MXPA97005968A - Baci proteases - Google Patents

Abstract

The present invention relates to the field of detergent proteases that can be obtained from strains of a new Bacillus sp. I 612. Moreover, the invention is directed towards a process for the preparation of the protease, the use of the protease as a detergent enzyme, and detergent compositions containing the protease of the invention.

Description

"PROTEASAS DE BACILOS" FIELD OF THE INVENTION c The present invention relates to detergent proteases that can be obtained from strains of bacillue sp. More specifically, the invention is directed towards a new alkaline protease derived from a strain of bacillus spp. I 612. In addition, the invention is directed towards A process for the preparation of the protease, the use of the protease as a detergent enzyme, and detergent compositions comprising the protease of the invention.

BACKGROUND OF THE INVENTION Detergent enzymes have been marketed for more than 20 years, and have been well established as normal detergent ingredients in both liquid detergents and powder detergents worldwide. Q The enzymes used in washing formulations comprise many different enzymes such as proteases, lipases, amylases, cellulases, as well as other enzymes, or mixtures thereof. Commercially, the most important enzymes are proteases. 25 Detergent proteases have been developed by - REF: 25167 isolation of proteases found in nature, followed by tests in detergent formulations. Most detergent proteases are obtained from members of the bacillus genus. Examples of commercial protease products are: ALCALASE ™, ESPERASE ™ and SAVINASE ™, all supplied by - TM Novo Nordisk A / S, Denmark. The ALCALASE protease is produced by strains of the Bacillus licheniformis species. The TM TM teasas ESPERASE and SAVINASE are obtained by cultivation of alkalophilic Bacilli strains. Washing traditions, especially the washing temperature used, the hardness of the water used, and the ingredients of the detergents, vary greatly from one country to another. Characteristic conditions are summarized below: - Low pH and low water hardness: liquid detergents in the US and Asia; - Low pH and high water hardness: liquid detergents in Europe; - High pH and low water hardness: powder detergents in £ U and Asia; and high pH and high water hardness: powder detergents in Europe. (A low pH in detergents is typically a pH in the range of 8.0-9.5, in particular of around 9, an al-to-pH in detergents, is typically a pH in the range of 10-11.5, in particular of about 10.5 A hardness of the low water is typically in the range of 3 - 6 ° dH, a hardness - of the high water is typically in the range of 15 - 20 ° dH, particularly around 18 ° dH). In addition, the detergent compositions have changed these years to make the washing process more favorable with the environment. All these differences and changes within the detergent industry make the field of action extremely complicated. There is, therefore, the need to find new proteases which optimally perform certain specific conditions.

SUMMARY OF THE INVENTION It is an object of the present invention to provide new detergent proteases with improved wash performance at moderate to low wash temperatures. Accordingly, in this first aspect, the invention provides a protease characterized by: - possessing immunochemical properties identical or partially identical to those of a protease derived from the bacillus strain Sp. I 612, DSM 9701. In In a second aspect, the invention relates to the isolation of a biologically pure culture of a strain of Bacillus sp. represented by the strain -facillus 3p * 612.

In a more specific aspect, the invention is related to a strain of bacillus sp. I 612, DSM 9701, or a mutant or a variant thereof. In a third aspect, the invention provides a process for the preparation of the protease, wherein the method comprises the cultivation of a protease that is produced in a strain of bacillus sp. I 612 in a suitable nutrient medium which contains carbon and nitrogen sources and inorganic salts, followed by the recovery of the desired enzyme. In a more specific aspect, bacillus sp. I 612, DSM 9701, or a mutant or a variant thereof, or other host organism carrying with it the gene of a protease having immunochemical properties identical or partially identical to those of the protease derived from bacillus sp. I 612 that is cultivated. In a fourth aspect, the use of the enzyme as a detergent enzyme is demanded. In more specific aspects, the invention provides detergent compositions and detergent additives containing the protease. In a fifth aspect, the invention provides a washing process which comprises the addition of the protease.

BRIEF DESCRIPTION OF THE FIGURES The present invention is further illustrated in relation to the accompanying figures, in which: Fig. 1 shows the relationship between the temperature and the proteolytic activity of an enzyme according to the invention (the preparation of the enzyme obtained in accordance with Example 1, with 1% casein as substrate and a pH of 9.5); Fig. 2 shows the relation between the pH and the proteolytic activity of an enzyme according to the invention (the preparation of the enzyme obtained according to Example 1, with 1% of casein as substrate and a temperature of 25 ° C).

DETAILED DESCRIPTION OF THE INVENTION The Microorganism The new microorganism of the invention, capable of -producing an enzyme of the invention, is represented by the-strain that is isolated from a soil sample. The bacillue sp. I 612 has been closed according to the Budapest Treaty on the International Recognition of Deposits - of Microorganisms for the Purpose of Patent Procedures, on January 30, 1995 at DSM - Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH - under the Access No. DSM 9701. The microorganism of this invention is an aerobic spore that forms a bacterium belonging to the genus Bacillus. Morphologically, it can be described as rods with a diameter of 0.8 - 1.0 microns, and a length of 2-5 microns. The spores are cylindrical to ellipsoid, not exalting the sporangium, central to subterminal. The optimum temperature for growth is within 25-35 ° C, and the optimum pH for growth is within 7 - 9.5, with a loose growth at a pH of 10.

Cultivation of the Microorganism The microorganism of the invention can be cultured under aerobic conditions in a nutrient medium containing nitrogen and carbon assimilable together with other e-sential nutrients; the medium is composed according to the principles known in the art. Suitable carbon sources are such carbohydrates -such as sucrose, glucose and starch, or carbohydrates containing materials such as cereal grains, malt, rice and sorghum. The concentration of the carbohydrate incorporated in the medium can vary greatly, e.g. up to 25% and up to 1 - 5%, but usually 8 - 10% will be adequate; the percentages are calculated as glucose equivalents.

The source of nitrogen in the nutrient medium can be of an inorganic and / or organic nature. Suitable inorganic sources of nitrogen are nitrates and ammonium salts. Among the organic sources of nitrogen a large number of these are regularly used in fermentation processes involving the cultivation of bacteria. Illustrative examples are: soybean meal, cottonseed meal, peanut flour, casein, corn, corn infusion liquor, yeast extract, urea, and albumin. In addition, the medium will also contain traces of usual substances. The new bacillus species of this invention have, for their growth, a neutral pH-optimum. Therefore, cultivation is preferably conducted with a pH of 7-9. For cultivation in fermenting tanks, it is necessary to use artificial aeration. The aeration index is similar to that used in conventional fermentation tanks. After fermentation, the liquid concentrates of the enzyme can be produced by the transfer of the raw material from the broth or, if desired, the concentration of the broth by, e.g. , evaporation at low temperature or by reverse osmosis. Finally, some condoms can be added to the concentrate. Preparations for the solid enzyme can be prepared from the purified and / or concentrated broth by pre-precipitation with salts, such as Na? S0 or water miscible solvents, such as ethanol or acetone. Removal of water in the broth by suitable drying methods, such as spray drying, can also be employed.

Test for Proteolytic Activity Proteolytic activity is determined with casein as substrate. A Casein Protease Unit (CPU) is defined as the amount of enzyme that releases 1 mM of non-primary arni groups (determined by comparison with a serine standard) per minute under standard conditions, i.e. incubation for 30 minutes at 25 ° C and pH 9.5. A prospectus AF 228, which describes the analytical method, is available by application to Novo Nordisk A / S, Denmark, where said prospectus is included herein for reference.

The Enzyme The enzyme of the invention is a new detergent protease. This is obtained by cultivation of a microorganism of the invention, preferably baciliu sp. I 612, DSM 9701, or a mutant or a variant thereof, in a suitable nutrient medium which contains carbon and nitrogen sources as well as inorganic salts. The enzyme can also be obtained by recombinant DNA technology. The protease of the invention can be characterized by the physico-chemical properties described below.

Physical-Chemical Properties A molecular weight of 31 kD, determined by SDS-PAGE A pl of about 8.0 will be determined by isoelectric convergence on LKB Ampholine PAG plates. The -protease activity is inhibited by PMSF, alpha-1-antitrypsin, and EDTA.

The soy protein inhibitor does not influence the protease activity. The result found indicates that the protease of the invention is inhibited by both PMSF and EDTA, which is very surprising; an inhibition by PMSF shows that the protease is a serine protease, but normally the serine proteases tolerate EDTA, and furthermore, it is extraordinary that the protease of the invention is inhibited by EDTA and at the same time it is a very good washing performer, as shown in Example 2. The ratio of activity to temperature is determined with 1% casein as substrate and with a pH of 9.5. The test for proteolytic activity previously described is used by modifying the incubation temperature - which varies in the range of about 10 to 70 ° C. The result is shown in Fig. 1. It is apparent from the Figure that the enzyme possesses proteolytic activity ranging from temperatures below 10 ° C to about 40 ° C, and has an optimum temperature of about 30 ° C . As can be seen from Figure 1, the protease has less than 20% activity at 40 ° C. The dependence of the activity on the pH is determined by the same procedure using regulators that adjust to predetermined pH values in the pH range -of around 6 to 11. The result is shown in Fig. 2. As apparent from the figure, the enzyme possesses proteolytic activity at pH values below 6 to about 10, with an optimum pH in the range of about pH 7 to pH 9.5. The protease of the invention possesses special potentials in detergents with medium ionic strength and medium pH, and moderate to low wash temperatures.

Immunochemical Properties The protease of the invention possesses identical or partially identical (i.e. at least -partially identical) immunochemical properties to those of a protease derived from the bacillus sp strain. I 612, DSM 9701. The immunochemical properties for several proteases - bacillus are, in fact, a very eminent characteristic: while the optimum pH, the optimum temperature, pl, etc. as they were defined above they are more or less the same, different immunochemical properties will result in a very different stability in various detergents. The immunochemical properties can be determined immunologically by identity tests of opposite reaction. The identity tests can be carried out by the well known Ouchterlony double immunodiffusion procedure, or by tandem crossed immunoelectrophoresis according to N. H. Axelsen; Handbook of Immunoprecipitation-in-Gel Techniques Black ell Scientific Publications (1983), chapters 5 and 14. The terms "antigenic identity" and "partial antigenic identity" are described in the same book, chapters 5, 19 and 20. The monospecific antiserum it is generated according to the aforementioned method, by the immunization of rabbits with the purified protease of the invention. The immunogen is mixed with Freund's adjuvant and injected subcutaneously - in the rabbits every second week. The antiserum is obtained after a total immunization period of 8 weeks, and the immunoglobulin is prepared from there as described by N. H. Axelsen, supra. Using the test double immunodiffusion Ouchterlo ny described above, the protease of the invention shows no known opposite reaction to the serine protease: TM - ALCALASE (available from Novo Nordisk A / S) TM - SAVINASE (available from Novo Nordisk A / S) TM - ESPERASE (available from Novo Nordisk A / S) - subtilisin Novo (available from Novo Nordisk A / S), TM - Kazusase (available by Showa Denko), - the Bacillus proteases described "WO 92/07067 in - the Bacillus proteases described in WO 92/17576, the Bacillus proteases described in WO 92/17577, - the Bacillus proteases described in WO 92/17578, - the Bacillus proteases described in WO 93/18140, - the Bacillus proteases described in WO 93/24623, - the Bacillus proteases described in WO 94/01532, and -. the Bacillus proteases described in WO 95/07350 Bacillus proteases tolerate various several detergents, with a great variety, and one of the best tools today for the differentiation between the Bacillus proteases, is the immunochemical identity tool.

Detergent Compositions In accordance with the invention, the protease can typically be a component of a detergent composition, e.g., a dishwashing detergent composition or a laundry detergent composition. As such, it can be included in the detergent composition in the form of a granular non-powder, a stabilized liquid, or a protected enzyme. Granular non-powders may be produced, e.g., as disclosed in US 4,106,991 and 4,661,452 (both by Novo Industri A / S) and may optionally be covered by known methods in the art. Examples of waxy layer materials are products -poly (ethylene oxide) (polyethylene glycol, PEG) with average molecular weights of 1000 to 20,000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are from 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids. Examples of film-forming layer materials suitable for application by fluid layer techniques are given in patent GB 1483591. Preparations for liquid enzyme can be, for example, stabilized by the addition of a polyol such as propylene glycol, a sugar or an alcohol of some sugar, lactic acid or boric acid according to established methods. Other stabilizers of the enzyme are well known in the art. The protected enzymes can be prepared according to the method disclosed in EP 238,216. The detergent composition of the invention may be in any convenient form, e.g. as dust, granules, pas ta or liquid. A liquid detergent can be aqueous, typically containing up to 70% water and 0-30% organic solvent, or non-aqueous solvent. The detergent composition comprises one or more surfactants, each of which may be anionic, nonionic, cationic, or ionic with positive and negative charge. The detergent will usually contain 0-50% anionic surfactant such as linear alkylbenzene sulfonate (LAS), alpha-olefinsulfonate (AOS), alkyl sulfate (fatty alcohol sulfate) (AS), alcohol ethoxysulfate (AEOS or AES), alkanesulfonates -secondary (SAS), esters of alpha-sulfo-methylic fatty acid, alkyl- or alkenyl-succinic acid, or soap. It may also contain 0-40% of nonionic surfactant such as alcoholic acid (AEO or AE), carboxylated alcohol ethoxylates, nonylphenyl ethoxylate, alkyl polyglycoside, alkyldimethylamine oxide, ethoxylated monoethanolamide fatty acid, fatty acid. of monoethanolamide, or polyhydroxy-alkyl amide fatty acid (eg as described in WO 92/06154). The detergent composition may further comprise one or more other enzymes, such as amylase, lipase, kinase, cellulase, peroxidase, and oxidase, e.g., laccase. The detergent may contain from 1 - 65% of a detergent builder or complex agent such as zeolite, diphosphate, triphosphate, phosphonate, citrate, nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), diethylenotriaminepentaacetic acid (DTMPA), alkyl acid - or alkenyl-cyclic, soluble silicates or silicates that form layers (eg SKS-6 of Hoechst). The detergent can also be a non-built detergent, i.e. essentially free of constructor -detergent. The detergent may comprise one or more polymers. Examples of these are carboxymethylcellulose (CMC), alc? Holl-.β-livinyl (PVA), polycarboxylates such as polyacrylates, maleic / acrylic acid copolymers and copolymers of the acid lauryl methacrylate / acrylic. The detergent may contain a bleaching system which may comprise an H 0 such as perborate or bicarbonate percarbonate and which may be combined with a peracid-forming bleach activator such as tetraacetylethylene diamine (TAED) or nonanoyloxybenzenesulfonate (NOBS). Alternatively, the subject of bleaching may comprise peroxyacids of, e.g., amide, imide, or sulfone type. Enzymes of the detergent composition of the invention can be stabilized using conventional stabilizing agents, e.g. a polyol such as propylene glycol or glycerol, a sugar or an alcohol of some sugar, lactic acid, boric acid, or a derivative of boric acid such as, eg, an aromatic borate ester, and the composition can be formulated as it is described in, eg, WO 92/19709 and WO 92/19708. The detergent may also contain other conventional detergent ingredients such as, .g., Texture conditioners including clays, foam boosters, foam suppressants, anti-corrosion agents, stain-suspending agents, anti-stain agents, stains, bactericides, optical brighteners, or perfume. The pH (measured in aqueous solution in use concentration) will usually be neutral or alkaline, e.g. in the range of 7-11. Particular forms of detergent compositions within the application range of the invention include: 1) A detergent composition formulated as a granular composition having a mass density of at least 600 g / 1 which comprises: Linear alkyl benzene sulfonate 7 - 12% (calculated as acid) Alcoholic ethoxy sulfate (eg alcohol C_. 1t-f 1-2 EO) or sulfate of 1 - 4% ¡.-LO alkyl (eg c16_18) Alcoholic ethoxylate (eg alcohol 5 - 9% C14-15 '7 E ° Sodium carbonate (as Na CO) 14 - 20% Soluble silicate (as Na ~ 0, 2SiO_) 2 - 6% Zeolite (as NaAlSio) 15 - 22% Sodium sulfate (as Na SO) 0 - 6% Sodium citrate / citric acid 0 - 15% (as C6H5Na307 / C6H807) Sodium perborate (as NaBO .H 0) 11 - 18% TAED 2 - 6% Carboxymethylcellulose 0 - 2% Polymers (eg acid copolymer 0 - 3% maleic / acrylic, PVP, PEG) Enzymes (calculated as protein of 0.0001 - 0.1% pure enzyme) Minor ingredients (eg foam suppressants, perfume, optical brighteners, 0 - 5% photo-bleach) 2) A detergent composition formulated as a granular composition having a mass density of at least 600 g / 1 which comprises: Linear alkyl benzene sulfonate 6 - 11% (calculated as acid) Alcoholic ethoxysulfate (e.g. alcohol C? _0, 1-2 E0 or alkyl sulfate 1-3% (e-g 'C16-18) Alcohol ethoxylate (e.g. alcohol 5-9%) C14-15 '7 E ° > Sodium carbonate (as Na C0) 15 - 21% Soluble silicate (as Na 0, 2SiO) 1 - 4% Zeolite (as NaAlSiO) 24 - 34% Sodium sulphate (as Na SO) 4 - 10% Sodium citrate / citric acid 0 - 15% (as C6H5Na307 / C6H807) Carboxymethylcellulose 0-2% Polymers (eg copolymer of acid 1 - 6% maleic / acrylic, PVP, PEG) Enzymes (calculated as 0.0001 - 0.1% protein pure enzyme) Minor ingredients (eg suppressors 0 - 5% foam, perfume) 3) A detergent composition formulated as a granular composition having a mass density of at least 600 g / 1 which comprises: Linear alkyl benzene sulfonate 5-9% (calculated as acid) Alcoholic ethoxylate (e.g. alcohol 7 - 14% C12-15 '7 E ° > Soap as a fatty acid (eg acid 1 - 3% fatty C16_22) Sodium carbonate (as Na C0 ,,) 10 - 17% Soluble silicate (as Na 0, 2SiO) 3 - 9% Zeolite (as NaAlSiO) 23 - 33% Sodium sulphate (as Na_S0.) 0 - 4% Sodium perborate (as NaBO.HO) 8 - 16% TAED 2 - 8% Phosphonate (eg EDTMPA) 0 - 1% Carboxymethylcellulose 0 - 2% Polymers (eg acid copolymer 0 - 3% maleic / acrylic, PVP, PEG) Enzymes (calculated as protein of 0.0001 - 0.1% pure enzyme) Minor ingredients (eg superabs. 0 - 5% foam, perfume, optical lightening) 4) A detergent composition formulated as a granular composition having a mass density of at least 600 g / 1 which comprises: linear alkylbenzenesulfonate 8-12% (calculated as acid) Alcoholic ethoxylate (e.g., alcohol 10-25%) C12-15 '7 E ° > Sodium carbonate (as Na C0) 14 - 22% Soluble silicate (as Na 0, 2SiO) 1 - 5% Zeolite (as NaAlSiO) 25 - 35% Sodium sulfate (as Na SO) 0 - 10% Carboxymethylcellulose 0 - 2 % Polymers (eg 1 - 3% maleic / acrylic acid copolymer, PVP, PEG) Enzymes (calculated as protein of 0.0001 - 0.1% pure enzyme) Minor ingredients (eg suppressors 0 - 5% foam, perfume) ) An aqueous liquid detergent composition which comprises: Linear alkylbenzenesulfonate 15-21% (calculated as acid) Alcoholic ethoxylate (eg alcohol 12 - 18% C12-15 '7 E0 ° C12_15 alcohol, 5 EO) Soap as fatty acid (eg 3 - 13% oleic acid) Alkenyl succinic acid (C) 0 - 13% Aminoethanol 8 - 18% Citric acid 2 - 8% Phosphonate 0 - 3% Polymers (eg PVP, PEG) 0 - 3% Borate (as B 0) 0 - 2% Ethanol 0 - 3% Propylene glycol 8 - 14% Enzymes (calculated as protein of 0.0001 - 0.1 pure enzyme) Minor ingredients (eg dispersants, suds suppressors, perfume, optical 0-5 clarifier) 6) An aqueous structured liquid detergent composition, which comprises: Linear alkylbenzenesulfonate 15-21% (calculated as acid) Alcoholic ethoxylate (e.g. alcohol 3-9% C12-15 '7 EO, or alcohol C_ _, _, 5 EO) li- X or Soap as a fatty acid (eg 3 - 10% oleic acid) Zeolite (as NaAlSi04) 14 - 22% Potassium citrate 9 - 18% Borate (as B 0) 0-2% Carboxymethylcellulose 0-2% Polymers (eg PEG, PVP) 0-3% Anchor polymers such as, eg, 0-3% lauryl methacrylate / acrylic acid copolymer; 25: 1 molar ratio; P.M. 3800 Glycerol 0 - 5% Enzymes (calculated as a protein of 0.0001 - 0.1% pure enzyme) Minor ingredients (eg dispersants, 0 - 5% foam suppressants, perfume, optical brightener) 7) A detergent composition formulated as a composition - granular It has a mass density of at least 600 g / 1 which comprises: Fatty alcohol sulfate 5 - 10% Fatty acid monoethanolamide 3 - 9% ethoxylated Soap as fatty acid 0 - 3% Sodium carbonate (as Na C0") 5 - 10% Soluble silicate (as Na 0, 2SiO) 1 - 4% Zeolite (as NaAlSiO) 20 - 40% Sodium sulphate (as Na SO) 2 - 8% Sodium perborate (as NaBO .H 0) 12 - 18% TAED 2 - 7% Polymers (eg 1 - 5% acid copolymer maleic / acrylic, PEG) Enzymes (calculated as protein of 0.0001 - 0.1% pure enzyme) Minor ingredients (eg 0 - 5% optical lightening, foam suppressors, perfume) 8) A detergent composition formulated as a granular composition which comprises: Linear alkylbenzenesulfonate 8 - 14% (calculated as acid 5 - 11% ethoxylated fatty acid monoethanolamide Soap as fatty acid 0 - 3% Sodium carbonate (as Na? C0) 4 - 10% Soluble silicate (as Na_0, 2SiO?) 1 - 4% Zeolite (as NaAlSiO.) 30 - 50% Sodium sulphate (as Na "S0) 3 - 11% Sodium citrate (as C_H_.Na 0") OD / 5 - 12% Polymers (eg PVP, 1 - 5% copolymer maleic / acrylic acid, PEG) Enzymes ( calculated as protein of 0.0001 - 0.1% pure enzyme) Minor ingredients (eg suppressors 0 - 5% foam, perfumes) 9) A detergent composition formulated as a granular composition which comprises: Linear alkylbenzenesulfonate 6 - 12% (calculated as acid) Nonionic surfactant 1-4% Soap as fatty acid 2-6% Sodium carbonate (as Na C0) 14 - 22% Zeolite (as NaAlSiO) 18 - 32% Sodium sulphate (as Na? SO) 5 - 20% Sodium citrate (as C ^ H-.Na ^ O.,) 3 - 8% 6 5 3 7 Sodium perborate (as NaB0".H 0) 4 - 9% Activator bleaching (eg NOBS or 1 - 5% TAED) Carboxymethylcellulose 0 - 2% Polymers (eg polycarboxylate or 1 - 5% PEG) Enzymes (calculated as protein of 0.0001 - 0.1% pure enzyme) Minor ingredients (eg clarifier 0 - 5% optical, perfume) ) An aqueous liquid detergent composition which comprises: Linear alkylbenzenesulfonate 15-23% (calculated as acid) Alcoholic ethoxysulfate (e.g. alcohol 8-15% C12-15 '^ - 3 EO) Alcohol ethoxylate (e.g. alcohol 3 - 9% C12-15 '7 E0, ° alcoho1 C12-15' 5 E0) Soap as fatty acid (eg 0 - 3% lauric acid) Aminoethanol 1 - 5% Sodium citrate 5-10% Hydrotrope (eg toluenesulfonate 2-6% of sodium) Borate (as ^> 7) 0-2% Carboxymethylcellulose 0-1% Ethanol 1-3% Propylene glycol 2-5% Enzymes (calculated as 0.0001-0.1% pure enzyme) Minor ingredients (eg polymers) , 0 - 5% dispersants, perfume, optical brighteners) 11) An aqueous liquid detergent composition which comprises: Linear alkylbenzenesulfonate 20-32% (calculated as acid) Alcoholic ethoxylate (e.g. alcohol 6-12% C12-15 '7 E0, or alcohol C, ", _, 5 E0) 1¿-15 Aminoethanol 2 - 6% Citric acid 8 - 14% Borate (as B40) 1-3% Polymer (eg copolymer of acid 0 - 3% maleic / acrylic, anchor polymer such as, eg, lauryl methacrylate / acrylic acid copolymer) Glycerol 3 - 8% Enzymes (calculated as protein 0.0001 - 0.1% pure enzyme) Minor ingredients (eg hydro- 0 - 5% tropos , dispersants, perfume, optical brighteners) 12) A detergent composition formulated as a granular composition having a mass density of at least 600 g / 1 which comprises: Anionic surfactant (alkylbenzenesulfo- 25-40% cream, alkyl sulfate, alpha-olefin sulphonate, alpha-sulfo methyl fatty acid esters, alkane sulphonates, soap) Nonionic surfactant (eg 1-10% alcoholic ethoxylate) Sodium carbonate ( as Na? C0") 8 - 25% Soluble silicates (as Na 0, 2SÍ0) 5 - 15% Sodium sulphate (as Na SO) 0 - 5% Zeolite (as NaAlSiO) 15 - 28% Sodium perborate (as NaBO , 4H 0) 0 - 20% Bleach activator (TAED or NOBS) 0 - 5% Enzymes (calculated as protein of 0.0001 - 0.1% pure enzyme) Minor ingredients (eg perfume, 0 - 3% optical brighteners) 13) Detergent formulations as described in 1) -12), wherein all or part of the linear alkylbenzene sulphonate is replaced by (C 1 -C) alkyl sulfate. What 14) A detergent composition formulated as a granular composition having a mass density of at least 600 g / 1 which comprises: C12-C18 alkyl sulfate 9-15% Alcohol ethoxylate 3-6% Polyhydroxy fatty acid amide 1-5% Zeolite alkyl (as NaAlSiO) 10-20% Laminated disilicate (eg SK56 10-20% Hoechst) Carbonate sodium (as Na C0) 3 - 12% Soluble silicate (as Na? 0, 2SiO?) 0 - 6% Sodium citrate 4-8% Sodium percarbonate 13 - 22% TAED 3 - 8% Polymers (eg polycarboxylates and 0 - 5% PVP) Enzymes (calculated as a protein of 0.0001 - 0.1% pure enzyme) Minor ingredients (eg 0 - 5% optical brightener, photo bleach, perfume, foam suppressors) ) A detergent composition formulated as a granular composition having a mass density of at least 600 g / 1 which comprises: Alkyl Sulfate (C-0-C-0) 4-8% Alcoholic ethoxylate 11 - 15% Soap 1 - 4% Zeolite MAP or Zeolite A 35 - 45% Sodium carbonate (as Na C0) 2 - 8 % Soluble silicate (as Na 0, 2SiO) 0 - 4% Sodium percarbonate 13 - 22% TAED 1 - 8% Carboxymethyl cellulose 0 - 3% Polymers (eg polycarboxylates and - 3% PP) Enzymes (calculated as protein of 0.0001 - 0.1% pure enzyme) Minor ingredients (eg 0 - 3% optical lightening, phosphonate, perfume) 16) Detergent formulations as described in 1) -15) which contain a stabilized or encapsulated peracid, each as an additional component or as a substitute for already specified bleaching systems. 17) Detergent compositions as described in 1), 3), -7), 9) and 12) wherein the perborate is replaced by percarbonate. 18) Detergent compositions as described in 1), 3), - 7), 9), 12), 14) and 15) which additionally contain - a manganese catalyst. The manganese catalyst may be, e.g., one of the compounds described in "Efficient manganese catalysts for low-temperature bleaching", Nature 369, 1994, pp. 637-639. 19) Detergent composition formulated as a non-aqueous detergent liquid which comprises a liquid non-ionic surfactant such as, e.g., linear alkoxylated primary alcohol, a builder system (e.g. phosphate), enzyme and alkali. The detergent may also comprise anionic surfactant and / or a bleaching system.

The protease of the invention can be incorporated in concentrations conventionally used in detergents. It was contemplated herein that, in the detergent composition of the invention, the protease may be added in an amount corresponding to 0.00001-1 mg (calculated as pure enzyme protein) of protease per liter of wash liquor. The invention is further illustrated by the following examples, which are not intended to limit in any way, the field of application of the claimed invention.

EXAMPLE 1 Bacillus sp. I 612, DSM 9701, is grown at 30 ° C -on a rotary vibrating table (300 rpm) in 500 ml Erlenmeyer flasks containing 100 ml of a medium of the following composition (per liter): Potato starch 100 g Barley 50 g Soybean meal 20 g Na HP0 / 1 x 12 H 9 g 2 4 2 n Pluronic Caseinate sodium 10 g The starch in the medium is liquefied with alpha-amylase, and the medium is sterilized by heating at 120 ° C for 45 minutes. After sterilization, the pH of the medium is a-just 9.0 by the addition of 10 ml of a 1 M solution of sodium bicarbonate. After cultivation (3 days), and after separation of the solid material, the protease is purified by a conventional chromatographic method. The production from 2.5 liters of culture broth was 50 ml with 45 CPU / liter. The purity was more than 90% as estimated by SDS-PAGE. The characteristics of the preparation prepared in accordance with this Example, have been previously referenced in this specification, with the attached reference.

EXAMPLE 2 Performance in washing the protease Bacillus sp. I 612 (_ a ° C) The wash performance tests were performed on cotton stained with grass juice, in a model washing system at 25 ° C, with a constant temperature for 10 minutes.

The tests are run at protease concentrations of 1, 2, 7.5, 20 and 50 nM. 2.0 g / 1 of an American type detergent powder composition are used in the test. The detergent does not contain any enzyme before the addition of the protease of the invention. The detergent dissolves in water of approximately 6 ° dH (German Hardness). The pH of the wash liquor was 10. The proportion of the textile liquor / wash was about 5 g of textile per liter of wash liquor. For each enzyme concentration, two independent tests were performed. Subsequent to washing the cloth, the clothes were cleaned with a water tap for 20 minutes and then dried in the air. The performance of the protease of the invention TM and of the Savinase, is evaluated by the change (deltaR) of the decrease (% R) at 460 nm measured in a Da-tacolor 2000 Elrefometer; the deltaR is the decrease after washing with the added protease, minus the decrease after washing with the non-addition of the protease. The results of these tests are shown in - Table 1 below (average of 2 tests): Table 1 Concentration of the Potease deltaR (nM) TM I 612 SAVINASE 1 1.9 1.7 2 4.2 2.4 7.5 9.1 5.5 20 14.4 11.1 50 17.8 14.3 As can be seen from Table 1, deltaR (Bacillus lll sp.I 612) is larger than deltaR (Savinase) in all measured protease concentrations, i.e. the -protease of the invention has a better washing performance in all concentrations measured at 25 ° C.

EXAMPLE 3 Wash performance of Bacillus sp. Protease. I 612 (at 15 ° C) The washing performance tests were performed on cotton stained with grass juice, in a model -15 ° C washing system, with a constant temperature for 10-minutes. The tests are run at pro-tease concentrations of 2, 7.5, 20 and 50 nM. 2.0 g / 1 of a detergent with the following composition Linear alkylbenzenesulfonate 0.3 g / 1 Alcohol ethoxylate 0.04 g / 1 Soap 0.1 g / 1 Na2S04 0.3 g / 1 Na2C03 0.4 g / 1 Zeolite 0.6 g / 1 Citrate-Na 0.08 3 g / 1 Carboxymethylcellulose 0.006 g / 1 Polycarboxylate 0.083 g / 1 they are used in the test. The detergent dissolves in water of approximately 6 ° dH (German Hardness). The pH of the wash liquor is adjusted to pH 10. The proportion of the textile liquor / wash was about 5 g of textile per liter of wash liquor. For each concentration of the enzyme, two independent tests were performed. Subsequent to washing the cloth, the clothes were cleaned with a water tap for 20 minutes and then dried in the air. The performance of the protease of the invention and of the Savinase TM is evaluated by the change (deltaR) of the decrease (% R) at 460 nm measured on a Datacolor 2000 Elrefometer; deltaR is the decrease after washing with the addition of the protease, minus the decrease after washing with the non-addition of the protease. The results of these tests are shown in -Table 2 below (average of 2 tests): Table 2 Concentration of protease deltaR (nM) TM I 612 SAVINASE 2 2.5 2.3 7.5 6.6 3.9 20 10.5 7.9 50 14.5 11.8 As can be seen from Table 2, deltaR (Bacillus lll sp.I 612) is larger than deltaR (Savinase) in all measured protease concentrations, i.e. the -protease of the invention has a better washing performance in all concentrations measured at 15 ° C.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, property is claimed as contained in the following:

Claims (12)

1. A protease characterized in that it possesses identical or partially identical properties to those of a pro-tease derived from the strain Bacillus sp. I 612, DSM 9701, further characterized by: (a) an apparent molecular weight of 31 kD; (b) a pl of about 8.0; (c) an optimum pH in the range of about pH 7 to pH 9.5 (at 25 ° C and with casein as the substrate); (d) an optimum temperature of about 30 ° C and less than 20% activity at 40 ° C (at pH 9.5 and with casein as the substrate); (e) the protease is inhibited by PMSF and EDTA.

2. The protease according to claim 1, characterized in that it is obtained from the strain Bacillus sp. I 612, DSM 9701.

3. An isolated, biologically pure culture of a strain of Bacillus sp., Represented by the strain Bacillus sp. I 612, DSM 9701.

4. A process for the preparation of a protease according to any of claims 1-2, characterized in that the process comprises the cultivation of a strain of Bacillus sp. producing a protease according to claim 3, in an a-decuado nutrient medium which contains carbon and nitrogen sources and inorganic salts, followed by the recovery of the desired enzyme.

5. A process according to claim 4, characterized in that the Bacillus sp. I 612, DSM 9701, or other host organism carrying the protease gene according to claim 1, is cultured.

6. The use of a protease according to any one of claims 1-2, as a detergent enzyme.

7. A detergent composition comprising a protease according to any of claims 1-2.

8. A detergent composition according to claim 7, characterized in that it further comprises one or more other enzymes, in particular an amylase, a lipase, a cellulase, a peroxidase, and an oxidase.

9. A detergent additive comprising a protease according to any of claims 1-2, -provided in the form of a granular non-powder, a liquid, in particular a stabilized liquid, a slurry, or a protected enzyme.

10. A washing process comprising the addition of a protease according to any one of claims 1 - 2.

11. A washing process according to claim 10, characterized in that it comprises the addition of a detergent composition according to any of claims 7-8.

12. A process of washing of the stringency with the claim 10, characterized in that it comprises the addition of a detergent additive according to claim 9.