JP2002509981A - Acid detergent containing acidic protease - Google Patents

Abstract

  (57) [Summary] The present invention relates to an acidic cleaning composition comprising an acidic protease that retains proteolytic activity in the presence of an inhibitor selected from the group of pepstatin, Pefabloc, PMSF or EDTA, and at least one nonionic surfactant. About things. The present invention further provides a method of cleaning a hard surface or garment, the method comprising contacting the hard surface or garment with the composition dissolved in an aqueous solution in an amount sufficient to exert a cleaning effect. Comprising.

Description

DETAILED DESCRIPTION OF THE INVENTION

FIELD OF THE INVENTION [0001] The present invention relates to cleaning compositions containing a resistant protease that is substantially insensitive to pepstatin and a non-ionic surfactant. The composition is suitable for cleaning hard surfaces or cellulosic and / or wool woven or knitted fabrics at acidic pH.

[0002] The cleaning composition of the majority which are used in the background household and industrial invention is alkaline. Industrial cleaning, such as CIP cleaning, for example, cleaning of membranes in the processing of dairy or other food products, includes mineral deposits, such as hard stones such as milk stone.
Two are often involved: acid treatment to remove ss salts (scale), and alkaline detergent treatment to remove organics such as fats, proteins and / or sugars. This process often comprises the following steps: Rinse → alkali → rinse → acid → rinse

[0003] The lack of an acidic protease available at acidic pH that can be produced industrially hinders the development of enzymatic cleaning of tableware, clothing, and industrial and domestic hard surfaces under acidic conditions. Have been. Only a few attempts have been made to develop proprietary acidic cleaning treatments, and there is a need for advantageous acidic cleaning compositions.

[0004] DE 3833047 A1 discloses an acidic detergent composition for automatic dishwashers, which contains a hydrolase enzyme. The hydrolase includes an amylase, protease or lipase. US 5,698,507 discloses a specific amount of a nonionic surfactant, citric acid, H ₂ O ₂ , at least one acid-resistant protease enzyme, at least one amylase enzyme, hydrotrope, CaCl ₂ , sodium formate, a gelling system. And a gelled dishwashing composition having a pH of 3-5, consisting essentially of water and water. Enzymes specifically mentioned are Bacill
α-amylase of us amyloliquefaciens (eg, Tenase 1200, Tenase L-1200 and
Tenase L-340) and Aspergillus niger or Aspergillus oryzae protease.

WO95 / 02044 discloses acidic aspartic proteases (proteases I and II) from A. aculeatus for the production of food, animal feed, drinking water, leather, and for cleaning contact lenses. I have. WO 96/29978 discloses an acidic composition for oral care containing an acidic protease. It is substantially inert in the slightly alkaline normal oral environment. WO 96/23579 comprises at least: a) treatment of the membrane with an aqueous solution containing β-glucanase, xylanase and cellulase enzymes; b) washing with an acidic detergent and c) washing with an alkaline solution containing peroxide. Cleaning of a beer filtration treatment membrane is disclosed.

SUMMARY OF THE INVENTION The present inventors have determined that proteases that retain proteolytic activity in the presence of an inhibitor selected from the group consisting of pepstatin, Pefabloc, PMSF or EDTA have similar pH-
It has been found that it exhibits surprisingly good detergency and / or activity efficiency under acidic conditions compared to other acidic proteases having active properties.

The present invention therefore offers the following advantages over the field of alkaline cleaning compositions: a) peroxygen which oxidizes or bleaches polyaromatic compounds present in soil or contamination. / Activator drift system, eg perboric acid or sodium percarbonate and T
The AED activator is more effective at low temperatures; b) the enzyme-enhancer drift system is for example peroxidase-PPT or laccase-P
PT can also be used at low temperatures; acidic conditions by itself have a blistering effect on certain soils, such as coffee and tea soils; c) Acidic cleaning compositions As inorganic dirt can be removed,
Industrial hard surface cleaning, eg alkali cleaning and rinsing steps in CIP (Cleaning In Place) can be omitted;

[0008] d) Omission of the alkaline cleaning step reduces hard surface damage; e) At acidic pH, the surfactant does not normally precipitate due to water hardness ions, so that alkaline clothing The builder system normally present in detergents can be reduced or eliminated in acidic garment detergents. Therefore, scale adhesion in a washing device, for example, an automatic washing machine can be avoided.

Accordingly, the present invention provides, as a first point, pepstatin, Pefabloc, PMSF, or EDTA.
An acidic cleaning composition for cleaning hard surfaces and clothes, comprising an acidic protease that retains proteolytic activity in the presence of an inhibitor selected from the group consisting of: and at least one nonionic surfactant. Offer things.

DETAILED DESCRIPTION OF THE INVENTION Definitions As used herein, the term "hard surface" refers to any surface that is essentially impermeable to water.
Examples include metals, such as stainless steel or other alloys, plastics /
There are surfaces composed of synthetic polymers, rubber, glass, wood, concrete, rock, marble, gypsum and ceramic, all of which may optionally be coated with paint, enamel, polymer and the like. As used herein, the term "inhibitor" refers to a compound that interacts with the protease, either competitively or non-competitively, thereby reducing and / or destroying the enzyme's activity on a substrate. As used herein, the term "retaining proteolytic activity" refers to the protease as 1
After treatment with an inhibitor of either mM pepstatin, 0.1% Pefabloc, 0.1% PMSF or 100 mM EDTA, the activity measured in HPU units at pH 5.5 (residual activity) is at least 75% of the protease activity. It means there is.

Protease The protease in the present invention is an acidic protease that retains proteolytic activity in the presence of an inhibitor selected from the group consisting of pepstatin, Pefabloc, PMSF, or EDTA. Inhibition by pepstatin having the formula: Muroa S. a
nd Oda K. (1985).

Embedded image

[0012] The inhibitory properties of this enzyme in the present invention are shown in WO95 / 02044. Inhibition studies show that protease I is not inhibited by pepstatin. Protease II is inhibited by pepstatin. According to Oda K. and Muroa S. (1991), two classes of papstatin-sensitive carboxyl or aspartic protease and pepstatin-insensitive carboxyl proteinase are described as acidic proteases. See also the publication Muroa S. and Oda K. (1985) in which this new subclass of acid protease was introduced.

[0013] PMSF is an inhibitor having the formula:

Embedded image On the other hand, PEFABLOC is a protease inhibitor having the following formula:

Embedded image

Preferred proteases are microorganisms, such as bacteria, such as Bacillus, Pseudomo
nas or Xanthomonas or fungi (including yeast), such as Aspergillus species (eg, A. aculeatus or A. niger) or Scytalidium species (eg, S. li)
gnicolum). In another embodiment, the protease is obtained from a genetically modified bacterium or fungus by transforming said bacterium or fungus with a DNA vector / construct containing the DNA encoding it. In a particularly preferred embodiment, the protease of the present invention contains one or more aspartic acid residues and / or carboxyl residues as a functional group of its active center.

In addition, the protease retains proteolytic activity in the presence of inhibitors present in meat, egg white, whole blood, plasma, milk, beer, potato or beans. Such inhibitors include ovomacroglobulin, ovomucoid or ovoglycoprotein. Inhibitors present in the soil that one wishes to wash (eg, competitive or non-competitive inhibitors, as the term is known to those skilled in the art), may play an important role in the washing process. Because they inactivate or reduce the activity of soil-hydrolyzing enzymes. This may be why it was difficult to find a suitable protease for use in acidic cleaning compositions. Further, the protease preferably has an optimum pH in the range of 2-7, more preferably 3-6, and even more preferably 4.5-5.5. The protease of the present invention has an optimum temperature in the range of 20 to 70 ° C, for example, 20 to 60 ° C, for example, 30 to 50 ° C.

In a more particular embodiment, the protease is an A. aculeat described in WO 95/02044.
us-derived protease I or II. The most preferred protease is protease I.

Nonionic Surfactants Nonionic surfactants are particularly suitable for acidic detergents. Because it is not functionally affected in a moderately acidic environment. Preferred nonionics are
Glycolipid, alcohol ethoxylate, alkylphenol ethoxylate, glucamide, alkyl polyglycoside (Stache & Kosswig, 1990).

Other Suitable Composition Ingredients If the water from which the wash fluid is prepared contains significant amounts of water-hardening ions, ie, calcium ions, that can affect the performance of the protease,
The composition may contain a sequestering agent. Suitable sequestering agents can sequester calcium ions at acidic pH. Preferred sequestering agents are methylglycine diacetate, nitroloacetic acid, citric acid, oligomers and polymeric (poly) carboxylic acids from polysaccharides (Kock
et al., 1993), dextrins or protein hydrolysates (DE 19547730 A1).

The composition may further comprise other ingredients that enhance the detergency of the composition, such as softeners, amylases (eg, Fungamyl (Novo Nordisk A / S, Denmark)), lipases (eg, Novocor AD (Novo Nordisk A / S, Denmark)), cellulases (eg Celluzyme
, Carezyme, Celluclast (Novo Nordisk A / S, Denmark)), xylanase (eg, Biofeed PLUS, Shearzyme (Novo Nordisk A / S, Denmark)), β-glucanase (
For example, Viscozyme, Ultraflo (Novo Nordisk A / S, Denmark)), pectinase (for example, Pectinex Ultra (Novo Nordisk A / S, Denmark)), peroxidase (for example, Guardzyme (Novo Nordisk A / S, Denmark)), laccase (for example, Myceliophtho
ra or Polyporus bacteria), enhancers for peroxidase / laccase (eg, PPT, methylsyringic acid, methylsyringate, or derivatives thereof), and / or buffers (eg, citric acid).

Finally, the composition can be a liquid or a powder. In the latter case, it may be appropriate to formulate the protease as stabilized granules. The preparation can be obtained in a customary manner.

Use of the Composition The composition of the present invention may suitably be used in a method for cleaning hard surfaces or clothing. The method preferably comprises contacting the hard surface or garment with an aqueous solution of the composition in a sufficient amount to provide a cleaning effect. Alternatively, the protease and other composition components can be added separately to the solution.

[0022] The composition may be prepared at 500-3000 HUT / L, preferably 500-1500 HUT / L, more preferably at 75
It is preferred to dissolve in an amount sufficient to provide an enzyme dose of 0-1250 HUT / L, eg, 1000 HUT / L wash. The hard surface to be cleaned by the method is, in one aspect, preferably an industrial treatment or household fixture.

Particularly preferred industrial equipment is heat exchangers, tanks, pipes, centrifuges, evaporators,
Filters, extruders, meat cleavers, cooking jars, beer and wine fermenters, beer and wine filters, filter aids used, coolers, storage tanks, sieves, hydroelectric cyclones, ultrafiltration equipment , Nanofiltration brace, hyperfiltration brace, microfiltration brace and milking machine.

A particularly suitable embodiment is to clean equipment or products for health care or animal care. Health care equipment includes diagnostic / analytical (eg, endoscopes, blood analyzers), procedural (eg, dialysis or blood processing equipment) or surgical procedures that come into contact with human or animal blood, other bodily fluids, or tissues. Equipment (e.g., scalpels, peen, clips or tweezers, forceps, and other patient care tools used by doctors, veterinarians, or dentists).

In certain embodiments, the method for cleaning industrial equipment comprises “in-situ cleaning (CI
P) "method. Particularly preferred household items include kitchen utensils for eating, dishes, cups, beakers,
There are glassware, pots, breads, appliances, toilets, washbasins or tiles.

Another suitable aspect of the present invention is the use of the industrial process used to remove salts, charged carbohydrates, residues of proteins and amino acids, and colored substances prior to crystallization in the mass production of sugars. Washing of the ion exchange column. Further, in syrup production from starch, the ion exchange columns used before and after the isomerization step can be washed with the composition of the present invention.

Another preferred embodiment of the present invention is the washing of a manufacturing ion exchanger covered by a protein or peptide and / or clogged with its resin by a proteinaceous substance. Normally, such clogged ion exchangers require washing treatment with excess alkali and heat (eg, recirculating hot alkali at pH 13-14 at 85 ° C. for about 1 hour), thereby reducing the resin And the efficiency of the regeneration process are reduced. However, the use of the composition according to the invention enables a gentle cleaning treatment which guarantees the efficient removal of soils which hinder the efficient regeneration of the ion exchanger.

[0028] Yet another particularly suitable aspect of the present invention is the washing of columns used in gel filtration or affinity chromatography to separate proteins. Many such columns contain a significant excess of separation and / or chromatographic material / resin and must be efficiently washed when used in large-scale processes.
Typically, harsh conditions are used to remove mucous contaminants, such as complex proteins and carbohydrates, which are deposited in the material. These harsh conditions often shorten the life of the material. However, the use of the composition of the present invention allows for a gentle cleaning process that ensures efficient cleaning of the column material and prolongs its life. A preferred washing treatment for washing the column containing the material for gel filtration and affinity chromatography is 500-3000 HUT / L, preferably 500-1500 HUT / L, more preferably 750-1250 HUT / L, for example Contains enzyme at a dose of 1000 HUT / L wash, pH
Recycling 2-7, preferably 3-6, for example 4-5 washing solutions, the temperature of which must be 10-65 ° C, preferably 30-50 ° C, for example 40 ° C . The washing time is, in a preferred embodiment, from 2 minutes to 20 hours, depending on the type of method.

In a particular embodiment, a method for cleaning household appliances is used in an automatic dishwasher. If the composition is used for washing clothes, the method is preferably used on an industrial or home-scale washing machine. Preferred garments are cellulosic woven and / or non-structural garments, such as silk, acetate, wool, ramie, or rayon garments. The cleaning of wool and silk in particular according to the invention is useful. This is because acidic washing conditions soften the garment and have an antimicrobial action (kill or inhibit microbial cells).

In one embodiment, the method of cleaning a hard surface or clothing comprises a pH of 2-7, preferably 3-6.
For example, at 4-5, while the temperature is 10-65 ° C., preferably 30-50 ° C., for example 4
Maintained at 0 ° C. In a preferred embodiment, the washing time is from 2 minutes to 20 hours, depending on the type of method. For example, for cleaning industrial membranes, up to overnight (20 hours)
Immersion in the (circulating) solution of the composition, while industrial dishwashing will be completed within 2-10 minutes.

Materials and Methods Determination of HUT Activity of Proteases HUT activity was determined according to the method of AF92 / 2 published by Novo Nordisk A / S, Denmark. One HUT is the amount of enzyme that digests denatured hemoglobin at 40 ° C. and pH 4.7 for 30 minutes to form a hydrolyzate corresponding to the absorbance at 275 nm in a solution of 1.10 μg / ml tyrosine in 0.006 N HCl. It is. Its absorbance is 0.0084.
The denatured hemoglobin substrate is digested by the enzyme under predetermined conditions in a 0.5 M acetate buffer. Undigested hemoglobin is precipitated with trichloroacetic acid and the absorbance at 275 nm of the hydrolyzate of the supernatant is measured.

Protease HPU Activity One hemoglobin protease unit (hpu) is defined as the amount of enzyme that releases 1 mmol of primary amino groups per minute under the following standard conditions (compared to standard serine): decide).

[0033] A 2% (w / v) hemoglobin (bovine, Sigma) solution was added to Britton and Robinson, J. C.
hem.Soc., 1931, p. 1451 using a universal buffer and adjusting the pH to 5.
Adjust to 5. Pre-incubate 2 ml of the substrate solution in a water bath at 25 ° C. for 10 minutes. Add 1 ml of enzyme solution containing a bg / ml enzyme preparation corresponding to about 0.2-0.3 hpu / ml universal buffer (pH 5.5). After incubation at 25 ° C. for 30 minutes, the reaction is stopped by the addition of a terminator (17.9 g of trichloroacetic acid, 29.9 g of sodium acetate and 5 ml of a final solution of 19.8 g of acetic acid with 500 ml of deionized water). A blank is prepared in a similar manner to the test solution except that a terminator is added before adding the enzyme solution. The reaction mixture is kept in the water bath for 20 minutes, after which it is filtered through Whatman 42 filter paper.

Color development with o-phthaldialdehyde (OPA) determines the primary amino group as follows. 7.62 g of disodium tetraborate decahydrate and 2.0 g of sodium dodecyl sulfate are dissolved in 150 ml of water. Next, 160 mg of OPA dissolved in 4 ml of methanol are added together with 400 ml of β-mercaptoethanol, after which the solution is adjusted to 200 ml with water. 400 ml of the filtrate obtained above is added to 3 ml of this OPA reagent while stirring. The optical density (OD) at 340 nm is measured after about 5 minutes. In addition, a universal buffer (pH 5.5)
This OPA test is performed using a serine standard solution containing 10 mg of serine in 100 ml. Use buffer alone as blank.

The protease activity is calculated from the OD measurement according to the following formula: (OD _t -OD _b ) × C _SER × Q hpu / ml Enzyme solution: ─────────── (OD _ser × _{OD B) × MW SER × t} i hpu / g enzyme preparation = hpu / ml: b However OD _t, OD _b, OD _ser and OD _B are each inspected solution, blank, at serine standard solution and buffer Optical density, C _ser is the concentration of serine in the standard (mg / ml) (0.1 mg / ml in this case) and MW _ser is the molecular weight of serine (105.09). Q is the dilution factor of the enzyme solution (in this case 8) and t _i is the incubation time (in minutes) (in this case 30 minutes).

Evaluation Method of the Composition in Automatic Dishwashing (ADW)
At 75 ° C. The efficiency of the protease was examined by standard laboratory techniques.
The dishes made of stainless steel were soiled as described below. Fifteen eggs (egg white + yellow) and 255 ml of whole milk were mixed for 2 minutes at minimum speed with a Braun UK 20 cooking mixer. Thereafter, the mixture was sieved through a sieve having a pore size of 0.5 mm. Five stainless steel dishes were slightly dipped in the egg / milk mixture and placed on a drying shelf. After drying overnight at room temperature, the dishes were baked at 120 ° C. for 1 hour in a ventilated heat oven. To test for amylase, stain five porcelain dishes with gelatinized 2% starch solution through overnight drying at room temperature.

For automatic dishwashing, a Cylinda Excellence Kompak + 770 machine for 6 people was used. This machine was equipped with an ion exchanger for removing calcium ions from the incoming water. In program number 4, the temperature was set to 55 ° C. This program worked as follows. a) The main wash starts with heating the wash solution from 25 ° C. to 55 ° C. in 7 minutes and ends with a 10 minute wash. b) Rinsing with cold water is performed. This rinsing takes 5 minutes and the temperature during rinsing varies between 35 and 45 ° C. depending on the negative charge. c) A second rinse with cold water is performed. At that time, over 8 minutes, add 20 water
Heat to 55 ° C. and pump. d) Dry at 55 ° C for about 5 minutes.

Before and after washing, the light reflection value is measured directly on the starch or protein coating stained with iodine (KI / I ₂ ). Staining was performed using a standard solution prepared as follows: potassium iodide (KI) 20.0 g (Merck art. No. 5043) and iodine (KI).
I ₂ ) 1.27 g (Merck art. No. 4763) was placed in a 2 liter beaker, and deionized water was added thereto to adjust the volume to 1.0 liter. The solution was stirred at room temperature for about 10 minutes. The dish was slowly passed through the iodine solution and then placed on a drying shelf.

The measurement was performed using a Minolta Chroura Meter (CR-300 type). For each dish, the average R value is determined from six single measurements. Film removal rate% (RPF% for protein,
The RSF% for starch) is calculated according to the following formula: RPF (%) [or RSF (%)] = [after R wash-before R wash] / [R clean dish-before R wash] * 100% The cleaning composition used in one cleaning machine and the obtained cleaning results are shown in the tables in the following Examples. The pH was varied by adding 2-7 ml of 4N HCl.

Method for evaluating the composition in washing clothes A washing machine commercially available in Europe (AEG model OKO-LAVAMAT JUBILEUM 40),
The cleaning performance was examined using 2.0 kg of adjustment clothing and artificially contaminated fabric. 10 pieces (5cm ×) stained with milk, blood and carbon black (EMPA116)
A commercially available “standard” cloth (5 cm) (standard cotton cloth) and 10 pieces of heat-treated at 70 ° C. for 30 minutes after dipping in spinach extract were fixed on the fabric for adjustment. Washing was carried out according to the instructions of the supplier according to the program “Klarvask” at 40 ° C. without final washing at a final centrifugation of 1400 rpm. During the washing process, a sample of the washing solution was withdrawn, and the pH was measured with a pH test paper Acilit pH0-6. Following the washing treatment, the plate was rinsed twice with a moderate centrifugation at 400 rpm. A total of 50 liters of water was used throughout the treatment.

The washed test cloth pieces were evaluated by measuring the intensity of 460 nm reflected light and% R (% regular reflectance) attenuated by the cloth pieces. For this purpose, a J & M Tidas MMS / 16 photometer equipped with a CLX 75W xenon lamp and a fiber optic was used. Each piece of fabric was measured individually on three or four fabric stacks (to reduce the amount of light passing through the fibrous structure without being absorbed or reflected). The value ΔR enzyme = R wash-no R enzyme wash reflects the contribution of the enzyme for each type of cloth. The results are expressed as an average value and a confidence interval, for example, [% R wash-W;% R wash + W
]. Where W is a 95% confidence value.

Chemicals / enzymes used a) NTA (nitrilotriacetic acid), available from Fluka Chemika (no. 72560). b) Trilon® A (NTA-Na ₃ , nitrilotriacetic acid trisodium salt), BASF
-Available from Germany. c) Trilon® M (MGDA-Na ₃ , methyl glycine diacetate trisodium salt), available from BASF-Germany. d) Dehyphon® LS 54 (non-ionic alcohol ethoxylate), Hen
Available from kel KGaA-Germany. e) Lutensol® AO 3 (nonionic alcohol ethoxylate), BASF
-Available from Germany. f) Lutensol® AO 7 (nonionic alcohol ethoxylate), BASF
-Available from Germany. g) Sokalan® HP25 (modified polycarboxylate), available from BASF-Germany; used as anti-redeposition agent. h) Protease I (1.05 KHUT / g), obtained as described in WO95 / 02044. i) Protease II (5.22 KHUT / g), obtained as described in WO95 / 02044. j) Flavorzyme® (65.2 KHUT / g), available from Novo Nordisk A / S, Denmark. k) Fungamyl® available from Novo Nordisk A / S, Denmark.

[0043] EXAMPLES Example 1: This example shows the effect of protease sample in ADW. The data is shown in Table 1.

[0044]

[Table 1]

From Table 1, it can be seen that Protease I shows the highest wash value, despite the fact that its HUT activity used is much lower than Flavorzyme and protease.

Example 2 This example demonstrates the effect of pH fluctuations on the performance of Protease I in ADW. The pH was varied from test to test by the addition of various amounts of 4N HCl. Table 2 shows the data.

[0047]

[Table 2]

From Table 2, it can be seen that Protease I shows almost the same wash value (RPF%) at pH 4 and 4.5. Under more acidic conditions (pH 3.2) in the absence of the enzyme, the dishes were not appreciably washed.

Example 3 In this example, Fungam was used as a detergent using a porcelain dish with a starch coating.
Check the effect of adding yl. Table 3 shows the data.

[0050]

[Table 3]

Table 3 shows that protease I, with or without amylase, has a prominent effect on protein removal. In the absence of Protease I, amylase showed little effect.

Example 4 In this example, the hydrolytic capacity of a wash solution containing a pepstatin-insensitive acidic protease was examined in the presence and absence of egg white. In addition, the effects of tap water (which is slightly hard) and ion-exchanged water were examined. Table 4 shows the data. Lyophilized hemoglobin (Novo Nordisk, Denmark) was dissolved in tap water (18 ° dH (German water hardness)) or demineralized water to a concentration of 20 g / L. A 1000 ml wash solution was made by suspending / dissolving the following ingredients in demineralized water: a) 12 g NTA b) 20 g Lutensol A07 c) 62 g Na ₂ SO ₄

In an Ehrenmeyer flask, 5 g of the washing solution was added to 100 ml of the hemoglobin solution while stirring. The pH was adjusted to 4.5 with NaOH. The flask was placed in a 40 ° C. water bath. Add 1000 μL of Protease I, withdraw sample at t = 1 min and t = 30 min, and measure osmotic pressure (mOSM / kg H ₂ O) with an osmometer (Wide Rauge Osm. 3 W 2, Advanced Instruments) did. The measurement result is the difference between the two measurements, ΔmOSM /
expressed as kg H ₂ O. To determine the inhibitory effect of egg white, 5 ml of gently homogenized egg white was added to the reaction mixture before addition of the enzyme.

[0054]

[Table 4]

Table 4 shows that protease I effectively hydrolyzes hemoglobin protein regardless of whether tap water or ion-exchanged water is used. Furthermore, it is clear that protease I is not at all inactivated by egg white. Protease I also hydrolyzes this protein when egg white is present, due to the higher value of ΔmOSM / kg H ₂ O. Approximately 0.6 g of protein is added by 5 ml of egg white, which is further hydrolyzed.

Example 5: In this example, the hydrolytic capacity of a wash solution containing different pepstatin-insensitive acidic proteases by the method described in Example 4 using tap water (slightly hard) and ion exchanged water. Was inspected. Table 5 shows the data.

[0057]

[Table 5]

From Table 5, it can be seen that when using ion-exchanged water, the HUT is considerably lower than in the case of Flavorzyme.
It can be seen that using activity, Protease I contributes to a higher hydrolysis effect. With slightly harder tap water, Protease I shows comparable hydrolytic capacity, even with a much lower HUT activity than with Flavorzyme.

Example 6 In this example, the clothing cleaning performance of Protease I in a suitable slightly acidic clothing cleaning composition was investigated.

[0060]

[Table 6]

From Table 6, it can be seen that under the above mild conditions, Protease I significantly contributes to the cleaning of the two types of cloth.

Example 7: In this example, four potential inhibitors (PEFABLOC are from Pentapharm, Bose
l, Switzerland, and others from Sigma) were tested for sensitivity of protease I and protease II. EDTA is an inhibitor of metalloenzymes, while PEFABLOC and PMSF are inhibitors of serine proteases. The proteolytic activity was measured in HPU / L in a protease solution at pH 5.5 before and after inhibitor treatment. The following results were obtained from this inhibitor test.

[0063]

[Table 7]

From this test, Protease I retained activity in the presence of any inhibitor, while Protease II retained activity in the presence of EDTA, PEFABLOC and PMSF, but was inhibited by pepstatin.

[0065]

[Table 8]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme court ゛ (Reference) (C12N 9/62 (C12N 9/62 C12R 1:66) C12R 1:66) (31) Priority number PA 1998 01637 (32) Priority date December 11, 1998 (December 11, 1998) (33) Priority claiming country Denmark (DK) (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD , GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU , ZA, ZWF term (reference) 4B050 CC03 CC07 CC08 DD02 DD03 DD04 KK03 KK11 KK18 LL04 4H003 AC05 AC08 AC11 BA09 BA12 DA01 DA05 DA06 DA12 DA14 DA17 DA18 DA19 EA03 EA12 EB08 EB13 EB15 EB42 FA02 EC47

Claims (33)

[Claims] An acidic protease that retains proteolytic activity in the presence of an inhibitor selected from the group consisting of pepstatin, Pefabloc, PMSF and EDTA;
An acidic cleaning composition comprising at least one nonionic surfactant. 2. The composition according to claim 1, wherein the protease is derived from bacteria or fungi including yeast. 3. The composition according to claim 2, wherein the bacterium is a bacterium belonging to the genus Bacillus, Pseudomonas or Xanthomonas. 4. The method according to claim 1, wherein the fungus is of the genus Aspergillus or Scytalidium.
3. The composition according to claim 2, which is a fungus of the genus idium. 5. The composition according to claim 4, wherein said fungus is A. aculeatus. 6. The bacterium or fungus is genetically modified by transformation with a DNA vector containing DNA encoding the protease.
The composition according to any one of claims 1 to 5. 7. The composition according to claim 1, wherein the protease contains an aspartic acid residue as a functional group of its active center. 8. The composition according to claim 1, wherein the protease contains a carboxyl residue as a functional group at its active center. 9. The composition of claim 1, wherein said protease further retains proteolytic activity in the presence of an ovomacroglobulin inhibitor. 10. The composition of claim 1, wherein said protease further retains proteolytic activity in the presence of an ovomucoid inhibitor. 11. The composition of claim 1, wherein said protease retains proteolytic activity even in the presence of an ovoglycoprotein inhibitor. 12. The protease according to claim 11, further comprising meat, egg white, whole blood, plasma, milk,
2. The composition of claim 1, wherein the composition retains proteolytic activity even in the presence of an inhibitor present in one selected from the group consisting of beer, potato or beans. 13. The protease, wherein the protease is protease I or protease II.
The composition according to claim 1, wherein the composition is: 14. The composition of claim 13, wherein said protease is Protease I. 15. The composition of claim 1, wherein said composition further comprises a sequestering agent. 16. The composition according to claim 1, wherein the nonionic surfactant is glycolipid, alcohol ethoxylate, alkylphenol ethoxylate, glucamide or alkyl polyglucoside. 17. The composition of claim 15, wherein said sequestering agent is capable of binding calcium ions at acidic pH. 18. The method according to claim 17, wherein the sequestering agent is an oligomer and a polymeric (poly) carboxylic acid, dextrin, or protein hydrolyzate obtained from methylglycine diacetate, nitroloacetic acid, citric acid, polysaccharide. Composition. 19. The composition further comprises a softener, an amylase, a lipase, a cellulase, a xylanase, a β-glucanase, a pectinase, a peroxidase, a laccase, an enhancer for the peroxidase / laccase, and / or a buffer. The composition of claim 1. 20. A method for cleaning a hard surface or clothing, wherein the hard surface or clothing is dissolved in an aqueous solution in an amount sufficient to exert a cleaning effect. A method comprising contacting with a composition according to any of the preceding claims. 21. 500-3000 HUT / L, 500-1500 HUT / L, 750-1250 HUT / L and 10
21. The method of claim 20, wherein the protease is in an aqueous solution to a range selected from the group consisting of a HUT / L wash solution. 22. The method of claim 20, wherein the hard surface is included in industrial or household equipment. 23. The industrial equipment may be a heat exchanger, tank, pipe, centrifuge, evaporator, filter, extruder, meat knife, cooking jar, beer and wine fermenter, beer and wine filter. , Consumable filtration aid, cooler, storage tank, sieve, hydroelectric cyclone, ultrafiltration device, nanofiltration device, hyperfiltration device, microfiltration device, ion exchange column, gel filtration column, or 23. The method according to claim 22, which is a milking machine. 24. The household equipment, wherein the kitchen equipment, a dish, a cup, a beaker,
23. The method of claim 22, wherein the method is a glassware, pot, pan, appliance, toilet, washbasin, or tile. 25. The method of claim 23, wherein said method is a cleaning in place (CIP). 26. The method of claim 24, wherein the household items are cleaned in an automatic dishwasher. 27. The method of claim 20, wherein the hard surface is included in a health care or animal care product. 28. The method of claim 27, wherein said health care or animal care products are selected from the group consisting of diagnostic, analytical, processing and surgical products. 29. The method of claim 20, wherein the garment is washed in an industrial or household scale washer. 30. The pH of the washing solution is 2 to 7, preferably 3 to 6, for example 4 to 5.
30. The method according to any of claims 20 to 29, wherein 31. The temperature of the washing solution is between 10 and 65 ° C., preferably between 30 and 50 ° C., for example
30. The method according to any of claims 20 to 29, wherein the temperature is 40 <0> C. 32. The method according to claim 20, wherein the washing time ranges from 2 minutes to 20 hours. 33. The method of claim 20, wherein said garment contains wool or silk.