concentration of chelant. In the present assay the concentration of chelant necessary to reduce the concentration of free calcium from 2.0 mM to 0.10 mM is measured at pH 8, at 21° C., in potassium chloride and 49 mM EPPS.

Solutions:

[0338] Electrolyte solution: 4 M potassium chloride in ultrapure water (Milli-Q water). pH 8 buffer: 50 mM EPPS (4-(2-Hydroxyethyl)piperazine-1-propanesulfonic acid) adjusted to pH 8.0 using minimum amounts of 1 N sodium hydroxide.

Calcium stock solution: 25 mM Ca.sup.2+ in pH 8 buffer, made from CaCl.sub.2.2H.sub.2O.
Chelant stock solution: 15 mM chelant (on a 100% dry chelator basis) in pH 8 buffer, re-adjusted to pH 8.0 using minimum amounts of 1 M NaOH or 1 M HCl.
Ultra pure water (Milli Q water) is used for preparation of all buffers and solutions.

Equipment:

[0339] Calcium Ion Selective Electrode from Thermo Scientific (cat. No. 9720BNWP) calibrated against a Calcium chloride standard solution. The electrode is calibrated as described by the guidelines following the electrode.

Procedure:

[0340] A series of vials are prepared, each containing 4 mL of the calcium stock solution (final concentration 2.0 mM), 1 mL electrolyte solution (final concentration 80 mM potassium chloride), chelant stock solution in various amounts (0-45 mL) and using the pH 8 buffer for adjusting the total volume to 50 mL. The final concentration of EPPS in the assay is 49 mM. ° mM free calcium ions to a value below 0.10 mM or the final chelant concentration in the assay is higher than 10.0 mM. A suitable number of data points are 8 or more. The chelant concentration required to lower the initial 2.0 mM free calcium ions to 0.10 mM is obtained from a plot of the measured free calcium ion concentration versus chelator concentration by interpolation.

[0341] The solutions are equilibrated to the desired temperature, which in the present assay is 21° C.

Example 2

Fermentation of Aspergillus Oryzae Transformants Expressing Thermomyces Lanuginosus Lipase Variants of the Invention

[0342] Shake flasks containing 200 ml MDU2 (see WO 1995017513 A1) were inoculate with the desired transformants. The shake flasks were grown for 150 hours shaking at 200 RPM at 30° C. The culture broths were passed on for testing.

Example 3

Flocculation and Centrifugation of Culture Broth Prior to Lipase Activity Testing

[0343] Aliquots of 40 mL culture broths were flocculated with 0.8 mL Aluminium chlorohydrate solution (Magnasol 4710 G from BASF, 30-60%), followed by centrifugation for 10 minutes at 13,000 rpm at 5° C. The supernatants were filtered through 0.45 μm sterile filter (Minisart NML, hydrophile from Sartorius) and stored at −20° C. until use.

Example 4

[0344] Lipase Activity Assay with pNP-Acyl Ester Substrate
This experiment was carried out to determine the lipase activity of lipase variants in the presence of one or more surfactants and other agents (see ‘Surfactant solutions” table below) as a model for wash relevant conditions.
Principle: Enzyme samples were incubated with pNP-acyl ester (pNP-palmitate or -valerate) in detergent solution of desired composition and water hardness at pH 8.0. The hydrolysis the substrate by the lipase generated p-nitrophenol, the formation of which was be followed spectrophotometrically at 405 nm. The rate of formation of p-nitrophenol is a measure of the lipase activity after correction for blank.
Lipase reference: Thermomyces lanuginosus lipase shown as SEQ ID NO: 1 (LIPOLASE™)

Chemicals:

[0345] HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid),
pNP-palmitate
pNP-valerate
AEO: alcohol ethoxylate, Bio-soft N25-7 from Stepan (average Mw 516 g/mol)
LAS: Sodium alkylbenzene sulfonate; Thonyl P 85 from Unger Fabrikker, Norway (average Mw 286 g/mol)
AEOS: Sodium lauryl ether sulfate, SLES 28 from Danlind/Linds (Denmark), (average Mw 385 g/mol)
EDTA: ethylenediaminetetraacetic acid (or 2,2′,2″,2′″-(Ethane-1,2-diyldinitrilo)tetraacetic acid),

Titriplex III

[0346] CaCl.sub.2.2H.sub.2O
NaOH: sodium hydroxide
Ethanol: ≥99.5% ethylalcohol

Reagents:

[0347] HEPES buffer: 56 mM HEPES prepared with ultrapure water (Milli-Q water), adjusted to pH 8.5 with 1 M NaOH
pNP-palmitate stock solution: 10 mM pNP-palmitate in ethanol
pNP-palmitate working solution: 0.33 mM pNP-palmitate in Surfactant solution
pNP-valerate stock solution: 62.7 mM in ethanol
pNP-valerate working solution: 0.33 mM in Surfactant solution
Lipase dilution solution: 0.1 mM AEO
LAS stock solution: 50 mM LAS
AEOS stock solution: 50 mM AEOS
AEO stock solution: 50 mM AEO
EDTA stock solution: 100 mM
Surfactant solutions

TABLE-US-00026 Assay type LAS AEOS LAS:AEO 1.91 1.96 LAS:AEO LAS:AEO LAS:AEO 1.9 mM LAS:AEO mM mM 1.9 mM 0.49 mM 0.19 mM EDTA 1.9 mM Surfactant A B C D E F G solution name Substrate pNP- pNP- pNP- pNP- pNP- pNP- pNP- palmitate valerate palmitate palmitate palmitate palmitate valerate Detergent 1 LAS AEOS LAS LAS LAS LAS LAS Detergent 2 AEO AEO AEO AEO AEO Detergent 1.91 mM 1.96 mM 1.28 mM 0.32 mM 0.13 mM 1.28 mM 1.28 mM conc 1 Detergent 0.64 mM 0.17 mM 0.06 mM 0.64 mM 0.64 mM conc 2 Ca2+   1 mM   1 mM   1 mM   1 mM   1 mM   1 mM EDTA   2 mM Water 5.6 °dH 5.6 °dH 5.6 °dH 5.6 °dH 5.6 °dH 0 °dH 5.6 °dH hardness equivalents pH 8.5 8.5 8.5 8.5 8.5 8.5 8.5 Temp RT RT RT RT RT RT RT Buffer  50 mM  50 mM  50 mM  50 mM  50 mM  50 mM   50 mM Measuring 0-120 sec 300 sec 0-120 sec 0-120 sec 0-120 sec 0-120 sec 300 sec time

Procedure

[0348] 1) Enzyme samples were diluted to 0.2 ppm with Lipase dilution solution. [0349] 2) Substrate working solution was prepared from pNP-palmitate or pNP-valerate stock solution and Surfactant solution [0350] 3) 20 μL diluted enzyme sample (from (1)) was transferred to 96 well microtiter plate 4) 180 μL Substrate working solution (from (2)) was added [0351] 5) Immediately after addition of Substrate working solution, the absorbance was measured at 405 nm every 20 seconds for up to 300 seconds at room temperature (25° C.), with 10 second shaking before first reading. The absorbance was measured on a SpectraMax M2 from Molecular Devices.

[0352] For blanks, diluted enzyme was replaced by 20 μL Lipase dilution solution.

[0353] Each sample was run in triplicates on each plate, which also includes 3 blanks, reference in triplicate in same concentration as enzyme sample, as well as a standard curve made from 0.1, 0.2, 0.4, and 0.6 ppm lipase (SEQ ID NO: 1—LIPOLASE™)

[0354] Activity was calculated as the slope of the plot of OD405 nm versus time and then corrected by subtracting blank. The activities are given relative to the activity of the reference.

[0355] Relative lipase activity in composition comprising surfactant solution A compared to SEQ ID NO: 1

TABLE-US-00027 1.91 mM LAS (pNP-palmitate) 0.02 ppm lipase variant Relative Sample activity to ID Substitutions SEQ ID NO: 1 LP-1 SEQ ID NO: 1 1.00 LP-4 E1C + H198T + N233C 3.08 LP-5 E1C + H198G + N233C 1.77 LP-6 E1C + L227F + N233C 2.65 LP-8 E1C + E210T + N233C 2.31 LP-9 E1C + E210N + N233C 2.33 LP-10 E1C + V176M + N233C 1.88 LP-11 E1C + K98T + N233C 1.88 LP-12 E1C + K98E + N233C 2.09 LP-13 E1C + E56S + N233C 1.94 LP-14 E1C + E56Q + N233C 2.50 LP-15 E1C + E56R + N233C 2.38 LP-16 E1C + F51M + N233C 2.63 LP-17 E1C + D27R + F51Y + N233C 2.82 LP-18 E1C + V2I + N233C 2.38 LP-19 E1C + V2N + N233C 1.16 LP-20 E1C + V2K + N233C 1.33 LP-21 E1C + V2A + N233C 1.89 LP-22 E1C + D96L + N233C 1.68 LP-23 E1C + L69R + N233C 2.17 LP-24 E1C + V2Y + N233C 1.16 LP-25 E1C + N233C + P256T 2.11 LP-26 E1C + N233C + D254S 2.41 LP-28 E1C + H198S + N233C 2.18 LP-29 E1C + D111A + N233C 2.06 LP-30 E1C + D96E + N233C 2.15 LP-31 E1C + G38A + N233C 3.39 LP-32 E1C + N33Q + N233C 3.21 LP-33 E1C + N33K + N233C 3.62 LP-34 E1C + V176L + N233C 3.61 LP-35 E1C + E56K + N233C 3.03 LP-36 E1C + E210A + N233C 2.81 LP-37 E1C + E210Q + N233C 2.93 LP-38 E1C + E210R + N233C 2.99 LP-40 E1C + K98R + N233C 2.97 LP-41 E1C + K98V + N233C 3.17 LP-42 E1C + F51L + N233C 3.45 LP-43 E1C + F51I + N233C 3.16 LP-44 E1C + L227G + N233C 3.82 LP-45 E1C + E210K + N233C 2.70 LP-46 E1C + K98Q + N233C 3.36 LP-47 E1C + Y220F + N233C 2.75 LP-49 E1C + H198G + N233C 2.68 LP-50 E1C + L69V + N233C 3.10 LP-51 E1C + L69T + N233C 3.09 LP-52 E1C + L69S + N233C 3.20 LP-53 E1C + L69H + N233C 3.24 LP-54 E1C + L69F + N233C 3.57 LP-55 E1C + L69C + N233C 2.83 LP-56 E1C + K237C 4.59 LP-58 E1C + K98I + N233C 3.46 LP-61 E1C + D27R + F51I + E56R + K98E + 1.11 T231R +N233C LP-62 E1C + D27R + F51I + E56R + K98E + 1.69 T231R + N233C + D254S LP-63 E1C + D27R + G38A + F51L + K98I + 1.89 D111A + G163S + H198S + Y220F + T231R + N233C + P256T LP-64 E1C + D27R + G38A + F51L + D96E + 2.16 K98I + D111A + G163K + H198S + Y220F + T231R + N233C + D254S + P256T

TABLE-US-00028 1.96 mM AEOS (pNP-valerate) 0.02 ppm lipase variant Relative activity to Sample SEQ ID No Substitutions NO: 1 LP-1 SEQ ID NO: 1 1.00 LP-3 E1C + H198Y + N233C 1.67 LP-4 E1C + H198T + N233C 1.26 LP-5 E1C + H198G + N233C 1.24 LP-28 E1C + H198S + N233C 1.48 LP-48 E1C + H198L + N233C 1.97 LP-49 E1C + H198G + N233C 1.40
Relative lipase activity in composition comprising surfactant solution B compared to SEQ ID NO: 1
Relative lipase activity in composition comprising surfactant solution C compared to SEQ ID NO: 1

TABLE-US-00029 LAS:AEO 2:1, 2 mM Ca2+, 1.9 mM detergent (pNP-palmitate) 0.02 ppm lipase variant Relative activity to Sample SEQ ID No Substitutions NO: 1 LP-1 SEQ ID NO: 1 1.00 LP-3 E1C + H198Y + N233C 6.75 LP-4 E1C + H198T + N233C 5.90 LP-5 E1C + H198G + N233C 5.30 LP-6 E1C + L227F + N233C 6.52 LP-7 E1C + L227R + N233C 1.80 LP-8 E1C + E210T + N233C 8.47 LP-9 E1C + E210N + N233C 7.56 LP-10 E1C + V176M + N233C 4.52 LP-11 E1C + K98T + N233C 5.01 LP-12 E1C + K98E + N233C 5.10 LP-13 E1C + E565 + N233C 6.02 LP-14 E1C + E56Q + N233C 6.36 LP-15 E1C + E56R + N233C 5.90 LP-16 E1C + F51M + N233C 6.46 LP-17 E1C + D27R + F51Y + N233C 6.19 LP-18 E1C + V2I + N233C 6.18 LP-19 E1C + V2N + N233C 2.75 LP-20 E1C + V2K + N233C 2.46 LP-21 E1C + V2A + N233C 5.84 LP-22 E1C + D96L + N233C 6.73 LP-23 E1C + L69R + N233C 5.81 LP-24 E1C + V2Y + N233C 2.99 LP-25 E1C + N233C + P256T 5.54 LP-26 E1C + N233C + D254S 5.54 LP-27 E1C + T231R + N233C 2.62 LP-28 E1C + H198S + N233C 7.10 LP-29 E1C + D111A + N233C 4.75 LP-30 E1C + D96E + N233C 4.28 LP-31 E1C + G38A + N233C 5.83 LP-32 E1C + N33Q + N233C 5.90 LP-33 E1C + N33K + N233C 5.96 LP-34 E1C + V176L + N233C 6.48 LP-35 E1C + E56K + N233C 5.65 LP-36 E1C + E210A + N233C 7.84 LP-37 E1C + E210Q + N233C 8.01 LP-38 E1C + E210R + N233C 7.51 LP-39 E1C + H198D + N233C 2.54 LP-40 E1C + K98R + N233C 6.10 LP-41 E1C + K98V + N233C 5.38 LP-42 E1C + F51L + N233C 7.00 LP-43 E1C + F51I + N233C 5.16 LP-44 E1C + L227G + N233C 4.79 LP-45 E1C + E210K + N233C 6.65 LP-46 E1C + K98Q + N233C 6.02 LP-47 E1C + Y220F + N233C 5.25 LP-48 E1C + H198L + N233C 5.77 LP-49 E1C + H198G + N233C 5.18 LP-50 E1C + L69V + N233C 5.37 LP-51 E1C + L69T + N233C 5.74 LP-52 E1C + L69S + N233C 5.56 LP-53 E1C + L69H + N233C 5.39 LP-54 E1C + L69F + N233C 6.58 LP-55 E1C + L69C + N233C 4.73 LP-56 E1C + K237C 3.15 LP-58 E1C + K98I + N233C 5.30 LP-61 E1C + D27R + F51I + E56R + K98E + T231R + 3.00 N233C LP-62 E1C + D27R + F51I + E56R + K98E + T231R + 3.34 N233C + D254S LP-63 E1C + D27R + G38A + F51L + K98I + D111A + 4.37 G163S + H198S + Y220F + T231R + N233C + P256T LP-64 E1C + D27R + G38A + F51L + D96E + K98I + 5.06 D111A + G163K + H198S + Y220F + T231R + N233C + D254S + P256T LP-65 E1C + D27R + G38R + F51L + D96E + K98I + 4.79 D111A + G163K + H198S + Y220F + T231R + N233C + D254S + P256T LP-66 E1C + D27R + F51L + D96I + K98I + D111A + 5.70 G163K + H198S + Y220F + T231R + N233C + P256T LP-67 E1C + D27R + F51L + D96E + K98I + D111A + 4.98 G163S + H198S + Y220F + T231R + N233C + P256T LP-68 E1C + D27R + F51L + D96E + K98I + D111A + 4.12 G163K + H198S + Y220F + T231R + N233C + P256T LP-69 E1C + D27R + G38A + F51I + D96E + K98I + 4.98 D111A + G163K + H198S + Y220F + T231R + N233C + D254S + P256T LP-70 E1C + D27R + G38A + F51V + D96E + K98I + 4.22 D111A + G163K + H198S + Y220F + T231R + N233C + P256T LP-71 E1C + D27R + F51V + D96E + K98I + D111A + 3.81 G163K + H198S + Y220F + T231R + N233C + P256T LP-72 E1C + D27R + F51V + D96E + K98I + D111A + 5.33 G163S + H198S + Y220F + T231R + N233C + D254S + P256T LP-73 E1C + D27R + F51V + D96I + K98I + D111A + 4.68 G163K + H198S + Y220F + T231R + N233C + D254S + P256T LP-74 E1C + D27R + F51V + K98I + D111A + 4.73 G163K + H198S + Y220F + T231R + N233C + D254S + P256T LP-75 E1C + D27R + F51V + D96E + K98I + D111A + 5.43 G163K + H198S + Y220F + T231R + N233C + D254S + P256T LP-76 E1C + D27R + G38A + F51V + D96E + K98I + 5.07 D111A + G163K + H198S + Y220F + T231R + N233C + D254S + P256T LP-77 E1C + F51V + D96E + K98I + D111A + G163S + 3.99 H198S + Y220F + T231R + N233C + P256T LP-79 E1C + G38A + F51V + D96E + K98I + D111A + 3.96 G163K + H198S + Y220F + T231R + N233C + P256T LP-80 E1C + F51V + D96E + K98I + D111A + 4.33 G163K + H198S + Y220F + T231R + N233C + P256T LP-81 E1C + F51V + D96I + K98I + D111A + G163K + 6.32 H198S + Y220F + T231R + N233C + D254S + P256T LP-82 E1C + F51V + K98I + D111A + G163K + 4.65 H198S + Y220F + T231R + N233C + D254S + P256T LP-83 E1C + F51I + D96E + K98I + D111A + G163K + 5.16 H198S + Y220F + T231R + N233C + D254S + P256T LP-84 E1C + D27R + F51L + D96E + K98I + D111A + 4.78 G163K + H198S + Y220F + T231R + N233C + D254S + P256T LP-85 E1C + D27R + N33K + G38A + F51V + D96E + 4.39 K98I + D111A + G163K + H198S + Y220F + T231R + N233C LP-86 E1C + G38R + F51V + D96E + K98I + D111A + 4.34 G163K + H198S + Y220F + T231R + N233C + D254S + P256T LP-87 E1C + F51V + D96E + K98I + D111A + 5.70 G163K + H198S + Y220F + T231R + N233C + D254S + P256T LP-88 E1C + G38A + F51V + D96E + K98I + D111A + 5.66 G163K + H198S + Y220F + T231R + N233C + D254S + P256T LP-89 E1C + D27R + G38R + F51V + D96E + K98I + D111A + G163K + H198S + Y220F + T231R + N233C + D254S + P256T 5.25
Relative lipase+activity in composition comprising surfactant solution 0 compared to SEQ+ID NO:1

TABLE-US-00030 LAS:AEO 2:1, 2 mM Ca2+, 0.49 mM detergent (pNP-palmitate) 0.02 ppm lipase variant Relative activity to Sample SEQ ID No Substitutions NO: 1 LP-1 SEQ + ID + NO: +1 1.00 LP-3 E1C + H198Y + N233C 5.17 LP-4 E1C + H198T + N233C 1.71 LP-5 E1C + H198G + N233C 1.97 LP-6 E1C + L227F + N233C 3.75 LP-8 E1C + E210T + N233C 9.96 LP-9 E1C + E210N + N233C 8.37 LP-10 E1C + V176M + N233C 2.59 LP-11 E1C + K98T + N233C 2.99 LP-12 E1C + K98E + N233C 3.13 LP-13 E1C + E56S + N233C 3.20 LP-14 E1C + E56Q + N233C 2.89 LP-15 E1C + E56R + N233C 3.92 LP-16 E1C + F51M + N233C 3.38 LP-17 E1C + D27R + F51Y + N233C 4.63 LP-18 E1C + V2I + N233C 3.59 LP-19 E1C + V2N + N233C 2.46 LP-20 E1C + V2K + N233C 2.80 LP-21 E1C + V2A + N233C 3.56 LP-22 E1C + D96L + N233C 3.48 LP-23 E1C + L69R + N233C 2.66 LP-24 E1C + V2Y + N233C 2.47 LP-25 E1C + N233C + P256T 3.25 LP-26 E1C + N233C + D254S 3.76 LP-27 E1C + T231R + N233C 5.84 LP-28 E1C + H198S + N233C 2.70 LP-29 E1C + D111A + N233C 1.91 LP-30 E1C + D96E + N233C 1.68 LP-31 E1C + G38A + N233C 2.30 LP-32 E1C + N33Q + N233C 2.71 LP-33 E1C + N33K + N233C 2.38 LP-34 E1C + V176L + N233C 2.79 LP-35 E1C + E56K + N233C 2.31 LP-36 E1C + E210A + N233C 9.68 LP-37 E1C + E210Q + N233C 7.17 LP-38 E1C + E210R + N233C 7.78 LP-40 E1C + K98R + N233C 2.94 LP-41 E1C + K98V + N233C 2.18 LP-42 E1C + F51L + N233C 2.30 LP-43 E1C + F51I + N233C 2.50 LP-44 E1C + L227G + N233C 1.33 LP-45 E1C + E210K + N233C 6.71 LP-46 E1C + K98Q + N233C 2.93 LP-47 E1C + Y220F + N233C 2.74 LP-48 E1C + H198L + N233C 2.58 LP-49 E1C + H198G + N233C 1.41 LP-50 E1C + L69V + N233C 2.07 LP-51 E1C + L69T + N233C 2.50 LP-52 E1C + L69S + N233C 2.33 LP-53 E1C + L69H + N233C 2.45 LP-54 E1C + L69F + N233C 3.04 LP-55 E1C + L69C + N233C 2.27 LP-56 E1C + K237C 1.49 LP-58 E1C + K98I + N233C 2.16 LP-61 E1C + D27R + F51I + E56R + K98E + T231R + 6.57 N233C LP-62 E1C + D27R + F51I + E56R + K98E + T231R + 11.24 N233C + D254S LP-63 E1C + D27R + G38A + F51L + K98I + D111A + 2.83 G163S + H198S + Y220F + T231R + N233C + P256T LP-64 E1C + D27R + G38A + F51L + D96E + K98I + 3.33 D111A + G163K + H198S + Y220F + T231R + N233C + D254S + P256T LP-65 E1C + D27R + G38R + F51L + D96E + K98I + 3.51 D111A + G163K + H198S + Y220F + T231R + N233C + D254S + P256T LP-66 E1C + D27R + F51L + D96I + K98I + D111A + 9.11 G163K + H198S + Y220F + T231R + N233C + P256T LP-67 E1C + D27R + F51L + D96E + K98I + D111A + 5.19 G163S + H198S + Y220F + T231R + N233C + P256T LP-68 E1C + D27R + F51L + D96E + K98I + D111A + 4.86 G163K + H198S + Y220F + T231R + N233C + P256T LP-69 E1C + D27R + G38A + F51I + D96E + K98I + 4.22 D111A + G163K + H198S + Y220F + T231R + N233C + D254S + P256T LP-70 E1C + D27R + G38A + F51V + D96E + K98I + 3.72 D111A + G163K + H198S + Y220F + T231R + N233C + P256T LP-71 E1C + D27R + F51V + D96E + K98I + D111A + 4.51 G163K + H198S + Y220F + T231R + N233C + P256T LP-72 E1C + D27R + F51V + D96E + K98I + D111A + 4.67 G163S + H198S + Y220F + T231R + N233C + D254S + P256T LP-73 E1C + D27R + F51V + D96I + K98I + D111A + 5.65 G163K + H198S + Y220F + T231R + N233C + D254S + P256T LP-74 E1C + D27R + F51V + K98I + D111A + 4.26 G163K + H198S + Y220F + T231R + N233C + D254S + P256T LP-75 E1C + D27R + F51V + D96E + K98I + D111A + 4.40 G163K + H198S + Y220F + T231R + N233C + D254S + P256T LP-76 E1C + D27R + G38A + F51V + D96E + K98I + 3.93 D111A + G163K + H198S + Y220F + T231R + N233C + D254S + P256T LP-77 E1C + F51V + D96E + K98I + D111A + 2.97 G163S + H198S + Y220F + T231R + N233C + P256T LP-79 E1C + G38A + F51V + D96E + K98I + D111A + 2.26 G163K + H198S + Y220F + T231R + N233C + P256T LP-80 E1C + F51V + D96E + K98I + D111A + 2.93 G163K + H198S + Y220F + T231R + N233C + P256T LP-81 E1C + F51V + D96I + K98I + D111A + G163K + 5.14 H198S + Y220F + T231R + N233C + D254S + P256T LP-82 E1C + F51V + K98I + D111A + G163K + 3.63 H198S + Y220F + T231R + N233C + D254S + P256T LP-83 E1C + F51I + D96E + K98I + D111A + G163K + 3.94 H198S + Y220F + T231R + N233C + D254S + P256T LP-84 E1C + D27R + F51L + D96E + K98I + D111A + 3.83 G163K + H198S + Y220F + T231R + N233C + D254S + P256T LP-85 E1C + D27R + N33K + G38A + F51V + D96E + 2.80 K98I + D111A + G163K + H198S + Y220F + T231R + N233C LP-86 E1C + G38R + F51V + D96E + K98I + D111A + 3.31 G163K + H198S + Y220F + T231R + N233C + D254S + P256T LP-87 E1C + F51V + D96E + K98I + D111A + 3.57 G163K + H198S + Y220F + T231R + N233C + D254S + P256T LP-88 E1C + G38A + F51V + D96E + K98I + D111A + 3.08 G163K + H198S + Y220F + T231R + N233C + D254S + P256T LP-89 E1C + D27R + G38R + F51V + D96E + K98I + 3.45 D111A + G163K + H198S + Y220F + T231R + N233C + D254S + P256T
Relative activity in composition comprising surfactant solution E compared to SEQ ID NO: 1

TABLE-US-00031 LAS:AEO 2:1, 2 mM Ca2+, 0.19 mM detergent (pNP-palmitate) 0.02 ppm lipase variant Relative activity to SEQ Sample ID NO: No Substitutions 1 LP-1 SEQ ID NO: 1 1.00 LP-3 E1C + H198Y + N233C 1.28 LP-6 E1C + L227F + N233C 2.74 LP-8 E1C + E210T + N233C 18.73 LP-9 E1C + E210N + N233C 14.28 LP-10 E1C + V176M + N233C 1.49 LP-11 E1C + K98T + N233C 1.20 LP-12 E1C + K98E + N233C 1.69 LP-13 E1C + E56S + N233C 1.59 LP-14 E1C + E56Q + N233C 2.13 LP-15 E1C + E56R + N233C 2.74 LP-16 E1C + F51M + N233C 2.12 LP-17 E1C + D27R + F51Y + N233C 3.26 LP-18 E1C + V2I + N233C 2.62 LP-19 E1C + V2N + N233C 1.66 LP-20 E1C + V2K + N233C 2.44 LP-21 E1C + V2A + N233C 2.34 LP-22 E1C + D96L + N233C 3.97 LP-23 E1C + L69R + N233C 2.17 LP-24 E1C + V2Y + N233C 1.87 LP-25 E1C + N233C + P256T 2.15 LP-26 E1C + N233C + D254S 5.97 LP-27 E1C + T231R + N233C 10.45 LP-29 E1C + D111A + N233C 1.34 LP-31 E1C + G38A + N233C 1.88 LP-32 E1C + N33Q + N233C 2.46 LP-33 E1C + N33K + N233C 2.20 LP-34 E1C + V176L + N233C 2.61 LP-35 E1C + E56K + N233C 2.61 LP-36 E1C + E210A + N233C 25.77 LP-37 E1C + E210Q + N233C 16.45 LP-38 E1C + E210R + N233C 20.99 LP-40 E1C + K98R + N233C 2.25 LP-41 E1C + K98V + N233C 1.73 LP-42 E1C + F51L + N233C 1.29 LP-43 E1C + F51I + N233C 1.63 LP-45 E1C + E210K + N233C 18.03 LP-46 E1C + K98Q + N233C 2.25 LP-47 E1C + Y220F + N233C 2.51 LP-48 E1C + H198L + N233C 1.85 LP-50 E1C + L69V + N233C 2.17 LP-51 E1C + L69T + N233C 2.12 LP-52 E1C + L69S + N233C 2.29 LP-53 E1C + L69H + N233C 2.42 LP-54 E1C + L69F + N233C 2.85 LP-55 E1C + L69C + N233C 2.06 LP-56 E1C + K237C 1.71 LP-58 E1C + K98I + N233C 1.68 LP-61 E1C + D27R + F51I + E56R + K98E + T231R + 25.40 N233C LP-62 E1C + D27R + F51I + E56R + K98E + T231R + 25.76 N233C + D254S LP-63 E1C + D27R + G38A + F51L + K98I + D111A + 2.95 G163S + H198S + Y220F + T231R + N233C + P256T LP-64 E1C + D27R + G38A + F51L + D96E + K98I + 5.55 D111A + G163K + H198S + Y220F + T231R + N233C + D254S + P256T LP-65 E1C + D27R + G38R + F51L + D96E + K98I + 7.93 D111A + G163K + H198S + Y220F + T231R + N233C + D254S + P256T LP-66 E1C + D27R + F51L + D96I + K98I + D111A + 22.39 G163K + H198S + Y220F + T231R + N233C + P256T LP-67 E1C + D27R + F51L + D96E + K98I + D111A + 8.12 G163S + H198S + Y220F + T231R + N233C + P256T LP-68 E1C + D27R + F51L + D96E + K98I + D111A + 6.23 G163K + H198S + Y220F + T231R + N233C + P256T LP-69 E1C + D27R + G38A + F51I + D96E + K98I + 10.04 D111A + G163K + H198S + Y220F + T231R + N233C + D254S + P256T LP-70 E1C + D27R + G38A + F51V + D96E + K98I + 3.71 D111A + G163K + H198S + Y220F + T231R + N233C + P256T LP-71 E1C + D27R + F51V + D96E + K98I + D111A + 4.97 G163K + H198S + Y220F + T231R + N233C + P256T LP-72 E1C + D27R + F51V + D96E + K98I + D111A + 10.36 G163S + H198S + Y220F + T231R + N233C + D254S + P256T LP-73 E1C + D27R + F51V + D96I + K98I + D111A + 14.61 G163K + H198S + Y220F + T231R + N233C + D254S + P256T LP-74 E1C + D27R + F51V + K98I + D111A + 10.71 G163K + H198S + Y220F + T231R + N233C + D254S + P256T LP-75 E1C + D27R + F51V + D96E + K98I + D111A + 12.15 G163K + H198S + Y220F + T231R + N233C + D254S + P256T LP-76 E1C + D27R + G38A + F51V + D96E + K98I + 9.77 D111A + G163K + H198S + Y220F + T231R + N233C + D254S + P256T LP-77 E1C + F51V + D96E + K98I + D111A + G163S + 2.32 H198S + Y220F + T231R + N233C + P256T LP-79 E1C + G38A + F51V + D96E + K98I + D111A + 1.32 G163K + H198S + Y220F + T231R + N233C + P256T LP-80 E1C + F51V + D96E + K98I + D111A + 2.59 G163K + H198S + Y220F + T231R + N233C + P256T LP-81 E1C + F51V + D96I + K98I + D111A + G163K + 20.02 H198S + Y220F + T231R + N233C + D254S + P256T LP-82 E1C + F51V + K98I + D111A + G163K + 7.79 H198S + Y220F + T231R + N233C + D254S + P256T LP-83 E1C + F511 + D96E + K98I + D111A + G163K + 9.43 H198S + Y220F + T231R + N233C + D254S + P256T LP-84 E1C + D27R + F51L + D96E + K98I + D111A + 10.26 G163K + H198S + Y220F + T231R + N233C + D254S + P256T LP-85 E1C + D27R + N33K + G38A + F51V + D96E + 2.51 K98I + D111A + G163K + H198S + Y220F + T231R + N233C LP-86 E1C + G38R + F51V + D96E + K98I + D111A + 7.57 G163K + H198S + Y220F + T231R + N233C + D254S + P256T LP-87 E1C + F51V + D96E + K98I + D111A + 6.53 G163K + H198S + Y220F + T231R + N233C + D254S + P256T LP-88 E1C + G38A + F51V + D96E + K98I + D111A + 5.26 G163K + H198S + Y220F + T231R + N233C + D254S + P256T LP-89 E1C + D27R + G38R + F51V + D96E + K98I + 6.84 D111A + G163K + H198S + Y220F + T231R + N233C + D254S + P256T
Relative activity in composition comprising surfactant solution F compared to SEQ ID NO: 1

TABLE-US-00032 LAS:AEO 2:1 EDTA 1.9 mM detergent (pNP-palmitate) 0.02 ppm Lipase variant Relative activity to SEQ Sample ID NO: No Substitutions 1 LP-1 SEQ ID NO: 1 1.00 LP-8 E1C + E210T + N233C 2.98 LP-9 E1C + E210N + N233C 2.32 LP-15 E1C + E56R + N233C 2.09 LP-22 E1C + D96L + N233C 1.45 LP-26 E1C + N233C + D254S 1.64 LP-35 E1C + E56K + N233C 1.30 LP-36 E1C + E210A + N233C 2.20 LP-37 E1C + E210Q + N233C 1.77 LP-38 E1C + E210R + N233C 2.26 LP-45 E1C + E210K + N233C 2.13 LP-61 E1C + D27R + F51I + E56R + K98E + T231R + 2.45 N233C LP-62 E1C + D27R + F51I + E56R + K98E + T231R + 2.29 N233C + D254S LP-64 E1C + D27R + G38A + F51L + D96E + K98I + 2.10 D111A + G163K + H198S + Y220F + T231R + N233C + D254S + P256T LP-65 E1C + D27R + G38R + F51L + D96E + K98I + 4.21 D111A + G163K + H198S + Y220F + T231R + N233C + D254S + P256T LP-66 E1C + D27R + F51L + D96I + K98I + D111A + 2.71 G163K + H198S + Y220F + T231R + N233C + P256T LP-69 E1C + D27R + G38A + F51I + D96E + K98I + 3.86 D111A + G163K + H198S + Y220F + T231R + N233C + D254S + P256T LP-72 E1C + D27R + F51V + D96E + K98I + D111A + 4.32 G163S + H198S + Y220F + T231R + N233C + D254S + P256T LP-73 E1C + D27R + F51V + D96I + K98I + D111A + 4.54 G163K + H198S + Y220F + T231R + N233C + D254S + P256T LP-74 E1C + D27R + F51V + K98I + D111A + G163K + 3.19 H198S + Y220F + T231R + N233C + D254S + P256T LP-75 E1C + D27R + F51V + D96E + K98I + D111A + 4.45 G163K + H198S + Y220F + T231R + N233C + D254S + P256T LP-76 E1C + D27R + G38A + F51V + D96E + K98I + 4.14 D111A + G163K + H198S + Y220F + T231R + N233C + D254S + P256T LP-81 E1C + F51V + D96I + K98I + D111A + G163K + 7.01 H198S + Y220F + T231R + N233C + D254S + P256T LP-82 E1C + F51V + K98I + D111A + G163K + H198S + 2.72 Y220F + T231R + N233C + D254S + P256T LP-83 E1C + F51I + D96E + K98I + D111A + G163K + 4.07 H198S + Y220F + T231R + N233C + D254S + P256T LP-84 E1C + D27R + F51L + D96E + K98I + D111A + 4.00 G163K + H198S + Y220F + T231R + N233C + D254S + P256T LP-86 E1C + G38R + F51V + D96E + K98I + D111A + 3.90 G163K + H198S + Y220F + T231R + N233C + D254S + P256T LP-87 E1C + F51V + D96E + K98I + D111A + G163K + 3.44 H198S + Y220F + T231R + N233C + D254S + P256T LP-88 E1C + G38A + F51V + D96E + K98I + D111A + 3.41 G163K + H198S + Y220F + T231R + N233C + D254S + P256T LP-89 E1C + D27R + G38R + F51V + D96E + K98I + 3.54 D111A + G163K + H198S + Y220F + T231R + N233C + D254S + P256T
Relative activity in composition comprising surfactant solution G compared to SEQ ID NO: 1

TABLE-US-00033 LAS:AEO 2:1 1.9 mM detergent (pNP-valerate) 0.02 ppm Lipase variant Relative activity to Sample SEQ ID No Substitutions NO: 1 LP-1 SEQ ID NO: 1 1.00 LP-3 E1C + H198Y + N233C 1.71 LP-4 E1C + H198T + N233C 1.33 LP-5 E1C + H198G + N233C 1.25 LP-28 E1C + H198S + N233C 1.52 LP-48 E1C + H198L + N233C 1.93 LP-49 E1C + H198G + N233C 1.26