IMPROVEMENTS IN AND RELATING TO AN ENZYMATIC DETERGENT ADDITIVE, A DETERGENT, AND A WASHING METHOD

Abstract: There is provided an enzymatic detergent additive the active component of which is a microbially produced lipase, characterised in that the lipase is producible by means of a lipase producing strain of Fusarium oxysporum, as well as a detergent comprising such an enzymatic additive and a wash­ ing process suing such a detergent.

Description: Improvements in and relating to an enzymatic detergent additive, a detergent, and a washing method The field of enzymatic additives has been rapidly growing during the last decades. Reference is mode to e.g. the article "How Enzymes Cot into Detergents", Vol. 12,.Developments in Industrial Microbiology, a publication of the Society for Industrial Microbiology, American Institute of Biological Sciences, Washington, D.C. 1971, by Claus Dambmann, Poul Holm, Villy Jensen, and Mogens Hilmer Nielsen. The most common enzymatic detergent additive is a proteolytic additive, but also lipolytic detergent additives are described, e.g. in U.S. Patent No. 4,011,116, column 4, line 65 to column 5, line 68, and British Patent No. 1,293,613, page 2, lines 6 to 29. Also, a comprehensive review article of lipases as detergent additives written by Hans Andree et al. is to be found in the Journal of Applied Biochemistry, 2, 218-229 (1980), entitled "Lipases as Detergent Components". If the washing process is conducted at high temperature and high alkalinity, the fat containing dirt will be dissolved by saponification. However, due to the energy crisis, low temperature washing processes (around 600C and below) are generally preferred, and at these low temperatures the known lipases are able to dissolve only a part of the fat containing dirt. - - The efficiency of lipolytic enzymatic detergent additives can conveniently be measured by means of EMPA (Eidgenössische MateriolprUfungsund Versuchsanstalt, St. Galleon, Switzerlond) swatches Nos. 101 (olive oil/cotton) and 102 (olive oil/wool) by odaptation of the procedure described in British Patent No. 1,36 1 ,386 (especially poges 4 and 7) and US patent No. 3,723,250 (especially ccl. 15-19). In this way it appears that the enzymes known as lipolytic detergent added tives are rather unsatisfactory in the sense that they exhibit an un- satisfactorily low lipolytic cleaning efficiency, as reflected in the low value of the differential remittance value iSR at economically reasonable lipase activities in the washing solution. Thus, a need exists for a lipolytic detergent additive which exhibits a considerably better lipolytic cleaning efficiency, corresponding to a considerably higher differential remittance value, AR, at economically reasonable lipase activities in the washing solution. Now, according to the first aspect of the invention, a lipolytic detergent additive has been found which exhibits a considerably better lipolytic cleaning efficiency, corresponding to a considerably higher differential remittance value, AR, at economically reasonable lipase activities in the washing solution - this lipolytic detergent additive being characterized by the fact that the lipase is producible by means of a lipase producing strain of rusarium oxvsDorum. The definition of the species Fusarium oxvsPorum has changed somewhat during the last decades. However, for the purposes of this invention the definition of the species Eusarium oxvsporum is the definition set forth in The Genus Fusarium", C. Booth, CMI, 1971. In relation to certain strains of Fusarium oxvsPorum a lipase formation is described, vide e.g. Agric. Biol. Chem. 43 (10) (1979), 2126, Table I where a Fusarium lini lipase is indicated (according to the above definition of Fusarium oxysporum, Fusarium lini now belongs to Fusarium oxysnorum), and Indian Journal of Experimental Biology, Vol. 11 (1973), p. 37-39 with the title "Lipids and Lipase Activity in Strains of Fusarium vasinfectum (according to the above defini titan of Fusarium oxysporum, Fusarium vasinfectum now belongs to Fusa- rium oxysporum). Some strains belonging to Fusarium oxYsPorum are poor lipase produ¯- ers. For the purposes of this invention, a lipase producing strain of Fusarlum oxvsnorum is defined as a strain which produces more than 10 LU/ml (the LU being the Lipase Units defined later in this specification) under the following conditions: A substrate intended for shaking flasks is prepared with the follows ing ingredients in grams per litre: Soy bean meal ......... 45 Glucose ............... 70 KH2P04 2 Na2HP04 3 Soy oil ............... 5. Sterilization took place at 121 C for 40 minutes. A 500 ml Erl-nmeyer flask with 100 ml of substrate was inoculated with spores from an agar slant previously inoculated with the strain of Fusarium oxyspcrum to be tested for lipase production. The flasks were shaken at 230 rpm and at 300C for 5 days whereafter the lipase yield was determined. Reference is made to example 29. Thus, it has surprisingly been found that the detergent additive according to the invention exhibits a drastically improved lipolytic cleaning efficiency which will appear from documentation presented later in this specification. With regard to the Fusarium oxvsorum strains DSM 2672, ATCC 7808, CBS 620.72, CBS 645.78, and CBS 794.70, it has been found that the average value of the pH activity optimum is around 9-11, and that the 0 average temperature activity optimum is around 35-50 C (time of analysis: 20 minutes). On the basis of crossed immunoelectrophoresis with antibodies produced from the lipase from DSM 2672 it appears that the lipases originating from the above indicated five strains are identical or partially identical. On the basis of the above findings the conclusion may be drawn that the active component of, the enzymatic detergent additive according to the invention is a group of closely related lipases. The lipase activity is determined according to the NOVO method for determination of lipase activity. This method is based on the hydrolysis of tributyrin by the enzyme. The butyric acid liberated is determined by titration with NaOH. One NOVO Lipase Unit (LU) is the amount of enzyme which1 in a pH-stat and under the standard conditions stated below, liberates titratable butyric acid equivalent to 1 pmol of NaOH per minute. Standard Conditions Temperature ...... 30.00C pH ; 7.0 Reaction time .... 20 minutes Substrate ........ tributyrin. Further details of this method are described in the leaflet AF 95/4 dated 1982-08-18, which is available on request from NOVO INDUSTRI A/S, Bagsvaerd, Denmark. In a specially preferred embodiment of the enzymatic detergent additive according to the invention, the lipase producing strain of Fusarium oxesDorum is DSM 2672. The strain Fusarium sP. DSM 2672 has been classified as Fusarium oxYsPorum Schlecht. ex Fries, emend. Snyder & BR< Hansen. The morphological properties of the strain DSM 2672 Fusarium oxvsDorum exactly correspond to the description of the species description of Fusarium oxvsporum in the Genus Fusarium, C. Booth, CMI, 1971. The pH activity curve of the lipase has been drawn up, the lipase activity being measured according to AF 95/4-GB, modified by adjustment of the pH value of the substrate to 4, 5, and 6, and 8, 9, and 10 besides the normal pH value of the substrate of 7. Due to the fact that tributyrin is decomposed without lipase at high pH values, the pH curve is corrected for such autodecomposition. Hereby a pH activity optimum of around pH 10 has been found. The stability of the enzyme is excellent over a wide pH interval, inasmuch as more than 80X residual activity after 18 hours can be observed in the pH interval of 4.5-11 at 50C and in the pH interval of 5-10 at 250C. Also, the temperature optimum of the lipase activity is around 400C. The enzyme is stable up to 400C for 30 minutes, which is very advantageous in relation to the previously mentioned low-temperature washing processes at around 600C and below. In a specially preferred embodiment of the enzymatic detergent additive according to the invention, the additive is provided as a non dusting granulate. These granulates can be produced in several different ways. Reference can be made to GB patent No. 1,362,365 which describes the production of enzyme-containing granulates used as detergent additives by means of an apparatus comprising an extruder and a spheronizer (sold as MARUMERIZER ), ), and to US patent No. 4,106,991 which describes the production of enzyme containing granulates used as detergent additives by means of a drum granulator. In a specially preferred embodiment of the enzymatic detergent additive according to the invention, the additive is provided as a liquid with an enzyme stabilizer. The stabilizer can be propylene glycol or other agents known as stabilizers for enzyme solutions. Liquid detergents exhibit a growing popularity due to the ease of application. In a specially preferred embodiment of the enzymatic detergent additive according to the invention, the lipase activity is between about 20,000 and about 100,000 LU/g of additive. In this manner, a convenient lipase activity is generated in the washing solution when the detergent additive is added to the detergent in an amount of 0.2-2 g/ 100 g of detergent, and when the detergent is added to the washing solution in an amount of 1-5 g of detergentll of washing solution. In a specially preferred embodiment of the enzymatic detergent additive according to the invention, the additive contains a proteolytic enzyme besides the lipase. Surprisingly it has been found that the proteolytic detergent additive does not break down the (protein) lipase, either in the additive, in the detergent, or in the washing solution. Thus, the proteolytic and the lipolytic detergent additives are compatible, and it has been found that this detergent additive has a very high cleaning efficiency, resulting in a very high AR value. The proteolytic enzyme ALCALASE &commat; from NOVO INDUSTRI A/S, manufactured microbially by means of Bacillus licheniformis, 'can be used with superior results. The mixed enzymatic additive can be prepared either by mixing a previously prepared granulate of proteinase with a previously prepared granulate of lipase1 or by mixing a concentrate of proteinase with a concentrate of lipase and then introducing this mixture into a granulating device, together with the usual granulating, aids. In a specially preferred embodiment of the enzymatic detergent additive according to the invention, the proteolytic activity is between about 0.5 and about 3.0 Anson Units/g of additive. In this manner, a convenient proteolytic activity is generated in the washing solution when the detergent additive is added to the detergent in'an amount of 0.2-2 g/100 g of detergent, and when the detergent is added to the washing solution in an amount of 1-5 g of detergent/l of washing solution. The second aspect of the invention comprises a detergent with an enzymatic detergent addictive, the active component of which is * mi- crobially produced lipase, wherein the enzymatic detergent additive is the enzymatic detergent additive according to the invention. In a specially preferred embodiment of the enzymatic detergent additive according to the invention, the detergent contains the enzymatic detergent additive according to the invention in an amount of between 0.2 and 2.0t w/w. In this manner, a reasonable balance between enzyme action and the action of the other detergent ingredients is generated. The third aspect of the invention comprises a washing process in which the detergent used is the detergent according to the invention, and in which the pH is between 7 and 11, and the temperature tis below 60 C. In a specially preferred embodiment of the enzymatic detergent additive according to the. invention, the washing solution contains the detergent according to the invention in an amount of between 1 and 5 g/l of washing solution. In this manner, a convenient enzyme activity is generated in the washing solution, i.e. typically between 1,000 and 5b,000 LU/l of washing solution. Under these circumstances, very high AR values are obtained for usual washing times, i.e. around 20 minutes. The invention will be illustrated by the following examples. Example l This example illustrates the production in shake flasks of the active component in the enzymatic detergent additive according to the invention. The substrate consists of the following ingredients in grams per liter. Soy bean meal 50 Glucose 50 KH2P04 2 Na2HP04 3 Soy oil 1 Sterilization took place at 1210C for 40 minutes. A 500 ml Erlenmeyer flask with 100 ml substrate was inoculated with 107 spores from an agar slant previously inoculated with Fusarium oxysporum DSM 2672. The flasks were shaken at 230 rpm and at 250 - 300 for 2 - 5 days. The yield was 30 - 80 LU/ml. Example 2 This example and examples 3 - 15 illustrate the production of the active component in the enzymatic detergent additive according to the invention in a 2 litre laboratory fermentor. A 500 ml Erlenmeyer flask with 260 ml of the following medium was prepared. Glucose 24 g per litre Corn steep liquor 24 g - Soy oil 3.8 g - Calcium carbonate 3.8 g - pH = 5.5 before addition of CaCO3 Sterilization took place at 121eC for 40 minutes. After inoculation with spores from an agar slant in the same manner as indicated in example 1 the flask was agitated at 230 rpm and at 300C for 2 days. 120 ml of this seed culture were transferred to a two litre fermentor with 1.3 litre medium sterilized for 1 hour at 1210C and of the following composition. Glucose 50 g per litre Soy bean meal 50 g - KH2PO4 2.0 g - - Na2HP04 2H20 3.0 g - Soy oil 10 g - Pluronic 0.3 ml Subsequent to the inoculation, aeration (700 ml/min) was initiated. Stirring was also initiated and the yelocity thereof was 600 rpm during the first 24 hours, and thereafter it was increased to 800 rpm. The temperature was maintained at 300C. Fermentation was maintained for 100 hours, during which time the pH value rose from 6.3 to 8.5. The supernatant was analysed, and the lipase activity was found to be 90 LU/ml. A series of experiments were performed in the same manner as indicated in example 2, except for the temperature, vide the following table Example No. 3 4 5 6 7 Temperature in fermentor, OC 28 30 32 34 36 LU/ml of supernatant 60 90 125 140 60 From the above table it appears that the optimum temperature is around 340C. In order to investigate the influence of pH two new experiments were performed at 340C and as indicated in example 6, except for pH: in no one of the two experiments pH was controlled during the first 24 hours of the fermentation in the fermentor; in the first experiment (example 8) the pH was maintained at 6.5 by means of H3PO4 after the first 24 hours of the fermentation in the fermentor, and in the second experiment (exampMeg VtSe¯pH was not monitored during the entire fermentation time. The results appear from the below indicated table. Example No 8 9 Temperature in fermentor, C 34 34 after 24 hours maintained at 6.5 x no no regulation x LU/ml of supernatant 180 140 From the above table it appears that the lipase yield is improved if the pH value is monitored in the manner indicated. In order to investigate the influence of the degree of agitation three experiments were performed in the same manner as indicated in example 8 except for the degree of agitation: the speed of the agitator was raised from 600 rpm to different levels during the entire fermentation time after the first 24 hours fermentation, vide the following table. Also the results appear from the following table. Example No 10 11 12 Temperature in fermentor, eC 34 34 34 After 24 h pH maintained at 6.5 x x x Speed of stirrer, rpm 700 800 900 LU/ml of supernatant 100 180 160 From the above table it appears that the optimum degree of agitation is around 800 rpm. In order to investigate the influence of the concentration of soy oil in the fermentation medium three experiments were performed in the same manner as indicated in example 11 (i.e. with optimal values for temperature, pH and degree Bf sgK8avl9in), except for the concentration of soy oil, vide the following table. Example No 13 14 15 Temperature in fermentor, C 34 34 34 After 24 hours pH maintained at 6.5 x x x Speed of stirrer, rpm 800 800 800 Soy oil, % 0 1 2 LU/ml of supernatant 50 170 170 Example 16 This example illustrates the production of the active component in the enzymatic detergent additive according to the invention in a pilot plant fermentor. In an inoculation tank with a volume of 300 1 the following medium was prepared. Corn steep liquor 7.2 kg Soy oil 1.5 Calcium carbonate 1.5 Pluronic 25 ml Water up to 290 1 The medium was sterilized at 121oC for 1 hour. A twelve liter solution containing 7.2 kg glucose and 7.2 g citric acid was sterilized at 1210C for 1/2 hour and added to the corn steep liquor mixture. After cooling to 300C the medium was inoculated with spores of DSM 2672 from a Fernbach flask containing yeast extract, phosphate, magnesium, glucose and agar and which had been incubated at 300C for 7 days. Aeration (300 1/min) was started immediately, and stirring (250 rpm) was initiated after 20 hours. The temperature was maintained at 300C and growth was maintained for 29 hours. 30 1 of this seed culture Mete transferred to the main fermentor containing: Soy bean meal 15 kg KH2P04 0.6 Na2HPO4 12H20 1.2 Soy oil 3 Pluronic 25 ml Water to a volume of 275 liters. The medium was sterilized for 1 hour at 1210C. 15 kg glucose and 15 g citric acid in 25 1 water were sterilized separately at 121"C for 30 minutes and added to the soy bean meal mixture. Following the inoculation, aeration (300 1/min) was started. Stirring was also started, and the velocity thereof was increased to 400 rpm during the first 10 hours of fermentation. The temperature was maintained at 340C. Fermentation was maintained for 60 hours, during which time the pH value rose from 6.64 to 8, and thereafter the tank was cooled and the mycelium separated by centrifugation. The supernatant liquid was analysed, and the lipase activity was found to be 90 LU/ml. Example 17 This example illustrates the production of the active component in the enzymatic detergent additive according to the invention in a pilot plant fermentor. The lipase was prepared by submerged aerobic fermeta- tion of Fusarium oxysporum DSM 2672 An agar substrate with the following composition was prepared in a Fernbach flask: Yeast extract Difco 4 g K2HPO4 1 g ¯ ¯ ¯ ¯ MgS04 . 7H20 0.5 g Glucose 15 g Distilled water ad 1000 ml Agar Merck 15 g The mixture was autoclaved for 40 min. at 1200C (The substrate is named YPG-agar) The strain DSM 2672 was cultivated at 300C for one week on an YPG-agar slant. The spores from the slant were suspended in sterilized skim milk, and the suspension was lyophilized in vials. The contents of one lyophilized vial was transferred to the Fernbach flask. The flask was then incubated for one week at 300C. A substrate with the following composition was prepared in a 500 liter seed fermentor: CaC03 1.5 kg Glucose 7.2 kg Corn steep liquor 7.2 kg Antifoam agent Pluronics 30 ml Tap water was added to a total volume of around 240 liters. pH was adjusted to around 5.5 before addition of CaCO3. The substrate was steam sterilized in the seed fermentor for 1 hour at 1210C. Final volume before inoculation was around 300 liters. The Fernbach flask spore suspension was transferred to the seed fermentor. Seed fermentation conditions were: Fermentor type: Conventional aerated and agitated fermentor with a height/diameter ratio of around 2 Agitation: 300 rpm (two turbine impellers) Aeration: 300 normal liter air per minute. Temperature: 300C Pressure: 0.5 ato. Time: Around 17 hours. In order to prevent excessive foaming reduction in agitation and aeration rate was possible. However, in the seed fermentation described here this possibility was not used. After 1/2 - 1 day when good growth was obtained, here around 17 hours after inoculation, 25 liters were transferred from the seed feMsstJgU & 4the main fermentor. A substrate with the following composition was prepared in a 500 liter main fermentor: Toasted, dehulled soy meal 39 kg Glucose 15 kg Soy oil 3 kg KH2P04 0.6 kg Na2HP04 1 H20 1.2 kg Antifoam agent Pluronic 30 ml Tap water was added to a total volume of around 250 liters. The soy bean meal was suspended in water. was adjusted to 8.0 with Na2C03, and the temperature was raised to 500C. Thereafter around 480 Anson Units of Alcalase&commat;L (4 AU/ml) was added to the suspension. The mixture was held for 4 hours at 500C and pH = 8.0 (Na2CO3 addition) with no aeration, zero ato and 150 200 rpm agitation. Thereafter the remaining substrate components were added and pH was adjusted to around 6.5 with phosphoric acid. The substrate was steam steril ized in the main fermentor for 1 hour at 121oC. After cooling to 340C pH was adjusted to around 6.0 with sterilized Na2CO3 solution (3 kg Na2CO3 in 20 liters total volume). Final volume before inoculation was around 280 liters. Then 25 liters of seed culture was added. Fermentation conditions were: Fermentor type: Conventional aerated and agitated fermentor with a height/diameter ratio of around 2 Agitation: 150 rpm (0 - 6 hours) and 400 rpm (6 hours to end) (2 turbine impellers). Aeration: 200 - 250 normal liter air per minute (0 - 22 hours) and 300 normal liter air per minute (22 hours to end). Temperature: 340C Pressure: 1.0 ato (0 - 34 hours) and 0.5 ato (34 hours to end) Time: Around 112 hours. In order to prevent excessive foaming it was possible to vary agitation, aeration, and pressure. In the fermentation described here this possibility was used as can be seen from the data given above. Fermentation pH was kept at around 6.5 with addition of phosphoric acid solution. In the fermentation described here dosage occurred from around 30 fermentation hours to the end. The phosphoric acid solution was prepared in a 500 liter dosage tank as follows: to 20 kg conc. phosphoric acid (80%) tap water was added to a total volume of around 160 liters. The solution was steam-sterilized in the dosing tank for 1 hour at 1210C. Final volume before start of dosage was around 200 liters. Only the amount of phosphoric acid solution required to keep fermentation pH at around 6.5 was added to the main fermentor. During the fermentation sterilized antifoam agent P2000 (polypropylene glycol) was added to the main fermentor to keep down foam formation. In the fermentation described here 3.5 liters of P2000 was used. After around 112 fermentation hours the fermentation process was stopped. The main fermentor contained around 315 liters of culture broth and the lipase yield obtained was around 200 LU per gram of broth, measured according to NOVO's LU-assay No t5/5-G using tributyrin as substrate. Example 18 The lipase was produced as described in example 17 but the fermentation was carried out in a 2500 liter main fermentor with correspondingly higher substrate amounts and volumes. Agitation: 250 rpm (two turbine impele'rL To prevent excessive foaming, aeration and pressure were varied as follows: Aeration: 750 Nl/min air (0 - 13 hours) 1200 Nl/min air (13 - 37 hours) 1500 Nl/min air (37 hours to end) Pressure: 0.7 ato (0 - 25 hours) 0.5 ato (25 hours to end) No antifoam agent P2000 was added in this fermentation. At the end of fermentation the fermentor contained around 1800 liters of culture broth and the lipase yield obtained was around 125 LU per gram of broth. Example 19 The lipase was produced as described in example 17 but the fermentation was carried out in a 2500 liter main fermentor with correspondingly higher amounts and volumes of substrate and the Alcalase treatment of the soy meal was omitted. 200 kg soy meal was used and the volume before inoculation was 1400 liters. The substrate was steam sterilized in the main fermenter for 1.5 hours at 1230C. agitation: 250 rpm (two turbine impellers) to prevent excessive foaming, aeration and pressure were varied as follows: aeration: 750 Nl/min of air (0 - 3 hours) 900 Nl/min of air (3 - 11 hours) 1100 Nl/min of air (11 - 24 hours) 1300 Nl/min of air (24 - 27 hours) 1500 Nl/min of air (27 hours to end) pressure: 1.0 ato (0 - 27 hours) 0.75 ato (27 - 40 hours) 0.5 ato (40 hours to end) No antifoam agent P2000 was added in this fermentation. At the end of the fermentation the fermentor contained around 1400 liters of culture broth and the lipase yield obtained was around 198 LU per gram broth. Fermentation time wasO64 hours. Example 20 The lipase was produced as described in example 17 but the fermentation was carried out in a 2500 liter main fermentor with correspondingly higher substrate amounts and volumes, and the Alcalase treatment of the soy meal was omitted. 170 kg soy meal was used and the volume before inoculation was 1400 liters. The substrate was steam sterilized in the main fermentor for 1.5 hours at 1230C. agitation: 250 rpm (two turbine impellers) to prevent excessive foaming, aeration and pressure were varied as follows: aeration: 750 Nl/min of air (0 - 11 hours) 1150 Nl/min of air (11 - 19 hours) 1300 Nl/min of air (19 - 27 hours) 1500 Nl/min of air (27 hours to end) pressure: 1.0 ato (0 - 27 hours) 0.5 ato (27 hours to end) This fermentation used 0.5 liter of antifoam agent P2000. At the end of the fermentation the fermentor contained around 1400 liters of culture broth and the lipase-yield obtained was around 221 LU per gram broth. Fermentation time was around 144 hours. Example 21 The lipase was produced as described in example 17 but the soy meal concentration in the main fermentor substrate was reduced to 5% and the Alcalase treatment of the soy meal was omitted. To prevent excessive foaming, agitation was varied as follows: Agitation: 0 rpm (0 - 22 hours) 100 - 150 rpm (22 - 33 hours) 300 rpm (33 hours to end) No antifoam agent P2000 was added in this fermeneaSion. At the end of fermentation the fermentor contained around 310 liters of culture broth and the lipase yield - :3 'obtained was around 146 LU per gram of broth. Example 22 The lipase was produced as described in example 17 but the soy meal concentration in the main fermentor substrate was reduced to 5%, Alcalase treatment was omitted, soy oil was removed from the initial fermentation medium and instead sterilized soy oil was added to the main fermentor 1 liter at a time at 48, 60, 72, and 84 fermentation hours, totalling 4 liters of soy oil. To prevent excessive foaming, agitation, aeration and pressure were varied as follows: Agitation: 200 - 300 rpm (0 - 24 hours) 400 rpm (24 hours to end) Aeration: 150 Nl/min air (0 - 17 hours) 275 Nl/min air (17 - 25 hours) 300 Nl/min air (25 hours to end) Pressure: 0.9 - 1.0 ato (0 - 33 hours) 0.5 ato (33 hours to end) This fermentation used 2 liters of antifoam agent P2000. At the end of fermentation the fermentor contained around 285 liters of culture broth and the lipase yield obtained was around 113 LU per gram of broth. Example 23 The culture broth from example 19 was adjusted to pH 8.0 with sodium hydroxide and flocculated with 1% calcium chloride, 1% Servamine KZA 346 (Servo), 0.03% Superfloc A-130 (American Cyanamid) and 0.58 Triton X-100 (Rohm and Haas). The flocculated culture broth was centrifuged on a disc centrifuge (Westfalia SAMS). The centrifugate was adjusted to pH 4.5 with acetic acid. At this pH a precipitate was formed. The precipitate was separated from the supernatant by centrifugation (Westfalia SAMS) and then treated with Triton X-100. The treated precipitate was then diluted with water and centrifuged again. The centrifugate from the two centrifugations were mixed and the sludge from the second centrifugation step was discarded. The centrifugate was diafiltered (DDS-module GR6oP membranes), and afterwards ultrafiltered and concentrated. The UF-concentrate was filtered on a polypropylene cloth with Hy-flo-supercell (Johns Manville) as filter aid. The filtrate was then adjusted to pH 8.0 with sodium hydroxide and filtered on Supra 100 filter sheets (Seitz) and at last germ filtered on Supra EKS (Seitz). The germ filtrate was precipitated with an equal quantity of acetone and washed with pure acetone. The precipitate was dried in a vacuum chamber. The dried concentrate obtained had an activity of about 90,000 LU/g. Example 24 Washing efficiency of Fusarium oxysporum lipase compared to other lipases. Test material: EMPA 101 (olive oil/cotton) Detergent solution: Detergent A *), 5g/l 10 dH water Washing machine: TERG-O-TOMETER Washing programme: 40eC for 20 minutes No of Swatches: 9/900 ml washing solution Lipase: Fusarium oxysporum lipase Asp. niger, AMANO AP6 Candida cylindracea, Sigma Mucor miehei, SP 225 Lipase conc.: 0, 750, 1500, 2250, 3000 LU/1 The Fusarium oxysporum lipase activity units were introduced as suitable volumes of the supernatant from the centrifugation of the culture broth, the production of which is described in example 1. The washing efficiency is expressed as R = Remission value, measured by the Elrepho photometer, filter R 46, average of 2 readings on 9 swatches. *) - Detergent A has the following composition: Linear alkyl benzene sulphonate (LAS, anionic surfactant) 17% Nonyl phenol ethoxylate, EO = 12 (non ionic surfactant) 3t Sodium tripolyphosphate (STPP) 30E Sodium silicate 4% Sodium carbonate 3% Sodium sulphate 37% Water 6% In the table the results appear as R and A R = R-Ro in which R0 is the remission value of swatches washed without lipase, and R is the remission value of swatches washed with lipase. Lipase concentration, LU/1 750 1500 2250 3000 Lipase R AR R AR R 4R R AR None, R0 46.1 0.0 46.1 0.0 45.1 0.0 45.2 0.0 Fusarium oxysporum lipase - - 53.2 7.1 53.4 8.3 54.0 8.8 A. niger, AMANO AP6 47.0 0.9 48.2 2.1 48.6 3.5 49.8 4.6 C. cylindracea, Sigma 46.2 0.1 46.4 0.3 46.4 1.3 45.6 0.4 M. miehei, SP 225 49.6 3.5 48.1 2.0 49.9 4.8 49.6 4.4 Example 25 In order to demonstrate the lipolytic effect on other textiles than cotton, test material was prepared on polyester/cotton and acrylic as well. Test soiling: 10% olive oil 1.5% emulsifier 0.48 earthen colour 0.3% indian ink 87.8% water The solution was emulsified in a Rannie homogenizer. Pieces of cotton, polyester/cotton and acrylic were immersed in the homogenized solution. The excess soiling was squeezed off and the pieces of cloth were dried in a spindrier for 30 minutes. Washing conditions Test material: Olive oil on: I cotton II polyester/cotton III acrylic Detergent solution: detergent B**), 3.25 g/l, 100C dH water detergent C***), 1 g/l, 100 dH water Washing machine: Launder-O-Meter Washing programme: heating from 150C to 400C, heating time 37 minutes, washing at 400C for 12 minutes No of swatches: 3/300 ml washing solution Lipase: Fusarium oxysporum lipase Mucor miehei, SP 225 C. cylindracea, Sigma Pancreatic lipase, Sigma Lipase concentration: varied The Fusarium oxysporum lipase units were introduced as suitable quantities of a freeze dried powder produced in the following manner. A fermentation in a 1 liter laboratory flask was carried out in much the same manner as indicated,in example 1. The culture broth was centrifuged, concentrated by ultrafiltration and freeze dried. **) Detergent B has the following composition: Linear alkyl benzene sulphonate (LAS, anionic surfactant) 34% Nonyl phenol ethoxylate, EO = 12 (non ionic surfactant) 3% Sodium tripolyphosphate (STPP) 62% Carboxymethylcellulose (CMC) 1% **t) Detergent C is 100% nonyl phenol ethoxylate, EO = 12 (non ionic surfactant). The washing efficiency is expressed as R = Remission value, measured by the Elrepho photometer, filter R 46, average of 2 readings on 3 swatches. In the table, R0 andA R have the same meaning as indicated in Example 24. Active component in enzymatic detergent additive according to the invention Detergent B Detergent C Test Lipase concentration, LU/1 Lipase concentration, LU/1 material 0 84 167 333 1000 0 84 167 333 1000 R0 OR #R AR 6R #R R AR #R AR bR o o cotton 32.2 2.8 6.7 7.9 10.7 27.9 4.9 4.7 7.9 11.4 polyester/ cotton 44.8 2.9 3.5 3.9 3.6 31.6 4.4 5.0 4.6 5.2 acrylic 56.7 3.9 5.6 7.0 6.7 33.7 5.8 10.5 11.0 10.2 Mucor miehei lipase Detergent B Detergent C Test Lipase concentration, LU/1 Lipase concentration, LU/1 material 0 750 3000 6000 10000 0 750 3000 6000 10000 R0 a AR AR OR R0 AR # R R 4R R cotton 35.3 4.1 4.8 7.5 8.8 25.4 1.9 2.0 4.3 4.9 polyester/ cotton 44.3 2.3 3.1 3.7 1.6 31.4 1.8 2.0 1.8 1.0 acrylic 54.4 3.6 4.4 5.7 5.7 33.8 4.4 5.8 6.5 6.3 C. cylindracea, pancreatic lipase Test material: cotton C. cylindracea lipase Pancreatic lipase Detergent Lipase concentration, LU/1 Lipase concentration, LU/1 0 250 500 750 3000 0 750 3000 6000 10000 R0 #R #R #R #R Ro #R #R #R #R B 36.4 0.0 0.0 1.1 0.0 34.5 2.7 2.2 3.0 3.8 C 26.0 0.0 0.0 0.4 0.5 24.4 3.4 3.8 3.5 3.6 Example 26 This example demonstrates the compatibility of a proteolytic enzyme and the lipase in the enzymatic detergent additive according to the invention. The test material was cotton soiled as indicated in Example 25. Washing conditions Test material: Cotton soiled as indicated above. Detergent solution: Detergent B 3.25 g/l, 100 dH water Detergent C 1.0 g/l, 100 dH water Washing machine: Launder-O-meter Washing programme: Heating from 150C to 400C, heating time 37 minutes, washing at 400C for 12 minutes. No of swatches: 3/300 ml washing solution Enzymes: Fusarium oxysporum lipase Alcalase 2.0 T Lipase concentration: 0; 250; 500; 750; 3000 LU/1 Proteolytic concn.: 0; 0.06 or 0.01; 0.02; 0.04; 0.08 AU/1 The Fusarium oxysporum lipase units were introduced as suitable volumes of the supernatant from the centrifugation of the culture broth, the production of which is described in example 1. The proteolytic activity is measured in Anson Units/l (AU/1) and is determined according to the modified Anson method described in NOVO ENZYME INFORMATION 1B No. 058 e-GB (the original Anson method is described in J.Gen.Physiol., 22, 79 - 89 (1939)). The washing efficiency is expressed as indicated in Example 25. The meaning of the symbols in the table is the same as indicated in Example 24. The results appear from the following table. Lipase Proteinase Remission Detergent Detergent concen- concen- value B C tration tration LU/1 AU/1 0 0 R0 35.1 27.0 250 0 #R 3.5 2.7 500 0 hR 3.8 3.2 750 0 bR 5.0 3.5 3000 0 # R 7.5 6.5 250 0.06 bR 2.8 3.5 500 0.06 R 3.4 5.1 750 0.06 R 3.8 5.6 3000 0.06 QR 5.4 6.4 0 0.01 R 1.5 1.9 0 0.02 R 1.2 2.4 0 0.04 A R 1.1 1.4 0 0.08 #R 1.8 1.5 Example 27 This example illustrates the superior washing properties of the lipolytic detergent additive according to the invention in comparison to known lipolytic detergent additives. Two sets of washing conditions were used: 1: 0 - 3000 LU/1, EMPA 101 Terg-O-tometer: 400C for 20 minutes Detergent ALL + Na2SO4: 3.75 + 1.25 g/l, 100dH, pH = 9.5 2: 0 - 3000 LU/1, 10% olive oil/cotton Launder-O-meter: European wash 400C Berol wasc (a nonionic tenside) 1.0 g/l;NOVO detergent 3.25 g/l, 10 dH, pH 8.5/9.5 The 3.25 g NOVO detergent consists of 1 gO1S3OêO9S4 STPP, 0.05 g CMC, 0.1 g Berol wasc. The following table shows the washing effect as values for different prior art lipolytic detergent additives in comparison to the lipolytic detergent additive according to the invention. More than one value with a slash in between indicates that two measurements were made. Microorganism or Company or com- Washing conditions origin mercial name 1 2 NOVO 3 A. niger Lipase A o AMANO AP G 5 Rhizopus rhizo- NOVO 0 podiformis SIGMA 0 0/0 C. cylindracea Lipase MY 0 Lipase OF 0/0 M. miehei NOVO 8 5/8 M. javanicus AMANO MAP-10 0 2/3 F. oxysporum NOVO 10 10/8 Bacillus circulons NOVO 6/5 (100 LU/1) Geotrichum Ip. NOVO 0 Pancreatin SIGMA 4/4 The superiority of lipolytic detergent additive based on Fusarium oxysporum in comparison to the prior art lipolytic detergent additives clearly appears from the table. Example 28 This example illustrates the washing characteristics of lipases from different lipase producing Fusarium pxysporum strains. The test material was acrylic fabric soiled in the following manner Test soiling: 3%olive oil 1.5% emulsifier 0.4% earthen colour 0.3% indian ink 87.8% water The solution was emulsified in a Rannie homogenizer. Pieces of acrylic were immersed in the homogenized solution. The excess soiling was squeezed off and the pieces of cloth were dried in a spindrier for 30 minutes. Experiment I Presoaking: 5 swatches (5 x 10 cm) are soaked for 2 hours at room temperature in a solution containing 50 ml diluted fermentation broth and 5 ml of solution A. Solution A: Berol wasc (nonyl phenol ethoxylate) in 100dH water (0.00603% MgCl2. 6H20, 0.0165% Cad2 and 0.035% NaHCO3 in deionized water). Wash: The swatches are rinsed for 10 minutes after soaking and washed in a Terg-O-tometer at 300C for 10 minutes in a detergent solution containing 1.33 g TOP/litre of water of l0odH. The swatches are rinsed in tap water for 10 minutes after the washing has been finished and dried. The detergency is expressed as 4 R. Results Ro AR Enzyme dosage LU/1 of presoak 0 750 1500 3000 4500 9000 solution C 597 5.7 6.2 6.7 8.4 8.4 30.9 MAl74 3.9 5.1 6.0 6.7 Experiment II A procedure similar to the one described above (Experiment 1) was used to test culture broth from some more lipase producing Fusarium oxysporum strains. Results Ro a R Enzyme dosage LU/I of presoak 0 750 1500 3000 4500 9000 solution C 597 0.9 3.2 3.7 4.5 A 1714 2.1 3.6 3.3 5.1 A 1755 36.8 3.5 2.8 3.1 5.4 A 1756 3.0 4.7 4.7 6.1 A 1760 2.0 3.7 5.3 5.9 Example 29 This example illustrates the fact that some Fusarium oxysporum strains are lipase producers according to the definition put forword in this specification, whereas other Fusarium oxysporum strains are not lipase producers according to the definition put forward in this specification. The substrate for lipase production consists of the following ingredients in grams/l: Soy bean meal 45 Glucose 70 KH2PO4 2 Na2HPO4 3 Soy oil 5 Sterilization took place at 121 0C for 40 minuteS. Eleven 500 ml Erlenmeyer flasks, each with 100 ml of substrate, were inoculated with 107 spores from agar slants previously inoculated with eleven Fusarium species. The flasks were shaken at 230 rpm and at 300C for 5 days. The yields are shown below: Scientific identifying designation Internal Official of strain identifi- identifi cotion cotion designa designa- Yield, tion tion LU/ml Fusarium oxysporum Schlecht. C597 DSM 2672 122.5 - f.sp. vasinfectum A1714 ATCC 7808 18.5 - f.sp. chrysanthemi CBS 127.81 1.8 - f.sp. cyclaminis CBS 159.57 5.1 - f.sp. gladioli Awl 755 CBS 620.72 15.2 - f.sp. lycopersici A 1756 CBS 645.78 14.6 - f.sp. narcissi CBS 196.65 7.6 - f.sp. opunciarum CBS 743.79 3.3 - - f.sp. passiflora CBS 744.79 7.9 - f.sp. perniciosum A1760 CBS 794.70 10.9 - f.sp. lini ATCC 10960 1.3 All strains indicated in this specification are available to the public. The feotures disclosed in the foregoing description, and/or in the following claims may, both separately and in any combination thereof, be material for realising the invention in diverse forms thereof.

Claims: CLAIMS 1. Enzymatic detergent additive the active component of which is a microbially produced lipase, characterised in that the lipase is producible by means of a lipase producing strain of Fusarium oxysporum. 2. Enzymatic detergent additive according to Claim I, wherein the lipase producing strain of Fusarium oxysporum is DSM 2672. 3. Enzymatic detergent additive according to Claim J or 2, in the form of a non-dusting granulate. 4. Enzymatic detergent additive according to Claim I or 2, in the form of a liquid with an enzyme stabilizer. 5. Enzymatic detergent additive according to Claim 3 or 4, wherein the lipase activity is in the range of from 20,000 to 100,000 LUig of additive. 6. Enzymatic detergent additive according to any preceding claim, wherein the additive contains, besides the lipase, a proteolytic enzyme. 7. Enzymatic detergent additive according to Claim 6, wherein the proteolytic activity is in the range of from 0.5 to 3.0 Anson Units/g of additive. 8. Detergent comprising an enzymatic additive, the active component of which is a microbially produced lipase, characterised in that the enzymatic detergent additive is an enzymatic additive according to any one of Claims I to 7. 9. Detergent according to Claim 8, wherein the detergent contains an enzymatic detergent additive according to Claim I in an amount of from 0.2 to 2.0% w/w. 10. Washing process, choracterised in that the detergent is a detergent according to Claim 8 or 9, the pH is in the range of from 7 to 11, and the temperature is below 600C. I I. Washing process according to Claim 10, wherein the washing solution contains the detergent according to Claim 8 or 9 in an amount in the range of from I to 5 g per litre of washing solution. 12. The use of the enzymatic detergent additive of Claim I, mixed with a detergent component, for washing.

Inventors: NIELSEN, RUBY IONE
Applicants: NOVO INDUSTRI A/S
Application year: 1984
Main classification: C11D3
IPC codes: C12N9, C11D3, C12R1