DETERGENT COMPOSITIONS

Abstract

The present invention relates to polypeptides having trypsin enzyme activity, detergent composition comprising such polypeptides and uses of the composition and/or polypeptide in methods of washing, such as laundering of textiles.

Claims

1. A detergent composition, comprising a polypeptide having trypsin enzyme activity, wherein the polypeptide is derived from Fusarium and wherein the polypeptide, has at least 80% sequence identity to the mature polypeptide shown in SEQ ID NO: 2 (ii).

2. The detergent composition according to claim 1, wherein the polypeptide is present at a concentration of 0.01-100 ppm enzyme protein.

3. The detergent composition according to claim 1, further comprising at least one detergent adjunct ingredient.

4. The detergent composition according to claim 3, wherein the detergent adjunct ingredient is selected from the group consisting of surfactants, builders, flocculating aid, chelating agents, dye transfer inhibitors, enzymes, enzyme stabilizers, enzyme inhibitors, catalytic materials, bleach activators, hydrogen peroxide, sources of hydrogen peroxide, preformed peracids, polymeric dispersing agents, clay soil removal/anti-redeposition agents, brighteners, suds suppressors, dyes, perfumes, structure elasticizing agents, fabric softeners, carriers, hydrotropes, builders and co-builders, fabric huing agents, anti-foaming agents, dispersants, processing aids, and pigments.

5. The detergent composition according to claim 1, wherein the composition is for use in washing an item.

6. The detergent composition according to claim 1, wherein the composition is for use in preventing, reducing or removing one or more of: (a) biofilm from an item, (b) stickiness from an item, (c) adherence of soil to an item, (d) deposition or re-deposition of soil on an item during a wash cycle; or (e) mal-odour of an item.

7. The detergent composition according to claim 6, wherein the soil is a pigment soil.

8. The detergent composition according to claim 1, wherein the composition is for use in pre-treating stains on an item.

9. The detergent composition according to claim 1, wherein the composition is for use in maintaining or improving whiteness of an item.

10. The detergent composition according to claim 5, wherein the item is a textile.

11. The detergent composition according to claim 1, wherein said detergent composition is a liquid detergent composition or a powder detergent composition.

12-16. (canceled)

17. A method of washing an item, comprising the steps of: a. exposing an item to the detergent composition of claim 1; b. completing at least one wash cycle; and c. optionally rinsing the item.

18. The method according to claim 17, wherein the item is a textile.

19. The method according to claim 17, wherein the wash cycle is performed at a temperature of 40° C. or less.

20-22. (canceled)

Description

EXAMPLES

[0491] Examples embodying an aspect of the invention will now be described with reference to the following figures:

[0492] FIG. 1. pH-activity on Protazyme AK at 37° C.

[0493] FIG. 2. pH-stability (residual activity after 2 hours at 37° C.).

[0494] FIG. 3. Temperature-activity on Protazyme AK at pH 9.0.

[0495] FIG. 4. Specificity on Suc-AAPX-pNA substrates at pH 9.0.

EXAMPLE 1—PURIFICATION OF FUSARIUM TRYPSIN

[0496] (a) Protazyme AK Assay

[0497] Substrate: Protazyme AK tablet (cross-linked and dyed casein; from Megazyme) Temperature: controlled (assay temperature).

[0498] Assay buffer: 100 mM succinic acid, 100 mM HEPES, 100 mM CHES, 100 mM CABS, 1 mM CaCl.sub.2), 150 mM KCl, 0.01% Triton X-100 adjusted to pH-values 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, and 11.0 with HCl or NaOH.

[0499] A Protazyme AK tablet was suspended in 2.0 ml 0.01% Triton X-100 by gentle stirring. 500 μl of this suspension and 500 μl assay buffer were dispensed in an Eppendorf tube and placed on ice. 20 μl protease sample (diluted in 0.01% Triton X-100) was added. The assay was initiated by transferring the Eppendorf tube to an Eppendorf thermomixer, which was set to the assay temperature. The tube was incubated for 15 minutes on the Eppendorf thermomixer at its highest shaking rate (1400 rpm.). The incubation was stopped by transferring the tube back to the ice bath. Then the tube was centrifuged in an ice cold centrifuge for a few minutes and 200 μl supernatant was transferred to a microtiter plate. OD.sub.650 was read as a measure of protease activity. A buffer blind was included in the assay (instead of enzyme).

[0500] (b) Suc-AAPX-pNA Assay

[0501] pNA substrates: Suc-AAPR-pNA (Bachem L-1720) [0502] Suc-AAPF-pNA (Bachem L-1400) [0503] Suc-AAPA-pNA (Bachem L-1775) [0504] Suc-AAPD-pNA (Bachem L-1835) [0505] Suc-AAPE-pNA (Bachem L-1710) [0506] Suc-AAPI-pNA (Bachem L-1790) [0507] Suc-AAPL-pNA (Bachem L-1390) [0508] Suc-AAPK-pNA (Bachem L-1725) [0509] Suc-AAPM-pNA (Bachem L-1395) [0510] Suc-AAPV-pNA (Bachem L-1770)

[0511] Temperature: Room temperature (25° C.)

[0512] Assay buffer: 100 mM succinic acid, 100 mM HEPES, 100 mM CHES, 100 mM CABS, [0513] 1 mM CaCl.sub.2), 150 mM KCl, 0.01% Triton X-100, pH 9.0.

[0514] 20 μl protease (diluted in 0.01% Triton X-100) was mixed with 100 μl assay buffer. The assay was started by adding 100 μl pNA substrate (50 mg dissolved in 1.0 ml DMSO and further diluted 45× with 0.01% Triton X-100). The increase in OD.sub.405 was monitored as a measure of the protease activity.

[0515] Purification of Fusarium Trypsin Derived from Fusarium oxysporum

[0516] The Fusarium trypsin is derived from Fusarium oxysporum but is expressed in Fusarium venenatum host cells.

[0517] The culture broth was centrifuged (20000×g, 20 min) and the supernatant was carefully decanted from the precipitate. The supernatant was filtered through a Nalgene 0.2 μm filtration unit in order to remove the rest of the Fusarium host cells. The pH of the 0.2 μm filtrate was adjusted to pH 6.0 with 20% CH.sub.3COOH and the 0.2 μm filtrate was diluted with deionized water to a conductivity below 1.0 mS/cm.

[0518] The adjusted 0.2 μm filtrate was applied to a SP-sepharose FF column (from GE Healthcare) equilibrated in 100 mM H.sub.3BO.sub.3, 10 mM Succinic acid, 2 mM CaCl.sub.2), pH 6.0. After washing the column extensively with the equilibration buffer the protease was eluted with a linear gradient over five column volumes between the equilibration buffer and 100 mM H.sub.3BO.sub.3, 10 mM Succinic acid, 2 mM CaCl.sub.2), 300 mM NaCl, pH 6.0.

[0519] Fractions from the column were analysed for protease activity (Protazyme AK activity assay at pH 9) and active fractions were further analysed by SDS-PAGE. Fractions, where only one band was seen on the coomassie stained SDS-PAGE gel, were pooled. The pool was the purified preparation and was used for further characterization.

EXAMPLE 2—CHARACTERIZATION OF THE FUSARIUM TRYPSIN: PH-ACTIVITY, PH-STABILITY, Temperature-Activity and P1-Specificity on Suc-AAPX-pNA Substrates

[0520] The Protazyme AK assay was used for obtaining the pH-activity profile at 37° C., the pH-stability profile (residual activity after 2 hours at 37° C. at the indicated pH-values) and the temperature-activity profile at pH 9. For the pH-stability profile the protease was diluted 10× in the different Assay buffers to reach the pH-values of these buffers and incubated for 2 hours at 37° C. After incubation, the pH of the protease incubations was transferred to pH 9, before assay for residual activity, by dilution in the pH 9 Assay buffer.

[0521] The Suc-AAPX-pNA assay was used for obtaining the P1-specificity at pH 9 on 10 Suc-AAPX-pNA substrates.

[0522] The results are shown in Tables 1-4 below. For Table 1, the activities are relative to the optimal pH for the enzyme. For Table 2, the activities are residual activities relative to a sample, which was kept at stable conditions (5° C., pH 9). For Table 3, the activities are relative to the optimal temperature at pH 9 for the enzyme. For Table 4, the activities are relative to the optimal substrate (Suc-AAPR-pNA) for the enzyme.

TABLE-US-00005 TABLE 1 pH-activity profile at 37° C. (also see FIG. 1) Fusarium trypsin from pH Fusarium oxysporum 2 0.00 3 0.00 4 0.00 5 0.00 6 0.07 7 0.35 8 0.74 9 1.00 10 0.76 11 0.61

TABLE-US-00006 TABLE 2 pH-stability profile (residual activity after 2 hours at 37° C.) (also see FIG. 2) Fusarium trypsin from pH Fusarium oxysporum 2 0.43 3 0.37 4 0.58 5 0.95 6 0.91 7 0.95 8 1.01 9 0.99 10 0.83 11 0.06 After 2 hours 1.00 at 5° C. (at pH 9)

TABLE-US-00007 TABLE 3 Temperature activity profile at pH 9.0 (also see FIG. 3) Temp Fusarium trypsin from (° C.) Fusarium oxysporum 15 0.09 25 0.15 37 0.44 50 1.00 60 0.20 70 0.08

TABLE-US-00008 TABLE 4 P1-specificity on Suc-AAPX-pNA at pH 9.0 (also see FIG. 4) Fusarium trypsin from Suc-AAPX-pNA Fusarium oxysporum Suc-AAPR-pNA 1.00000 Suc-AAPK-pNA 0.53071 Suc-AAPD-pNA 0.00000 Suc-AAPI-pNA 0.00000 Suc-AAPM-pNA 0.00002 Suc-AAPV-pNA 0.00000 Suc-AAPL-pNA 0.00000 Suc-AAPE-pNA 0.00000 Suc-AAPA-pNA 0.00000 Suc-AAPF-pNA 0.00003

EXAMPLE 3—CHARACTERIZATION OF THE FUSARIUM TRYPSIN: OTHER PROPERTIES

[0523] The relative molecular weight as determined by SDS-PAGE was approx. Mr=25 kDa.

[0524] The N-terminal sequence determined by EDMAN degradation was: IVGGTSAS.

[0525] The measured Intact molecular weight was 22180.0 Da.

[0526] The mature sequence (from EDMAN N-terminal sequencing data and Intact MS data):

TABLE-US-00009 IVGGTSASAGDFPFIVSISRNGGPWCGGSLLNANTVLTAAHCVSGYAQS GFQIRAGSLSRTSGGITSSLSSVRVHPSYSGNNNDLAILKLSTSIPSGG NIGYARLAASGSDPVAGSSATVAGWGATSEGGSSTPVNLLKVTVPIVSR ATCRAQYGTSAITNQMFCAGVSSGGKDSCQGDSGGPIVDSSNTLIGAVS WGNGCARPNYSGVYASVGALRSFIDTYA.

[0527] The calculated molecular weight from this mature sequence was 22180.3 Da.

EXAMPLE 4

[0528] Preparation of Swatches

[0529] Textile overgrown with biofilm of a.o. Brevundimonas vesicularis gives a very slimy and sticky surface that draws soils of different kinds to the surface in a wash situation. A model wash with model A detergent where standard pigment soil 09V is added to the wash solution is used to demonstrate the de-gluing effect achieve by enzyme addition. The wash results show that the Fusarium trypsin can inhibit deposition of soil on textile overgrown with biofilm in a MiniLOM wash. Biofilm was grown on Polyester/cotton (65% Polyester and 45% cotton wfk 20A). The Biofilm swatches were made by overgrowth of Brevundimonas vesicularis during 48 hours. The Brevundimonas vesicularis was taken from a −80° C. freeze culture and plated out on TSA Agar plates. After 3 days 4 tubes with 10 mL TSB (from Oxoid) was inoculated with bacteria culture from the plates. The TSB tubes were thoroughly mixed by whirl mixer before they were incubated overnight at 30° C. with shaking applied at 200 rpm. The overnight culture was then collected in a 50 ml centrifuge tube and centrifuged at 3000 rpm for 5 min. the supernatant was discarded and the pellet was dissolved in 5 ml TSB per tube (20 ml in total). 150 ml of the culture was diluted 10× with 1350 μl 100% TSB in an Eppendorf tube. A 20× dilution was made by taking 500 μl of the 10× dilution to 500100% TSB in an Eppendorf tube. 4×100 μL of the 10× and 20× dilutions and blank (100% TSB) was added to a 96 well nunc micro titter plate. The plate was measured at 600 nm on Fluostar Omega securing that one of the dilutions was below 0.6. The OD600 of the Overnight culture was determined and diluted to an OD Of 0.03 in 50% TSB. Prewashed Wfk 10A cotton (100% woven cotton) was cut in circular swatches with a 2 cm diameter and placed in autoclave bags and autoclaved at the “liquid program” on the Systec autoclave with 121° C. in 15 min and then cooling down to 80 before it is opened. Sterile 30A swatch was transferred with a sterile tweeze to each well in a 12 well micro titter plate. 1.62 ml of the diluted 0/N culture was added to each of the well. The swatches were incubated at 15° C. with 70 rpm shaking applied on a Innova 2100 Platform shaker. After incubation in 48 h the swatches were rinsed 2× in 3 ml 0.9% NaCl. The swatches were used immediately after the rinse for wash trial. Pre-washed Wfk 10A is cut in circular swatches with a diameter of 2 cm. The swatches were marked by scissors cut to differentiate them from the 1 day biofilm swatches after the washes.

[0530] Preparation of “Dirty Detergent”

[0531] Weigh of the 3.33 g Model A detergent in a weighing boat add 0.7 g add 09V pigment soil on top of the Model A detergent. This was done in a fume hood using disposable gloves. The weighing boat was added to 1 L of 15° dH water. The soil was dissolved at maximum speed for at least 10 minutes, to dissolve the soil completely.

[0532] Test of Fusarium Trypsin in MiniLOM

[0533] Turn on a heating cabinet to 30° C. Prepare 1 l of Dirty detergent according to preparation method. Five 50 mL Plastic tubes were prepared, each tube represented a wash. Five circular (2 cm diameter) biofilm swatches were added to the beakers prepared according to the Biofilm preparation method, described above. Five circular (2 cm diameter) clean pieces of PO (tracers) were added to each of the tubes. Enzymes prepared in correct dilutions were prepared and kept on ice. 10 ml of “Dirty detergent” (see preparation method) was added to the first tube and enzymes added immediately after. The lid was then added to the tube, and the tube was put on the Mini-LOM rotator (Stuart Rotator SB3). Rotation speed of MiniLOM rotator was 20 rpm. When the first tube was placed on the Mini-LOM rotator the time measurement of the wash was started. Dirty detergent and enzymes were then added to the other tubes, and placed on the Rotator. When all of the tubes were added to the Rotator, it was placed the 30° C. heating cabinet for 60 minutes from the time that the lid was added to the first tube. Rinse procedure was done by removing the rotator from the cabinet and placing it on the operating table with the rotation on. Tube no 1 was taken and the wash water discarded with the swatches left behind in the tubes. 20 ml of 15° dH was added to the tube and it was added back to the rotator. This was then done for all the test tubes in the trial. The rinse was done at ambient room temperature for 10 minutes. A second rinse was done repeating the above described steps. After the second wash the swatches were removed from each of the tubes and added to a piece of paper and dried overnight at room temperature in darkness. Evaluation was done in color eye measuring at 460 nm.

TABLE-US-00010 TABLE 5 Average and Δremission (Enz-blank) PPM Fusarium Trypsin blank 0.5 ppm 1.5 ppm 2 ppm 3 ppm 5 ppm 10 ppm Biofilm Avg 60.5 59.9 60.3 62.4 62.8 63.7 65.1 swath STDEV 0.1 1.3 2.3 0.6 1.1 1.3 1.2 Delta (Enz-blank) −0.7 −0.2 1.9 2.2 3.1 4.6 polyester Avg 69.5 68.6 69.5 69.7 71.7 71.1 70.2 tracer STDEV 1.5 0.7 0.8 1.5 0.8 0.5 1.1 Delta (Enz-blank) −0.9 0.0 0.2 2.2 1.6 0.7

[0534] When clean textile is washed in the “dirty” detergent there is a general background uptake of dirt that changes the color from white to light grey. If the surface is overgrown for 2 days with Brevundimonas then the surface takes up more dirt than the background. The original whiteness of the unwashed swatch is 83 rem units. Uptake results in a deep gray appearance with a remission value at 460 nm of 60.5 which means that it gives at delta of 22.5 compared to the background uptake. Adding increasing amounts of Fusarium trypsin to the washing solution result in significant increase in the cleaning ability by keeping the soil away from the surface. The effect can be perceived by the eyes down to a dosing of 2 ppm where it gives a delta performance of 1.9 meaning a remission at 460 nm of 62.4. The more we increase the dosage the more we get can avoid extra dirt from adhering to the textile surface. A dosage of 10 ppm gives a wash performance lift of 4.6 rem units.