A DETERGENT ADDITIVE CONTAINING A CELLULASE AND A PROTEASE

Abstract: A detergent additive comprises a cellulase and a protease with a higher degree of specificity than Bacillus lentus serine protease. In particular, the protease may be selected from subtilisin Novo or a variant thereof, a protease derivable from Nocardiopsis dassonvillei NRRL 18133, a serine protease specific for glutamic and aspartic acid, producible by Bacillus licheniformis, or a trypsin-like protease producible by Fusarium sp. DSM 2672.

Description: A DETERGENT ADDITIVE CONTAINING A CELLULASE AND A PROTEASE FIELD OF INVENTION The present invention relates to a detergent additive and detergent composition comprising a cellulase and a protease i the presence of which the cellulase exhibits improved stabilit properties. BACKGROUND OF THE INVENTION It has previously been observed that cellulases are sensitiv to the action of proteases, i.e. that in the presence o proteases commonly employed in detergents, cellulases ar degraded to lower molecular weight polypeptides resulting i inactivation of the cellulase enzymes. When cellulases ar employed in liquid detergent compositions, it has typicall been necessary to protect them from proteolytic degradation. It is therefore an object of the present invention to identif a protease towards which cellulases are less sensitive with view to improving the storage stability of cellulases i detergent additives or detergent compositions containing bot types of enzyme. SUMMARY OF THE INVENTION The present invention relates to a detergent additiv comprising a cellulase and a protease, the protease being on which has a higher degree of specificity than Bacillus lentu serine protease. According to the invention, it has been found that when th protease has such a higher degree of specificity, an increase storage stability of the cellulase is obtained. For the presen purpose, the term "higher degree of specificity" is defined a a protease which conditions degrades human insulin to fewe components than does the B_j_ lentus serine protease under t following conditions: 0.5 ml of a 1 mg/ml solution of hum insulin in B and R buffer, pH 9.5, is incubated with 75 enzyme solution of 0.6 CPU (cf. Novo Nordis Analysis Meth No. AF 228/1) per litre for 120 min. at 37"C, and the reactio is quenched with 50 μl IN HCl. In another aspect, the present invention relates to a detergen composition comprising a cellulase and a protease as define above. DETAILED DISCLOSURE OF THE INVENTION Examples of suitable proteases with a higher degree o specificity as defined above are subtilisin Novo or a varian thereof (e.g. a variant described in US 4,914,031) , a proteas derivable from Nocardiopsis dassonvillei NRRL 18133 (describe in WO 88/03947) , a serine protease specific for glutamic an aspartic acid, producible by Bacillus licheniformis (thi protease is described in detail in co-pending Internationa patent application No. PCT/DK91/00067) , or a trypsin-lik protease producible by Fusarium sp., DSM 2672 (this proteas is described in WO 89/06270) . The cellulase included in the detergent additive or detergent composition of the invention may be any enzyme exhibiting cellulolytic activity and may, for instance be derived from a strain of Humicola, Fusarium, Myceliophthora, Thermonospora, Bacillus or Strepto yces. For detergent purposes, it is, however, preferred that the cellulase is an endoglucanase, i.e. an enzyme which shows affinity for cellulose and which attacks amorphous regions of low crystallinity in cellulose fibres. A particular endoglucanase which it has been found favourable to combine with a protease as defined above is is one which is immunoreactive with an antibody raised against a highly purified ~43 kD endoglucanase derived from Humicola insolens, DSM 1800. One such enzyme which is decribed in detail in co- pending Danish patent application No. DK 1159/90 has the ami acid sequence shown in the appended Sequence Listing ID# 1. F the present purpose, this enzyme is referred to in t following as " ~ 43 kD endoglucanase". The detergent additive according to the invention may suitab be in the form of a non-dusting granulate, stabilized liqu or protected enzyme. Non-dusting granulates may be produc e.g. according to US 4,106,991 and 4,661,452 (both to No Industri A/S) and may optionally be coated by methods known the art. Liquid enzyme preparations may, for instance, stabilized by adding a polyol such as propylene glycol, a sug or sugar alcohol, lactic acid or boric acid according established methods. Other enzyme stabilizers are well kno in the art. Protected enzymes may be prepared according to t method disclosed in EP 238,216. The detergent additive may suitably contain 0.001-1.0 mg cellulase and 0.001-1.0 mg of protease per gram of t additive. It will be understood that the detergent additive m further include one or more other enzymes, such as a lipas peroxidase or amylase, conventionally included in deterge additives. The detergent composition of the invention additional comprises a surfactant which may be of the anionic, non-ioni cationic, amphoteric, or zwitterionic type as well as mixtur of these surfactant classes. Typical examples of anion surfactants are linear alkyl benzene sulfonates (LAS) , alp olefin sulfonates (AOS) , alcohol ethoxy sulfates (AES) a alkali metal salts of natural fatty acids. Detergent compositions of the invention may contain oth detergent ingredients known in the art as e.g. builders bleaching agents, bleach activators, anti-corrosion agents sequestering agents, anti soil-redeposition agents, perfumes enzyme stabilizers, etc. The detergent composition of the invention may be formulate in any convenient form, e.g. as a powder or liquid. The enzym may be stabilized in a liquid detergent by inclusion of enzym stabilizers as indicated above. Usually, the pH of a solutio of the detergent composition of the invention will be 7-12 an in some instances 7.0-10.5. Other detergent enzymes such a proteases. Upases or amylases may be included the detergen compositions of the invention, either separately or in combined additive as described above. In the detergent composition, the cellulase is typicall present in a concentration corresponding to 0.0005-20 mg o enzyme protein per litre of washing liquor, and the proteas is typically present in a concentration corresponding t 0.0005-20 mg per litre of washing liquor. The detergen composition of the invention is usually employed i concentrations of 0.5 - 20 g/1 in the washing solution. I general, it is most convenient to add the detergent additiv in amounts of 0.1 - 5% w/w or, preferably, in amounts of 0.2 - 2% of the detergent composition. The detergent additive or detergent composition of the invention may be used to reduce the rate at which cellulose- containing fabrics become harsh or to reduce the harshness of cellulose-containing fabrics, or to provide colour clarification of coloured cellulose-containing fabrics in a manner known per se (cf. for instance WO 89/00069) . The present invention is described in further detail in the following example which is not in any way intended to limit the scope of the invention as claimed. EXAMPLE Stability of " 43 kD endoglucanase in the presence of proteases The storage stability of " 43 kD endoglucanase in liquid detergent in the presence of different proteases was determined under the following conditions: Enzymes "43 kD endoglucanase as identified above Glu/Asp specific B_j_ licheniformis serine protease Trypsin-like Fusarium sp. , DSM 2672, protease B. lentus serine protease Subtilisin Novo Detergent US commercial liquid detergent not containing any opacifie perfume or enzymes (apart from those added in the experiment) +/- 1% (w/w) boric acid as enzyme stabiliser. Dosage Endoglucanase: 12 CMCU/g of detergent Proteases: 0.2 mg/g of detergent Incubation 7 days at 35°C Residual activity The residual activity of the endoglucanase after 7 days o incubation with the respective proteases was determined i terms of its CMCase activity (CMCU) . The CMCase activity was determined as follows: A substrate solution of 30 g/1 CMC (Hercules 7 LFD) i deionized water was prepared. The enzyme sample to b determined was dissolved in 0.01 M phosphate buffer, pH 7.5 1.0 ml of the enzyme solution and 2.0 ml of a 0.1 M phosphat buffer, pH 7.5, were mixed in a test tube, and an enzym reaction was initiated by adding 1.0 ml of the substrat solution to the test tube. The mixture was incubated at 40" for 20 minutes, after which the reaction was stopped by addin 2.0 ml of 0.125 M trisodiu phosphate.12H 2 0. A blind sample wa prepared without incubation. 2.0 ml of a ferricyanide solution (1.60 g of potassiu ferricyanide and 14.0 g of trisodium phosphate.12H 2 0 in 11 o deionized water) was added to a test sample as well as to blind immediately followed by immersion in boiling water an incubation for 10 minutes. After incubation, the samples wer cooled with tap water. The absorbance at 420 n was measured and a standard curve was prepared with glucose solution. One CMCase unit (CMCU) is defined as the amount of enzym which, under the conditions specified above, forms an amoun of reducing carbohydrates corresponding to 1 μmol of glucos per minute. Results The storage stability of the endoglucanase of the invention was determined as its residual activity (in CMCU%) under the conditions indicated above. Protease Residual Activity (%) + boric acid - boric acid Glu/Asp specific 105 93 Trypsin-like 77 63 B. lentus serine 57 24 Subtilisin Novo 63 55 These results indicate that the storage stability in liquid detergent of the endoglucanase of the invention is improved when a protease with a higher degree of specificity than B. lentus serine protease is included in the detergent composition. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: NOVO NORDISK A/S, (ii) TITLE OF INVENTION: (iii) NUMBER OF SEQUENCES: (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: NOVO NORDISK A/S, Patent Department (B) STREET: Novo Alle (C) CITY: Bagsvaerd (E) COUNTRY: DENMARK (F) ZIP: DK-2880 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Thalsoe-Madsen, Birgit (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: +4544448888 (B) TELEF-.X: +4544493256 (C) TELEX: 37304 (2) INFORMATION FOR SEQ ID N0:1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1060 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Humicola insolens (B) STRAIN: DSM 1800 (ix) FEATURE: (A) NAME/KEY: mat_peptide (B) LOCATION: 73..927 5 (ix) FEATURE: (A) NAME/KEY: sig_peptide (B) LOCATION: 10..72 (ix) FEATURE: 0 (A) NAME/KEY: CDS (B) LOCATION: 10..927 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:1: GGATCCAAG ATG CGT TCC TCC CCC CTC CTC CCG TCC GCC GTT GTG GCC Met Arg Ser Ser Pro Leu Leu Pro Ser Ala Val Val Ala -21 -20 -15 - -10 GCC CTG CCG GTG TTG GCC CTT GCC GCT GAT GGC AGG TCC ACC CGC TAC Ala Leu Pro Val Leu Ala Leu Ala Ala Asp Gly Arg Ser Thr Arg Tyr -5 1 5 TGG GAC TGC TGC AAG CCT TCG TGC GGC TGG GCC AAG AAG GCT CCC GTG Trp Asp Cys Cys Lys Pro Ser Cys Gly Trp Ala Lys Lys Ala Pro Val 10 15 20 AAC CAG CCT GTC TTT TCC TGC AAC GCC AAC TTC CAG CGT ATC ACG GAC 1 Asn Gin Pro Val Phe Ser Cys Asn Ala Asn Phe Gin Arg He Thr Asp 25 30 35 40 TTC GAC GCC AAG TCC GGC TGC GAG CCG GGC GGT GTC GCC TAC TCG TGC 2 Phe Asp Ala Lys Ser Gly Cys Glu Pro Gly Gly Val Ala Tyr Ser Cys 45 50 55 GCC GAC CAG ACC CCA TGG GCT GTG AAC GAC GAC TTC GCG CTC GGT TTT 2 Ala Asp Gin Thr Pro Trp Ala Val Asn Asp Asp Phe Ala Leu Gly Phe 60 65 70 GCT GCC ACC TCT ATT GCC GGC AGC AAT GAG GCG GGC TGG TGC TGC GCC 3 Ala Al Thr Ser He Ala Gly Ser Asn Glu Al Gly Trp Cys Cys Ala 75 80 85 TGC TAC GAG CTC ACC TTC ACA TCC GGT CCT GTT GCT GGC AAG AAG ATG 3 Cys Tyr Glu Leu Thr Phe Thr Ser Gly Pro Val Ala Gly Lys Lys Met 90 95 100 GTC GTC CAG TCC ACC AGC ACT GGC GGT GAT CTT GGC AGC AAC CAC TTC 43 Val Val Gin Ser Thr Ser Thr Gly Gly Asp Leu Gly Ser Asn His Phe 105 110 115 120 GAT CTC AAC ATC CCC GGC GGC GGC GTC GGC ATC TTC GAC GGA TGC ACT 48 Asp Leu Asn He Pro Gly Gly Gly Val Gly He Phe Asp Gly Cys Thr 125 130 135 CCC CAG TTC GGC GGT CTG CCC GGC CAG CGC TAC GGC GGC ATC TCG TCC Pro Gin Phe Gly Gly Leu Pro Gly Gin Arg Tyr Gly Gly lie Ser Ser 140 145 150 5 CGC AAC GAG TGC GAT CGG TTC CCC GAC GCC CTC AAG CCC GGC TGC TAC Arg Asn Glu Cys Asp Arg Phe Pro Asp Ala Leu Lys Pro Gly Cys Tyr 155 160 165 TGG CGC TTC GAC TGG TTC AAG AAC GCC GAC AAT CCG AGC TTC AGC TTC 10 Trp Arg Phe Asp Trp Phe Lys Asn Ala Asp Asn Pro Ser Phe Ser Phe 170 175 180 CGT CAG GTC CAG TGC CCA GCC GAG CTC GTC GCT CGC ACC GGA TGC CGC Arg Gin Val Gin Cys Pro Ala Glu Leu Val Ala Arg Thr Gly Cys Arg 15 185 190 195 200 CGC AAC GAC GAC GGC AAC TTC CCT GCC GTC CAG ATC CCC TCC AGC AGC Arg Asn Asp Asp Gly Asn Phe Pro Ala Val Gin He Pro Ser Ser Ser 205 210 215 20 ACC AGC TCT CCG GTC AAC CAG CCT ACC AGC ACC AGC ACC ACG TCC ACC Thr Ser Ser Pro Val Asn Gin Pro Thr Ser Thr Ser Thr Thr Ser Thr 220 225 230 25 TCC ACC ACC TCG AGC CCG CCA GTC CAG CCT ACG ACT CCC AGC GGC TGC Ser Thr Thr Ser Ser Pro Pro Val Gin Pro Thr Thr Pro Ser Gly Cys 235 240 245 ACT GCT GAG AGG TGG GCT CAG TGC GGC GGC AAT GGC TGG AGC GGC TGC 30 Thr Ala Glu Arg Trp Ala Gin Cys Gly Gly Asn Gly Trp Ser Gly Cys 250 255 260 ACC ACC TGC GTC GCT GGC AGC ACT TGC ACG AAG ATT AAT GAC TGG TAC Thr Thr Cys Val Ala Gly Ser Thr Cys Thr Lys He Asn Asp Trp Tyr 35 265 270 275 • 280 CAT CAG TGC CTG TAGACGCAGG GCAGCTTGAG GGCCTTACTG GTGGCCGCAA His Gin Cys Leu 285 40 CGAAATGACA CTCCCAATCA CTGTATTAGT TCTTGTACAT AATTTCGTCA TCCCTCCAGG GATTGTCACA TAAATGCAAT GAGGAACAAT GAGTAC 5

Claims: CLAIMS 1. A detergent additive comprising a cellulase and a protease the protease being one which has a higher degree of specificit than Bacillus lentus serine protease. 2. A detergent additive according to claim 1, wherein th protease is subtilisin Novo or a variant thereof, a proteas derivable from Nocardiopsis dassonvillei NRRL 18133, a serin protease specific for glutamic and aspartic acid, producibl by Bacillus licheniformis, or a trypsin-like proteas producible by Fusarium sp. DSM 2672. 3. A detergent additive according to claim 1 or 2, wherein the cellulase is one'producible by a strain of Humicola, Fusarium, Myceliophthora, Thermonospora, Bacillus or Streptomyces. 4. A detergent additive according to claim 3, wherein the cellulase is one which is immunoreactive with an antibody raised against a highly purified " 43 kD endoglucanase derived from Humicola insolens, DSM 1800. 5. A detergent additive according to any of claims 1-4, which further comprises a lipase, peroxidase and/or an amylase. 6. A detergent additive according to any of claims 1-5, which is in the form of a non-dusting granulate, stabilized liquid or protected enzyme. 7. A detergent additive according to any of claims 1-6, which contains 0.001-1.0 mg of cellulase per gram of the additive and 0.001-1.0 mg of protease per gram of the additive. 8. A detergent composition comprising a cellulase and a protease, the protease being one which as a higher degree of specificity than Bacillus lentus serine protease. 9. A detergent composition according to claim 8, wherein th protease is subtilisin Novo or a variant thereof, a proteas derivable from Nocardiopsis dassonvillei NRRL 18133, a serin protease specific for glutamic and aspartic acid, producibl by Bacillus licheniformis. or a trypsin-like proteas producible by Fusarium sp. DSM 2672. 10. A detergent composition according to claim 8 or 9, wherei the cellulase is one producible by a strain of Humicola Fusarium, Myceliophthora. Thermonospora. Bacillus o Streptomyces. 11. A detergent composition according to claim 10, wherein th cellulase is one which is immunoreactive with an antibod raised against a highly purified " 43 kD endoglucanase derive from Humicola insolens, DSM 1800. 12. A detergent composition according to any of claims 8-11 which further comprises a lipase, peroxidase and/or an amylase 13. A detergent composition according to any of claims 8-12 wherein the cellulase is present in a concentratio corresponding to 0.0005-20 mg of enzyme protein per litre o the washing liquor, and wherein the protease is present in concentration corresponding to 0.0005-20 mg of enzyme protei per litre of the washing liquor.

Inventors: TSUCHIYA RIE, WAGNER PETER, TSUCHIYA, RIE, WAGNER, PETER
Applicants: NOVONORDISK AS, NOVO NORDISK A/S
Application year: 1992
Main classification: C11D3
IPC codes: C11D3