EP1075505A1

EP1075505A1 - An acidic cleaning composition comprising an acidic protease

Info

Publication number: EP1075505A1
Application number: EP99914445A
Authority: EP
Inventors: Hans Sejr Olsen
Original assignee: Novozymes AS
Current assignee: Novozymes AS
Priority date: 1998-03-27
Filing date: 1999-03-25
Publication date: 2001-02-14
Also published as: JP2002509981A; CN1295610A; CN100360651C; AU3326999A; WO1999050380A1

Abstract

This invention relates to an acidic detergent composition comprising an acidic protease which retains proteolytic activity in the presence of an inhibitor selected from the group consisting of pepstatin, Pefabloc, PMSF, or EDTA and at least one nonionic surfactant. The composition further provides methods for cleaning or washing a hard surface or laundry comprising contacting said hard surface or laundry with the composition according to invention dissolved in an aqueous solution in an amount sufficient for providing a cleaning effect.

Description

AN ACIDIC CLEANING COMPOSITION COMPRISING AN ACIDIC PROTEASE

FIELD OF THE INVENTION

The present invention relates to a cleaning composition comprising an acidic substantially pepstatin-insensitive protease and a nonionic surfactant . The composition is suitable for cleaning hard surfaces or cellulosic and/or woolen fabrics at acidic pH.

BACKGROUND OF THE INVENTION

The majority of cleaning compositions used in household and industry today is alkaline.

Industrial cleaning like CIP cleaning, e.g. cleaning of membranes in dairies or other food processing industries, often involves both an acid treatment whereby mineral deposits, e.g. hardness salts (scale) like milk-stone, are removed and an alkaline detergent treatment removing organic matter, e.g. fats, proteins and/or sugars. The process often comprises the following steps:

RINSING → ALKALI → RINSING → ACID → RINSING

Lack of industrially producible acidic proteases effective at acidic pH has obstructed the development of enzymatic cleaning processes for dishes, laundry and industrial and household hard surfaces under acidic conditions, and only few attempts have been made to develop an exclusively acidic cleaning process although there is a desire for such advantageous acidic cleaning compositions.

DE 3833047 Al discloses acidic AD (Automatic Dish Washing) detergent compositions comprising a hydrolase enzyme, wherein the hydrolase may be an amylase, a protease or a lipase.

US 5,698,507 discloses a gelled dishwashing composition having a pH of 3-5 consisting essentially of specified amounts of nonionic surfactant, citric acid, H₂0₂, at least one acid resistant protease enzyme, at least one amylase enzyme, hydrotrope, CaCl₂, sodium formate, a gelling system and water. Specifically named enzymes were Bacillus amyloliquefaciens α- amylases (e.g. Tenase 1200, Tenase L-1200 and Tenase L-340) and Aspergillus niger or Aspergillus oryzae proteases. WO 95/02044 discloses acidic aspartic proteases obtainable from A . aculeatus (denoted protease I and protease II) for use in the production of food, animal feed, beverages, leather and for contact lens cleaning.

WO 96/29978 discloses an acidic oral care composition comprising acidic protease, which in the normal, slightly alkaline oral environment is substantially inactive.

WO 96/23579 discloses cleaning of membranes in a beer filtration process comprising at least a) treatment of the membrane with an enzyme-containing aqueous solution with beta- glucanase, xylanase and cellulase; b) cleaning with an acidic cleaning agent and c) cleaning with a peroxide containing alkaline solution.

SUMMARY OF THE INVENTION We have found that proteases which retain proteolytic activity in the presence of an inhibitor selected from the group consisting of pepstatin, Pefabloc, PMSF, or EDTA) exhibit a surprisingly good cleaning and/or activity performance at acidic conditions compared to other acidic proteases with a similar pH- activity profile. Accordingly, the invention provides advantages over the art of alkaline detergent compositions such as: a) a peroxygen/activator bleach system, e.g. sodium-perborate or percarbonate and TAED activators that can oxidize or bleach poly-aromatic compounds present in soiling or stains becomes more effective even at low temperatures, b) an enzyme-enhancer bleaching system, e.g. peroxidase-PPT or laccase-PPT may be used even at low temperatures.

The acidic condition has in itself a bleaching effect on some types of stains, e.g. coffee and tea, c) an alkaline cleaning step and a rinsing step in industrial hard surface cleaning, e.g. CIP (Cleaning In Place), may be omitted as the acidic detergent composition may remove organic soils as well inorganic soil or stains, d) omission of an alkaline cleaning step will reduce damaging of the hard surface. e) Builder systems usually present in alkaline laundry detergents may in acidic laundry detergents be lowered or even omitted as surfactants usually are not precipitated by water hardness ions at acidic pH. This in turn means that scaling in cleaning equipment, e.g. a automatic laundry washing machine, may be avoided.

The invention provides thus in a first aspect an acidic detergent composition comprising an acidic protease which retains proteolytic activity in the presence of an inhibitor selected from the group consisting of pepstatin, Pefabloc, PMSF, or EDTA) and at least one nonionic surfactant,

for hard surface and laundry cleaning.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The term "hard surface" as used herein relates to any surface which is essentially non-permeable for water. Examples of hard surfaces are surfaces made from metal, e.g. stainless steel or other alloys, plastics/synthetic polymers, rubber, glass, wood, concrete, rock, marble, gypsum and ceramic materials all which optionally may be coated, e.g. with paint, enamel, polymers and the like.

The term "inhibitor" as used herein relates to compounds which competitively or non-competitively interact with the protease thereby reducing and/or destroying the enzyme activity towards the substrate of the enzyme.

The term "retain proteolytic activity" is to be construed as the protease having the property of retaining at least 75% of its activity (residual activity) measured in HPU units at pH 5.5 after treatment of the protease with inhibitor, the inhibitor being either 1 M pepstatin, 0.1% Pefabloc, 0.1% PMSF or 100 mM EDTA. The protease

The protease in the context of the invention is an acidic protease which retains proteolytic activity in the presence of an inhibitor selected from the group consisting of pepstatin, Pefabloc, PMSF, or EDTA. Inhibition by Pepstatin which has the formula:

,CH. o ³ Chiral

O^_/OH .0

HO, CH, H₃C ^*C ^H CH 3

CH,

H₃C NH HN ^O

H

O

CH, O OH , H₃C ^{^}CH,

is described by Muroa S. and Oda K. (1985) .

A characterization of the protease with regard to inhibition in the context of the invention is shown in WO 95/02044. An inhibition test shows that Protease I is not inhibited by pepstatin. Protease II is inhibited by pepstatin. According to Oda K. and Murao S. (1991), acidic proteases are described in two classes as carboxyl or aspartic proteases sensitive to pepstatin and pepstatin-insensitive carboxyl proteinases. Reference is also given to Muroa S. and Oda K. (1985), where this new subclass for acidic proteases was introduced.

PMSF is an inhibitor of the formula:

while PEFABLOC is a protease inhibitor of the formula:

Preferred proteases are obtainable from a micro-organism, e.g. a bacterial strain, e.g. Bacillus , Pseudomonas or

Xanthomonas or a fungal strain (including yeasts) such as species of the genus Aspergillus (e.g. A . aculeatus or A . niger) or

Scytalidium (e.g. S . lignicolum) .

The protease may in a further embodiment be obtainable from a bacterium or fungus, which has been genetically modified by transforming said bacterium or fungus with a DNA vector/construct comprising DNA encoding said protease.

The protease of the invention may in a particular preferred embodiment comprise one or more aspartic and/or carboxylic residues as functional groups in the active center.

Further the protease retains proteolytic activity in the presence of inhibitors present in meat, egg white, whole blood, blood plasma, milk, beer, potatoes or beans. Such inhibitors may be ovomacroglobulin, ovomucoid or ovoglycoprotein. It is presently contemplated that inhibitors (such as competitive or non competitive inhibitors - terms that are known to the skilled person) present in soiling which is desired to be cleaned play an important role in the cleaning process as they may inactivate or reduce activity of enzymes which would otherwise hydrolyze the soiling. This may be the reason why it has been difficult to find suitable proteases for use in acidic cleaning compositions. The protease may further have a preferred pH optimum between 2- 7, more preferred between 3-6 or even more preferred between 4.5-5.5. Also the protease according to the invention may have a temperature optimum between 20-70°C, such as between 20-60°C, e.g. 30-50°C. In a further particular embodiment the protease is Protease I or Protease II obtainable from A. aculeatus as described in WO 95/02044, which -is hereby incorporated by reference. A most preferred protease is Protease I .

The nonionic surfactant

Nonionic surfactants are especially suitable for acidic detergents as they are not functionally affected in a moderate acidic environment. Preferred nonionics are glycolipids, alcoholethoxylates, alkylphenolethoxylates , glucamides, alkyl- polyglucosides (Stache & Kosswig, 1990) .

Other suitable composition components

The composition may optionally comprise a sequestering agent, when water for preparing a washing liquor contains considerable water hardness, i.e. calcium ions which may effect the performance of the protease. Suitable sequestering agents should be capable of sequestering calcium ion at acidic pH. Preferred sequestering agents are methylglycinediacetic acid, nitroloacetic acid, citric acid, oligo and polymeric (poly) carboxylic acid derived from polymer sugars (Kock et al . 1993) , dextrin or protein hydrolysates (DE 19547730 Al) .

The composition may further comprise other components enhancing the detergency of the composition such as softening agents, an amylase (e.g. Fungamyl^® from Novo Nordisk A/S,

Denmark), a lipase (e.g. Novocor^® AD from Novo Nordisk A/S,

Denmark), a cellulase (e.g. Celluzyme^®, Carezyme^®, and/or

Celluclast^®, all from Novo Nordisk A/S, Denmark), a xylanase

(e.g. Biofeed^® PLUS or Shearzyme™ from Novo Nordisk A/S, Denmark), a beta-glucanase (e.g. Viscozyme^® or Ultraflo™ from Novo Nordisk A/S, Denmark), a pectinase (e.g. Pectinex™ Ultra from Novo Nordisk A/S, Denmark), a peroxidase (e .g . Guardzyme™ from from Novo Nordisk A/S, Denmark), a laccase (e.g. obtained from Myceliophthora or Polyporus) , an enhancing agent for the peroxidase/laccase (e.g. PPT or methylsyringic acid methylsyringate or derivatives thereof) and/or a buffer (e.g. citric acid) Finally the composition may be liquid or a powder. In the latter case the protease may suitably be formulated as a stabilized granulate. The formulation may be obtained using conventional methods.

Uses of the composition

The composition according to the invention may suitably be used in methods for cleaning or washing a hard surface or laundry. The methods may preferably comprise contacting said hard surface or laundry with the composition dissolved in an aqueous solution in an amount sufficient for providing a cleaning effect. The protease and the other composition components may alternatively be added to the solution separately. The composition may preferably be dissolved in an amount sufficient for providing an enzyme dosage of 500-3000 HUT/L, preferably 500-1500 HUT/L, more preferably 750-1250 HUT/L, e.g. 1000 HUT/L wash liquor.

The hard surface to be cleaned or washed by said method is in one embodiment preferably industrial process equipment or household equipment .

Specific preferred industrial process equipment may be heat exchangers, tanks, pipes, centrifuges, evaporators, filters, extruders, meat choppers, cooking jars, beer and wine fermentors, beer and wine filters, spent filter aids, coolers, storage tanks, sieves, hydrocyclones, ultrafiltration units, nanofiltration units, hyperfiltration units, microfiltration units and milking machines.

A particular suitable embodiment is cleaning of health care or animal care equipment or products. Health care equipment may comprise diagnostical/analytical (e.g. endoscopes, blood analyzers), processing (e.g. dialysis or blood treatment equipment) or surgical equipment (e.g. scalpels, peens, clips or tweezers, forcepses etc. used by doctors, veterinarians or dentists for treatment of patients) which has been in contact with blood, other body fluids or tissues from humans or animals. With respect to cleaning of industrial process equipment the methods may in particular embodiments be "Cleaning in Place" (CIP) methods.

Specific preferred household equipment may be eating utensils, plates, cups, beakers, glasses, pots, pans, electric appliances, toilet bowls, lavatories or tiles.

Another suitable embodiment of the invention is cleaning of industrial ion exchange columns used in bulk sugar production for removing salts, residues of charged carbohydrates, protein and amino acids, and colored material before crystallization. Furthermore ion exchange columns used in the starch based syrup production before and after the isomerization process may be cleaned using the composition of the invention.

An additional preferred embodiment of the invention is cleaning of ion exchangers used in manufacturing processes where proteins or peptides may cover and/or clog up the resin material with proteinaceous material. Usually such clogged up ion exchangers needs a cleaning process based on excessive alkaline and heat conditions, which reduces the lifetime of the resin and reduces the efficiency of the regeneration process (e.g. by using 85°C hot alkali at pH=13-14 recirculated for about an hour) . Use of the composition of the invention however provides a mild cleaning process, which may secure an efficient removing of soil that hinders an efficient regeneration process of such ion exchangers.

Yet another particularly suitable embodiment of the invention is cleaning of columns used for protein separation by gelfiltration or affinity chromatography . Such columns often contain rather expensive separation and/or chromatography material/resin, which needs to be cleaned effectively if it is used for scaled up processes. Harsh conditions are normally used for removing slimy material such as combination products of protein and carbohydrates, which deposits in the material and these harsh conditions often reduce the life time of the material. Use of the composition of the invention however provides a mild cleaning process, which may secure an efficient cleaning and a prolonged life time of the column material. A preferred cleaning process for cleaning columns containing gelfiltration and affinity chromatography material may comprise re-circulating a cleaning solution containing an enzyme dosage of 500-3000 HUT/L, preferably 500-1500 HUT/L, more preferably 750-1250 HUT/L, e.g. 1000 HUT/L wash liquor at a pH between 2-7, preferably 3-6, e.g. 4-5, while the temperature should be kept between 10-65°C, preferably 30-50°C, e.g. 40°C. The cleaning or washing time is in a preferred embodiment kept between 2 minutes and 20 hours depending on the type of method.

With respect to cleaning of house hold equipment the methods may in particular embodiments be performed in an automatic dishwashing machine.

When using the composition for cleaning or washing of laundry the method may preferably be performed in an industrial scale or household scale washing machine. Preferred laundry is cellulosic fabric and/or non- structured garments such as silk, acetate, wool, ramie, or rayon garments. Cleaning of especially wool and silk according to the invention is useful, as acidic cleaning conditions softens the garments as well as having a antimicrobial effect (i.e. kills or inhibits microbial cells) . The methods for cleaning or washing of hard surfaces or laundry may in one embodiment be performed at a pH between 2-7, preferably 3-6, e.g. 4-5, while the temperature should be kept between 10-65°C, preferably 30-50°C, e.g. 40°C. The cleaning or washing time is in a preferred embodiment kept between 2 minutes and 20 hours depending on the type of method. For instance cleaning of an industrial membrane may provide soaking in a (circulating) solution of the composition for up to over night (up to 20 hours) , while industrial dishwashing should be completed within 2-10 minutes.

MATERIALS AND METHODS

Determination of protease HUT activity:

The HUT activity was determined according to the AF92/2 method published by Novo Nordisk A/S, Denmark. 1 HUT is the amount of enzyme which, at 40°C and pH 4.7 over 30 minutes forms a hydrolysate from digesting denatured hemoglobin equivalent in absorbancy at 275 nm to a solution of 1.10 μg/ml tyrosine in 10

0.006 N HC1 which absorbancy is 0.0084. The denatured hemoglobin substrate is digested by the enzyme in a 0.5 M acetate buffer at the given conditions. Undigested hemoglobin is precipitated with trichloroacetic acid and the absorbance at 275 nm is measured of the hydrolysate in the supernatant .

Proteolytic of protease HPU activity:

1 hemoglobin protease unit (hpu) is defined as the amount of enzyme liberating 1 millimole of primary amino groups (determined by comparison with a serine standard) per minute under standard conditions as described below:

A 2% (w/v) solution of hemoglobin (bovine, supplied by Sigma) is prepared with the Universal Buffer described by Britton and Robinson, J. Chem. Soc . , 1931, p. 1451), adjusted to a pH of 5.5.

2 ml of the substrate solution are pre- incubated in a water bath for 10 min. at 25°C. 1 ml of an enzyme solution containing b g/ml of the enzyme preparation, corresponding to about 0.2-0.3 hpu/ml of the Universal Buffer (pH 5.5) is added. After 30 min. of incubation at 25°C, the reaction is terminated by the addition of a quenching agent (5 ml of a solution containing 17.9 g of trichloroacetic acid, 29.9 g of sodium acetate and 19.8 g of acetic acid made up to 500 ml with deionized water) . A blank is prepared in the same way as the test solution with the exception that the quenching agent is added prior to the enzyme solution. The reaction mixtures are kept for 20 min. in a water bath after which they are filtered through Whatman 42 paper filters.

Primary amino groups are determined by their color development with o-phthaldialdehyde (OPA), as follows: 7.62 g of disodium tetraborate decahydrate and 2.0 g of sodium dodecylsulfate are dissolved in 150 ml of water. 160 mg of OPA dissolved in 4 ml of methanol were then added together with 400 ml of β- mercaptoethanol after which the solution is made up to 200 ml with water. To 3 ml of the OPA reagent is added 400 ml of the filtrates obtained above, with mixing. The optical density (OD) at 340 nm is measured after about 5 min. The OPA test is also performed with a serine standard containing 10 mg of serine in 11

100 ml of Universal Buffer (pH 5.5) . The buffer alone is used as a blank. The protease activity is calculated from the OD measurements by means of the following formula:

5 (OD_t - 0D_b) x C_SEH x Q hpu/ml enzyme solution :

(OD_SER - OD_B) x MW_SER x t,

hpu/g of enzyme preparation = hpu/ml: b

10 wherein OD_t, OD_b, OD_SΞR and 0D₃ is the optical density of the test solution, blank, serine standard and buffer, respectively, C_SER is the concentration of serine (mg/ml) in the standard (in this case 0.1 mg/ml), and MW_SER is the molecular weight of serine (105.09) . 15 Q is the dilution factor for the enzyme solution (in this case 8) and t₁ is the incubation time in minutes (in this case 30 minutes) .

Method for evaluating the composition in automatic 20 dishwashing (ADW)

Stainless steel plates and porcelain dishes had been pre- washed by 75 °C at high alkaline pH before soiling. Efficiency of proteases was tested using a standard laboratory procedure where stainless steel plates were soiled in the following way:

25 15 eggs (white + yolk) and 255 ml of whole milk were mixed at the lowest speed in a Braun UK 20 kitchen mixer machine for 2 minutes. Thereafter the mixed solution passed through a 0.5 mm pore size sieve. Five stainless steel plates were dipped into the egg/milk mixture and placed in a drying rack. After drying

30 over night at room temperature, the plates were baked for 1 hour at 120 °C in a ventilated thermostated oven. To test amylases, five porcelain plates are soiled using a 2% suspension of gelatinized starch solution which is dried overnight at room temperature .

35 For the automatic dishwashing procedure, a 6 persons Cylinda Excellence Kompakt type 770 machine was used. The machine had an ion-exchanger installed binding calcium ions from 12

the inlet water. Temperature was set to 55°C at -program no. 4. This program performed the following: a) Main wash starting with 7 minutes of heating of the cleaning solution from 25°C to 55°C and ending with 10 minutes of washing. b) Cold rinsing water is taken in. This rinsing takes 5 minutes and the temperature during rinsing varies between 35 and 45°C dependent on loading. c) A second rinsing made with cold water which is taken in. During the second rinsing the water is heated from 20°C to 55°C for 8 minutes and then pumped out . d) Drying at 55°C for about 5 minutes.

Before and after washing, light reflection values are measured directly for protein film or for starch films stained with iodine (KI/I₂) . The staining was made using a standard solution which was prepared the following way: 20.0 grams of potassium iodide (KI) Merck art. No. 5043 and 1.27 gram of iodine (I₂) Merck art. No. 4763 was weighed out in a 2 litre beaker and added up to 1.0 litre using ion exchanged water. The solution was stirred for approx. 10 minutes at room temperature.

The plates were pulled slowly through the iodine solution and afterwards placed in drying racks.

Measurements are made using a Minolta Chroma Meter (type CR-300) . Six single values for each plate are used for an average R-value . Calculations of % removed film - RPF% for protein or RSF% for starch - are calculated by the formula:

RPF ( % ) [or RSF ( % ) ] = [R_, after wash - R ',before was hi / [Rciean plate -^before wash ] *100%

Detergent composition used for one machine and the cleaning results obtained are shown in each of the tables belonging to the examples described below. pH was varied by addition of 2-7 ml of 4 N HCl . 13

Method for evaluating the composition in laundry

Test of washing performance was carried out in an commercial European washing machine (AEG model OKO-LAVAMAT JUBILEUM 40) with 2.0 kg of ballast laundry and artificially soiled fabrics. 10 pieces (5x5 cm) of a commercial "standard" swatch (standard cotton fabric) soiled with milk, blood and carbon black (EMPA 116) and 10 pieces of a swatch impregnated with an extract of spinach leaves and later on heat treated at 70°C for 30 minutes were fixed onto the ballast cloth. The washing process was performed at 40°C without pre-wash using a program called "Klarvask" according to the instructions by the vendor, with a final centrifugation at 1400 rpm. During the washing process, a sample of the washing liquor was taken and the pH was measured using a pH-indicator strip type Acilit^® pH 0-6. The washing process was followed by 2 rinsings with intermediate centrifugations at 400 rpm. In the total process a total volume of 50 liter water was used.

Assessment of washed test swatches was done by. measuring the intensity of reflected light, % R, (% remission) remitted from the swatches at 460 nm using a J&M Tidas MMS/16 photometer equipped with a CLX 75W Xenon lamp and fibre optics. Each swatch was measured individually at the top of a stack with 3 or 4 other swatches (in order to diminish the amount of light which may penetrate the textile structure without being absorbed or reflected) .

The value AR_Enz = R_washβd - R_{waεhed vi thout enzyr}._e reflects the contribution of the enzyme for each type of swatch. The results are illustrated as mean values and as confidence intervals e.g. as [% R_washed-W; %R_washed ^+w] / where W is the 95 % confidence value.

Used chemicals/enzymes: a) NTA (nitrilotriacetic acid) available from Fluka Chemika no. 72560) . b) Trilon^® A (NTA-Na₃ - nitrilotriacetic acid trisodium salt) available from BASF - Germany. c) Trilon^® M (MGDA-Na₃ - methylglycinediacetic acid tri sodium salt) available from BASF - Germany 14

d) Dehyphon^® LS 54 (a non-ionic alcoholethoxylate) available from Henkel KGaA - Germany e) Lutensol^® A03 (a non-ionic alcoholethoxylate) available from BASF - Germany f) Lutensol^® AO 7 (a non-ionic alcoholethoxylate) available from BASF - Germany) g) Sokalan^® HP25 (a modified polycarboxylate) available from

BASF - Germany; used as anti-redeposition agent, h) Protease I (1.05 kHUT/g) obtained as described in WO 95/02044. i) Protease II (5.22 kHUT/g) obtained as described in WO

95/02044. j) Flavourzyme^® (65.2 kHUT/g) available from Novo Nordisk A/S, Denmark . k) Fungamyl^® available from Novo Nordisk A/S, Denmark.

EXAMPLES

Example 1 : In this example the effect of the protease samples in ADW is demonstrated. The data are shown in Table 1.

Table 1

Detergent Trial Trial 1.2 Trial 1.3 Trial 1.4 1.1

Citric acid 3.0 g 3.0 g 3.0 g 3.0 g

NTA-Na₃ 5.0 g 5.0 g 5.0 g 5.0 g (Trilon A)

Dehyphon LS 54 1.6 g 1.6 g 1.6 g 1.6 g

Na₂S0₄ 10 g 10 g 10 g 10 g

Water: Volume : Volume : Volume : Volume :

Ion-exchanged 4 L 4 L 4 L 4 L in machine to 2-3° dH

4 N HC1 0 (pH 0 (pH was 0 (pH was 0 (pH was was 4.5) 4.5) 4.5) 4.5)

Proteases : Activity dosed 2.1 kHUT 130.4 kHUT 10.44 kHUT No enzyme and enzyme from from from 15

Detergent Trial Trial 1.2 Trial 1.3 Trial 1.4 1.1 type Protease Flavourzyme Protease II I

% RPF: 60 16 20 12

Table 1 shows that Protease I gives the highest cleaning value (RPF%) even though the dosed HUT-activity is considerably less than for Flavourzyme and Protease II.

Example 2 :

In this example the effect of variation of pH on the performance of Protease I in ADW is demonstrated. pH was varied in the trials by dosing variable amounts of 4 N HC1. The data are shown in Table 2.

Tabel 2

Detergent Trial 2.1 Trial 2.2 Trial 2.3

Citric acid 3.5 g 3.5 g 3.5 g

NTA-Na₃ (Trilon A) 3.0 g 3.0 g 3.0 g

Dehyphon LS 54 1.6 g 1.6 g 1.6 g

Na₂S0₄ 10 g 10 g 10 g

Water: 4 L 4 L 4 L

Ion-exchanged in machine to 2-3° dH

Mass of 4 N HC1 1.64 g 3.48 g 6.88 pH 4.5 4.0 3.2

Protease : 4.2 kHUT .2 kHUT No enzyme

Activity dosed and from from enzyme type Protease I Protease I

% RPF: 88 85 12

Table 2 shows that Protease I gives nearly the same cleaning value (RPF%) at pH 4 and pH 4.5. Even more acidic conditions (pH 3.2) without enzymes could not clean the plates to any visible degree. 16

Example 3 : '

In this example the effect of additionally adding Fungamyl to a detergent and using porcelain plates coated with starch is tested. The data are shown in Table 3.

Table 3

Detergent Trial Trial Trial Trial 3.1 3.2 3.3 3.4

Citric acid 3.2 g 3.2 g 3.2 g 3.2 g

NTA-Na₃ 3.0 g 3.0 g 3.0 g 3.0 g (Trilon A)

Dehyphon LS 54 1.6 g 1.6 g 1.6 g 1.6 g

Na₂S0₄ 10 g 10 g 10 g 10 g

Water: 4 L 4 L 4 L 4 L

Ion-exchanged in machine to 2-3° dH

Mass of 4 N HC1 3.5 g 3.5 g 3.5 g 3.5 g pH 4.0 4.0 4.0 4.0

Protease : Activity dosed 4.2 kHUT 4.2 kHUT No No and enzyme type from from

Protease Protease I I

Amylase :

Mass of Fungamyl 0.12 g 0 0.12 0

800 L

% RPF 76 75 3 9

% RSF 55 14 17 12

Table 3 shows the clear effect of Protease I on removal of protein whether the amylase was included or not. The amylase showed a minor effect when the Protease I was not included.

Example 4:

In this example the hydrolytic effects of detergent solutions with acidic pepstatine-insensitive proteases were tested with and without egg white. Also the effect of using tap 17

(slightly hard) water and ion exchanged water was tested. The data are shown in Table 4.

Freeze dried hemoglobin (Novo Nordisk - Denmark) , was dissolved in tap water (18° dH (German degree water hardness) , or in demineralized water to a 20 g/1 solution.

1000 ml of a detergent solution was made by suspending/dissolving the following ingredients: a) 12 g NTA b) 20 g Lutensol® A07 c) 62 g Na₂S0₄ in demineralized water to 1000 ml.

To 100 ml of the hemoglobin solution was added 5 g of the detergent solution in an Erlenmeyer flask with magnet stirring. pH was adjusted to 4.5 using NaOH. The flask was placed in a thermostated water bath at 40 °C . 1000 μL of Protease I was added and samples were taken at t=l minute and at t=30 minutes for measurement of osmolality (mOSM/kg H₂0) using an Osmometer, Type:Wide Range Osm.3 W 2 from Advanced Instruments. The result of the measurement may be presented as the difference of the two measurements as ΔmOSM/kg H₂0.

To test the inhibitory effect of e.g. egg white 5 ml of gently homogenised egg white was added to the reaction mixture before enzyme addition.

Table 4

Detergent Trial 4.1 Trial 4.2 Trial 4.3

NTA(Fluka) 0.06 g 0.06 g 0.06 g

Lutensol^® AO 7 0.10 g 0.10 g 0.10 g

Na₂S0₄ 0.31 g 0.31 g 0.31 g

Water:

Ion-exchanged 4.53 g 4.53 g 4.53 g

Substrate :

1) Hemoglobin 2.0 g 2.0 g 2.0 g

2) Egg white 0 0 5 ml

Substrate-Water : o g 95.0 g o g From the tap Detergent Trial 4.1 Trial 4.2 ^'Trial 4.3

Substrate-Water : Ion exchanged 95.0 g 0 95.0 g pH 4.5 4.5 4.5

Protease : 3.0 kHUT 3.0 kHUT 3.0 kHUT

Activity dosed and from from from enzyme type Protease I Protease I Protease I OSM/kg H₂0 at t=l minute 56 67 63 at t=30 mnutes 66 75 78 ΔmOSM/kg H₂0 10 8 15

Table 4 shows that Protease I effectively hydrolyzes the hemoglobin protein whatever the water used is from the tap or it is ion exchanged. Furthermore it is shown clearly that protease I is not to any degree inactivated by egg white. I fact because a higher value of ΔmOSM/kg H₂0 was found when the egg white was present, Protease I also hydrolyzes this protein. The 5 ml egg white add ca. 0.6 g of protein which is further hydrolyzed.

Example 5 :

In this example the hydrolytic effects of detergent solutions with different acidic pepstatin-insensitive proteases were tested by the method in example 4 using tap (slightly hard) water and ion exchanged water. The data are shown in Table 5.

Table 5

Detergent Trial 5.1 Trial 5.2 Trial 5.3 Trial 5.4

NTA-Na₃ 0.04 g 0.04 g 0.04 g 0.04 g (Trilon^® A)

NTA(Fluka) 0.02 g 0.02 g 0.02 g 0.02 g

Lutensol^® AO 3 0.03 g 0.03 g 0.03 g 0.03 g

Lutensol^® AO 7 0.07 g 0.07 g 0.07 g 0.07 g Sokalan^® HP 25 0.07 g 0.07 g 0.07 g 0.07 g 19

Detergent Trial 5.1 Trial 5.2 Trial 5.3 Trial 5.4

Water :

Ion-exchanged 4.77 g 4.77 g 4.77 g 4.77 g

Substrate :

1) Hemoglobin 2.0 g 2.0 g 2.0 g 2.0 g

Substrate- 0 0 95.0 g 95.0 g

Water:

From the tap

Substrate- 95.0 g 95.0 g 0 g 0 g

Water :

Ion exchanged pH 4.5 4.5 4.5 4.5

Protease : 3.0 kHUT 65.2 kHUT 3.0 kHUT 65.2 kHUT

Activity dosed from from from from and enzyme Protease I FlavourProtease I Flavourtype zyme^® zyme^® mOSM/kg H₂0 at t=l minute 56 40 58 60 at t=30 80 56 74 79 minutes

ΔmOSM/kg H₂0 24 16 16 19

Table 5 shows that Protease I contributes to a higher hydrolytic effect in ion exchanged water even though the dosed HUT-activity is considerably less than for Flavourzyme. In tap slightly harder water the hydrolytic effects are comparable even though the dosed HUT-activity is considerably less that for Flavourzyme .

Example 6 :

In this example the laundry washing performance of protease I was tested in a suitable slightly acidic laundry detergent composition. 20

Table 6

Detergent Trial 6.1 Trial 6.2

MGDA-Na₃ (Trilon^® M) 13.8 g 13.8 g

Lutensol^® AO 3 3.3 g 3.3 g

Lutensol^® AO 7 6.4 g 6.4 g

Sokalan^® HP 25 6.3 g 6.3 g

Citric acid 11.6 g 11.6 g

Na₂S0₄ 28.6 g 28.6 g

Cloth with test pieces 2 kg 2 kg

Water per wash 15 liter 15 liter pH in the wash water ca. 4 3.5-4

Protease : none 300 kHUT

Activity dosed and enzyme type from Protease I

Spinach:

Number of measurements: 12 18

Average % R₍₁₎ 18.7 20.8

W (95%) 0.5 0.6

[% R₍₁,-W; %R ₎+W] [18.2;19.2] [20.2,-21.4]

Δ-R_Enz 2.1

EMPA 116:

Number of measurements: 12 24

Average % R_(l) 16.1 20.2

W (95%) 0.9 0.7

[% R ₎-W; %R₍₁₎+W] [15.2,-17.0] [19.6;20.9] Δ-R_Enz 4.1

Table 6 shows that Protease I under these mild conditions significantly contributes to a cleaning of the two different swatches. The Δi?_enz-values obtained are visible

Example 7 :

In this example the sensitivity of Protease I and Protease II towards four different possible inhibitors all available from Sigma except for PEFABLOC which is available from Pentapharm, Basel, Switzerland were tested. EDTA is a metalloenzyme 21

inhibitor, while PEFABLOC and PMSF are serine protease inhibitors. Proteolytic activity was measured in HPU/1 at pH 5.5 protease solutions before and after treatment with the inhibitors.

Inhibition tests gave the following results:

% Residual activity

EDTA 100 Pepstatin PEFABLOC PMSF 0.1% mM 1 mM 0.1%

Protease I 104 91 83 92

Proteasell 97 9 90 108

The test showed that Protease I retained activity in the presence of any of the inhibitors, while Protease II retained activity in the presence of EDTA, PEFABLOC and PMSF, but was inhibited by Pepstatin.

REFERENCES

Muroa S. and Oda K. Pepstatin-insensitive acid proteinases, Asparctic proteinases and their inhibitors, Kostka V. (editor) pp 379-399, (1985) Walter de Gruyter, Berlin.

Kock H., Beck R. , Roper H. , Starch-Derived Products for Detergents, Starch/Starke, 45, pp 2-7, 1993.

Stache H., Kosswig K. , (editors) Tensid-Taschenbuch, 3 edition, Carl Hanser Verlag, Mύnchen, 1990.

Oda K. , and Murao S . ; Pepstatine-insensi tive carboxyl proteases; Structure and Function of the Aspartic Proteases; Dunn B.M. (Ed), Plenum Press, New York, 1991.

Claims

22CLAIMS

1. An acidic detergent composition comprising an acidic protease which retains proteolytic activity in the presence of an inhibitor selected from the group consisting of pepstatin, Pefabloc, PMSF, or EDTA and at least one nonionic surfactant.

2. The composition according to claim 1, wherein said protease is obtainable from a bacterium, or a fungus including yeasts

3. The composition according to claim 2, wherein said bacterium is of the genus Bacillus, Pseudo onas or Xanthomonas .

4. The composition according to claim 2, wherein said fungus is of the genus Aspergillus or Scytalidium .

5. The composition according to claim 4, wherein said fungus is A . aculeatus .

6. The composition according to claims 2-5, wherein said bacterium or fungus is genetically modified by transforming said bacterium or fungus with a DNA vector comprising DNA encoding said protease.

7. The composition according to claim 1, wherein the protease comprises an aspartic residue as a functional group in the active center.

8. The composition according to claim 1, wherein the protease comprises a carboxylic residue as a functional group in the active center.

9. The composition according to claim 1, wherein the protease further retains proteolytic activity in the presence of an ovomacroglobulin inhibitor. 23

10. The composition according to claim 1, wherein the protease further retains proteolytic activity in the presence of an ovomucoid inhibitor.

5 11. The composition according to claim 1, wherein the protease further retains proteolytic activity in the presence of an ovoglycoprotein inhibitor.

12. The composition according to claim 1, wherein said protease 10 further retains proteolytic activity in the presence of inhibitors present in products selected from the group consisting of meat, egg white, whole blood, blood plasma, milk, beer, potatoes or beans.

15 13. The composition according to any preceding claim, wherein said protease is protease I or protease II.

14. The composition according to claim 13, wherein said protease is protease I .

20

15. The composition according to claim 1, wherein said composition further comprises a sequestering agent.

16. The composition according to claim 1, wherein said nonionic 25 surfactant is a glycolipid, alcoholethoxylate, alkylphenol- ethoxylates, glucamides, or alkylpolyglucosides .

17. The composition according to claim 15, wherein said sequestering agent is capable of binding calcium ions at acidic

30 pH.

18. The composition according to claim 17, wherein said sequestering agent is methylglycinediacetic acid, nitroloacetic acid, citric acid, oligo and polymeric (poly) carboxylic acid

35 derived from polymer sugars, dextrin, or protein hydrolysates .

19. The composition according to claim 1 further comprising a softening agent, an amylase, a lipase, a cellulase, a xylanase, a beta-glucanase, a pectinase, a peroxidase, a laccase, an

40 enhancing agent for the peroxidase/laccase and/or a buffer. 24

20. A method for cleaning or washing a hard surface or laundry comprising contacting said hard surface or laundry with the composition according to any of the preceding claims dissolved in an aqueous solution in an amount sufficient for providing a cleaning effect .

21. The method according to claim 20, wherein the protease is present in the aqueous solution in a range selected from the group consisting of 500-3000 HUT/L, 500-1500 HUT/L, preferably 750-1250 HUT/L and 1000 HUT/L wash liquor.

22. The method according to claim 20, wherein the hard surface is comprised in industrial process equipment or household equipment .

23. The method according to claim 22, wherein said industrial process equipment is heat exchangers, tanks, pipes, centrifuges, evaporators, filters, extruders, meat choppers, cooking jars, beer and wine fermentors, beer and wine filters, spent filter aids, coolers, storage tanks, sieves, hydrocyclones, ultrafiltration units, nanofiltration units, hyperfiltration units, microfiltration units, ion exchanger columns, gel filtration columns or milking machines.

24. The method according to claim 22, wherein said household process equipment is eating utensils, plates, cups, beakers, glasses, pots, pans, electric appliances, toilet bowls, lavatories or tiles.

25. The method according to claim 23, wherein said method is a cleaning in place (CIP) method.

26. The method according to claim 24, wherein said house hold process equipment is cleaned in an automatic dish washing machine . 25

27. The method according to claim 20, wherein said hard surface is comprised in health care or animal care equipment .

28. The method according to claim 27, wherein said health care 5 or animal care equipment is selected from the group consisting of diagnostical , analytical, processing and surgical equipment

29. The method according to claim 20, wherein said laundry is cleaned in an industrial scale or household scale washing

10 machine.

30. The method according to claims 20-29, wherein the pH of the cleaning solution is between 2-7, preferably 3-6, e.g. 4-5.

15 31. The method according to claims 20-29, wherein the temperature of the cleaning solution is between 10-65┬░C, preferably 30-50┬░C, e.g. 40┬░C.

32. The method according to claims 20-29, wherein the cleaning 20 period is between 2 minutes and 20 hours.

33. The method according to claim 20, wherein the laundry comprises wool or silk.