Storage-Stable Liquid Detergent or Cleaning Agent Containing Protease and Lipase

Abstract

According to the invention, storage stability in terms of cellulolytic activity is to be improved in a liquid washing or cleaning agent which comprises a protease and cellulase. This is achieved by the use of a protease which comprises an amino acid sequence which is at least 80% identical to the amino acid sequence specified in SEQ ID NO. 1 and which has the amino acid glutamic acid (E) or aspartic acid (D) or the amino acid asparagine (N) or glutamine (Q) or the amino acid alanine (A) or glycine (G) or serine (S) at position 99 in the count according to SEQ ID NO. 1.

Claims

1. A method of formulating a liquid washing or cleaning agent for storage stability of cellulolytic activity, the method comprising: (1) mixing, to formulate the liquid washing or cleaning agent: (a) a protease comprising the amino acid sequence of SEQ ID NO: 2, wherein said amino acid sequence imparts a proteolytic activity to the protease, and wherein the protease imparts a proteolytic activity to the washing or cleaning agent; and (b) a cellulase; and (2) storing the liquid washing or cleaning agent for at least 4 weeks, wherein the liquid washing or cleaning agent exhibits increased storage stability of cellulolytic activity after storage as compared to a control liquid washing or cleaning agent that is the same liquid washing or cleaning agent but comprises a protease that differs from the protease of (1)(a) solely by having a protease comprising the amino acid arginine (R) at location 99 in the count according to SEQ ID NO: 1; wherein the liquid washing or cleaning agent exhibits increased storage stability of cellulolytic activity after storage for 4 weeks at 30° C. as compared to a control liquid washing or cleaning agent that is the same liquid washing or cleaning agent but comprises a protease that differs from the protease of (1)(a) solely by having a protease comprising the amino acid arginine (R) at location 99 in the count according to SEQ ID NO: 1, wherein, at the beginning of such storage, the liquid washing or cleaning agent and the control liquid washing or cleaning agent have the same concentration of protease and the same concentration of cellulase, and wherein the protease and the cellulase are in solution in the liquid washing or cleaning agent.

Description

EXAMPLE

Determination of the Storage Stability of the Liquid Washing Agent According to the Invention

[0165] The washing agent base formulations were [0166] a) a first liquid washing agent of the following composition (all data in wt %): 0.3-0.5% xanthane, 0.2-0.4% defoamer, 6-7% glycerin, 0.3-0.5% ethanol, 4-7% FAEOS (fatty alcohol ether sulfate), 24-28% non-ionic surfactants, 1% boric acid, 1-2% sodium citrate (dihydrate), 2-4% soda, 14-16% coconut fatty acids, 0.5% HEDP, (1-hydroxyethane-(1,1-diphosphonic acid)), 0-0.4% PVP (polyvinyl pyrrolidone) 0-0.5% optical brightener, 0-0.001% colorant, residue demineralized water. The comprised cellulase was 0.15 wt % Ecostone N400® (cellulase preparation from AB Enzymes company). [0167] b) a second liquid washing agent of the following composition:

TABLE-US-00001 Ingredient wt % C.sub.12-18 fatty alcohol with 7 EO 7.5 Lin. C.sub.10-C.sub.13 alkylbenzene sulfonate (Na salt) 8.5 Cocofatty acid (Na salt) 14.6 Lauryl ether sulfate with 2 EO (Na salt) 13.0 Citric acid (Na salt) 3.1 Boric acid (Na salt) 1.0 Polyacrylate thickener 0.4 Propylene glycol 2.1 silicone defoamer 0.2 Cellulase preparation Ecostone N400 ® (AB Enzymes) 0.1 Water ad 100

[0168] To the washing agent base formulations were added the following proteases for the various experimental approaches, wherein the data are based on active protein: [0169] Approach 1: Performance improved variant F49 of the protease from Bacillus lentus according to WO 95/23221 (Arg at position 99 (99R)): 0.4 mg/ml (0.04 wt %) into the liquid washing agents according to a) and b). [0170] Approach 2: Protease, disclosed in FIG. 2 or SEQ ID NO: 3 of the international patent application WO 03/057713 (Ser at position 99 (99S); identity to SEQ ID NO: 1<80%): 0.4 mg/ml (0.04 wt %) in the liquid washing agent according to a), 0.3 mg/ml (0.03 wt %) in the liquid washing agent according to b). [0171] Approach 3: Protease, containing an amino acid sequence according to SEQ ID NO: 2 (Glu at position 99 (99E)): 0.4 mg/ml (0.04 wt %) in the liquid washing agent according to a), 0.3 mg/ml (0.03 wt %) in the liquid washing agent according to b).

[0172] The storage stabilities of the washing agents according to each of the approaches 1, 2 and 3 were tested. The washing agents were stored at a temperature of 30° C. for the relevant time and the respective residual cellulolytic activity determined. The test samples were incubated under defined reaction conditions (100 mM sodium phosphate buffer pH 7.5, 40° C., 15 min) with a substrate (1.25% carboxymethyl cellulose). The reacted under alkaline conditions with p-hydroxybenzoic acid hydrazide (PAHBAH) in the presence of bismuth to afford a yellow dye that was determined photometrically at a wavelength of 410 nm. The quantity of released sugar corresponding to the coloration is a measure of the enzyme activity (see Lever, Anal. Biochem., 1972, 47 & 1977, 81). The measured residual cellulolytic activities are listed in Table 1.

TABLE-US-00002 TABLE 1 Washing a) b) agent 4 8 4 8 according to Initial weeks weeks Initial weeks weeks Approach 1 100% 42% 27% 100% 33% 21% Approach 2 100% 36% 24% 100% 42% 24% Approach 3 100% 46% 32% 100% 49% 26%

[0173] It is evident that inventive washing agents exhibit a significantly improved residual cellulolytic activity and consequently storage stability in comparison to the washing agents of the approaches 1 and 2.