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Opfindelsen angår et enzymatisk detergentadditiv, hvis aktive bestanddel er en mikrobielt fremstillet lipase, et detergent indeholdende sådanne additiver og en fremgangsmåde til vask af tekstiler, ved hvilken et sådan detergent 5 anvendes.The invention relates to an enzymatic detergent additive whose active ingredient is a microbially prepared lipase, a detergent containing such additives and a method for washing textiles using such a detergent 5.
Det område, som omfatter enzymatiske additiver i detergenter, har været i hastig vækst i de sidste årtier. Der kan f.eks. henvises til artiklen "How Enzymes Got into Detergents", bd. 12, Developments in Industrial Microbiology, en 10 publikation fra Society for Industrial Microbiology, American Institute of Biological Sciences, Washington D.C. 1971, af Claus Dambmann, Poul Holm, Villy Jensen og Mogens Hilmer Nielsen, og til P.N. Christensen, K. Thomsen og S. Branner: "Development of Detergent Enzymes", foredrag holdt den 9.The area that includes enzymatic additives in detergents has been growing rapidly in recent decades. For example, see article "How Enzymes Got into Detergents", vol. 12, Developments in Industrial Microbiology, a 10 publication by 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, and to P.N. Christensen, K. Thomsen and S. Branner: "Development of Detergent Enzymes", lecture held on the 9th.
15 oktober 1986 på "The 2nd World Conference on Detergents" i Montreux, Schweiz.October 15, 1986 at "The 2nd World Conference on Detergents" in Montreux, Switzerland.
Proteolytisk detergentadditiv anvendes i udstrakt grad i Europa, USA og Japan. I flere lande indeholder størstedelen af detergenterne, både flydende og i pulverform, 20 protease.Proteolytic detergent additive is widely used in Europe, the United States and Japan. In several countries, the majority of detergents, both liquid and powder, contain 20 proteases.
Anvendelsen af lipase som et detergentadditiv er kendt. En omfattende oversigt findes i H. Andree et al.: "Lipases as Detergent Components", Journal of Applied Biochemistry, bind 2, 218-229 (1980). Flere eksempler findes i US 25 patentskrift nr. 4,011,169 (spalte 4, linie 65 til spalte 5, linie 68) i GB patentskrift nr. 1,293,613 (side 2, linie 6-29) og i et foredrag af T. Fujii med titlen "Washing of Oil Stains with Lipase" (på japansk), der blev holdt på det 16. Symposium on Washing, der blev afholdt i Tokyo den 17.-18. september 30 1984.The use of lipase as a detergent additive is known. A comprehensive overview can be found in H. Andree et al: "Lipases as Detergent Components", Journal of Applied Biochemistry, Vol. 2, 218-229 (1980). Several examples are found in U.S. Patent No. 4,011,169 (column 4, line 65 to column 5, line 68) in GB patent no. 1,293,613 (page 2, lines 6-29) and in a lecture by T. Fujii entitled "Washing of Oil Stains with Lipase "(in Japanese), held at the 16th Symposium on Washing, held in Tokyo on April 17-18. September 30, 1984.
Blandt kendte lipaser, der er anvendt som detergentadditiver, har Fusarium oxysporum lipase så vidt vi ved de bedste lipolytiske egenskaber, betragtet ud fra et detergentanvendelsessynspunkt, jfr. europæisk patentansøgning med 35 publikåtionsnummer 0 130 064, især sammenligningstabellen på side 27.Among known lipases used as detergent additives, Fusarium oxysporum lipase has, as far as we know the best lipolytic properties, considered from a detergent application point of view, cf. European patent application with 35 public registration number 0 130 064, especially the comparison table on page 27.
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Hvis vaskeprocessen gennemføres ved høj temperatur og høj alkalinitet, vil størstedelen af det fedtholdige snavs under alle omstændigheder blive fjernet. Vaskeprocesser ved lav eller middel temperatur (ca. 60°C og derunder) anvendes 5 dog nu sædvanligvis, og ved disse lave temperaturer er de kendte lipaser kun i stand til at opløse en del af det fedtholdige smuds.If the washing process is carried out at high temperature and high alkalinity, most of the greasy dirt will be removed in any case. However, washing processes at low or medium temperature (about 60 ° C and below) are now commonly used, and at these low temperatures the known lipases are only capable of dissolving part of the fatty soil.
Hidtil er effektiviteten af lipolytiske detergentadditiver sædvanligvis blevet målt ved hjælp af EMPA-lapper 10 (EMPA er en forkortelse for Eidgenossische Materialprufungsund Versuchsanstalt, St. Gallen, Schweiz) nr. 101 (oliven-olie/bomuld) og 102 (olivenolie/uld) ved tillempning af den metode, der er beskrevet i engelsk patentskrift nr. 1.361.386 (især side 4 og 7) og US patentskrift nr. 3.723.250 (især 15 spalte 15-19). På denne måde kan den lipolytiske rensevirkning udtrykkes som forskellen i remissionsværdi delta R. Imidlertid blev her anvendt to direkte mål for den lipolytiske rensevirkning. For det første blev mængden af restfedt bestemt.To date, the effectiveness of lipolytic detergent additives has usually been measured using EMPA patches 10 (EMPA is an abbreviation of Eidgenossische Materialprufungsund Versuchsanstalt, St. Gallen, Switzerland) Nos. 101 (olive oil / cotton) and 102 (olive oil / wool). applying the method described in English Patent No. 1,361,386 (especially pages 4 and 7) and U.S. Patent No. 3,723,250 (especially 15 columns 15-19). In this way, the lipolytic purification effect can be expressed as the difference in the remission value delta R. However, two direct measures of the lipolytic purification effect were used here. First, the amount of residual fat was determined.
Derved vises den kombinerede virkning af detergent og lipase. For 20 det andet blev restfedtet analyseret for tilstedeværelsen af olie (triglycerid) og ned-brydningsprodukter (mono- og digly-cerid og fedtsyre), og antallet af ikke-hydrolyserede glyce-ridbindinger i olien blev beregnet; dette viser lipasevirk-ningen mere direkte. Ved anvendelse af disse metoder har det 25 vist sig, at selv den bedst kendte detergentlipase udviser en lipolytisk vaskevirkning, som kunne ønskes forbedret.This shows the combined effect of detergent and lipase. Second, the residual fat was analyzed for the presence of oil (triglyceride) and degradation products (mono- and diglyceride and fatty acid) and the number of non-hydrolyzed glyceride bonds in the oil was calculated; this shows the lipase effect more directly. Using these methods, it has been found that even the best known detergent lipase exhibits a lipolytic washing effect which could be desired to be improved.
Endvidere er det kendt, at lipaser, eftersom de er proteiner, har tendens til at blive angrebet af proteaser, og som tidligere nævnt indeholder mange detergenter i dag prote-30 aser. Der er intet publiceret, som fortæller om detergent-lipaser, der har tilfredsstillende stabilitet i tilstedeværelsen af protease. Tværtimod har det vist sig, at nogle kendte detergentlipaser har ringe stabilitet i detergentopløsninger i tilstedeværelsen af almindeligt anvendte deter-35 gentproteaser.Furthermore, since they are proteins, lipases are known to be attacked by proteases and, as previously mentioned, many detergents today contain proteases. Nothing has been published to tell us about detergent lipases that have satisfactory stability in the presence of protease. On the contrary, some known detergent lipases have been found to have poor stability in detergent solutions in the presence of commonly used detergent proteases.
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Der er således behov for et lipolytisk detergentadditiv, der udviser en betydeligt bedre lipolytisk detergentvirkning ved økonomisk rimelige lipaseaktiviteter i vaske-flotten, og som er stabil i detergentopløsninger, der inde-5 holder detergentprotease.Thus, there is a need for a lipolytic detergent additive that exhibits a significantly better lipolytic detergent effect on economically reasonable lipase activities in the wash fleet and which is stable in detergent solutions containing detergent protease.
Opfindelsen angår et lipolytisk detergentadditiv, der er ejendommelig ved, at lipasen kan fremstilles ved hjælp af en stamme af slægten Humicola, inklusive slægten Thermomyces.The invention relates to a lipolytic detergent additive, characterized in that the lipase can be prepared by a strain of the genus Humicola, including the genus Thermomyces.
10 Opfindelsen angår endvidere et detergent omfattende ovennævnte lipolytiske detergentadditiv og en fremgangsmåde til vask af tekstiler, der anvender dette detergent ved pH 7-12.The invention further relates to a detergent comprising the aforementioned lipolytic detergent additive and a method for washing fabrics using this detergent at pH 7-12.
Detergentlipaserne som anvendes ifølge opfindelsen 15 udviser en vaskevirkning, der er langt bedre sammenlignet med tidligere kendte detergentlipaser. Endvidere er de lipaser, som er anvendt i opfindelsen, stabile i detergentopløsning ved tilstedeværelsen af almindeligt anvendte detergentproteaser, modsat kendte detergentlipaser.The detergent lipases used according to the invention 15 exhibit a washing effect that is far better compared to previously known detergent lipases. Furthermore, the lipases used in the invention are stable in detergent solution in the presence of commonly used detergent proteases, as opposed to known detergent lipases.
20 Det er beskrevet i japansk patentpublikation nr.It is described in Japanese Patent Publication No.
48-62990 (ikke prøvet), at Humicola lanuginosa er en lipase-producent. Imidlertid nævnes det ikke i denne japanske patentansøgning, at Humicola lanuginosa lipasen er velegnet som aktiv komponent i et enzymatisk detergentadditiv. Tværtimod 25 fremgår det af fig. 1 i den japanske patentansøgning, at Humicola lanuginosa S-38 lipasens pH optimum ligger på ca. 8, og at aktiviteten falder hurtigt når pH-værdien stiger til over 8. Det måtte således forventes, at denne lipase ville være uegnet som detergentadditiv, da pH i vaskeflotter sædvanligvis 30 er langt over 8. Det har dog overraskende vist sig, at48-62990 (not tried) that Humicola lanuginosa is a lipase manufacturer. However, it is not mentioned in this Japanese patent application that the Humicola lanuginosa lipase is suitable as an active component of an enzymatic detergent additive. On the contrary, as shown in FIG. 1 of the Japanese patent application that the pH optimum of the Humicola lanuginosa S-38 lipase is approx. 8, and that the activity decreases rapidly when the pH increases above 8. Thus, it was to be expected that this lipase would be unsuitable as a detergent additive, since the pH of washing floats is usually well above 8. However, it has surprisingly been found that
Humicola lanuginosa S-38 lipasen har et pH optimum langt over 8, se eksempel 1 senere i denne beskrivelse.The Humicola lanuginosa S-38 lipase has a pH optimum well above 8, see Example 1 later in this specification.
Det er endvidere beskrevet i Current Science, aug.It is also described in Current Science, Aug.
5, 1981, bind 50, nr. 15, s. 680, at Humicola lanuginosa lipa-35 sen kan anvendes til kemisk rensning. Da pH optimummet, der udelukkende vedrører vandige medier, er helt uden betydning5, 1981, Volume 50, No. 15, p.680, that the Humicola lanuginosa lipase can be used for chemical purification. As the pH optimum, which relates exclusively to aqueous media, is completely irrelevant
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4 for en lipase til kemisk rensning, betyder ovenstående redegørelse intet for egnetheden af Humicola lanuginosa lipasen som et lipolytisk detergentadditiv.4 for a chemical purification lipase, the above statement does not mean the suitability of the Humicola lanuginosa lipase as a lipolytic detergent additive.
Desuden ses det af Agr.Biol.Chem. 37 (11), side 2488 5 (1973), at H. lanuginosa lipasen hæmmes meget ved tilsætning af visse anioniske overfladeaktive stoffer. Ifølge vore opdagelser er H. lanuginosa lipasen dog overraskende fortrinligt kompatibel med LAS, der er et almindeligt anvendt anionisk overfladeaktivt stof.Moreover, it is seen by Agr.Biol.Chem. 37 (11), page 2488 5 (1973) that H. lanuginosa lipase is greatly inhibited by the addition of certain anionic surfactants. However, according to our findings, the H. lanuginosa lipase is surprisingly excellently compatible with LAS, a commonly used anionic surfactant.
10 Fig. 1-4 viser pH-aktivitetskurverne, fig. 1-2 i er for DSM 3819 lipase, og fig. 3-4 for DSM 1800 lipase.FIG. 1-4 show the pH activity curves; 1-2 in is for DSM 3819 lipase, and figs. 3-4 for DSM 1800 lipase.
Fig. 1 og 3 er ved tributyrinmetoden, og fig. 2 og 4 ved olivenolie/PVC-metoden. jFIG. 1 and 3 are by the tributyrin method, and FIGS. 2 and 4 by the olive oil / PVC method. j
Lipaser som anvendes ifølge opfindelsen opnås j 15 fortrinsvis fra stammer af thermofil Humicola sp., inklusive thermophil Thermomyces sp., såsom H. lanuginosa (Griffon and Maublanc) Bunce, H. stellata Bunce, H. grisea var, thermoidea,Lipases used in the invention are preferably obtained from strains of thermophilic Humicola sp., Including thermophilic Thermomyces sp., Such as H. lanuginosa (Griffon and Maublanc) Bunce, H. stellata Bunce, H. grisea var,
Cooney & Emerson, H. insolens, Cooney & Emerson, Thermomyces ibadanensis, Apinis & Eggins, H. hyalothermophila Moubasher, 20 Mazen og Abdel-Hafez, H. grisea var, indica Subrahmanyam, H. brevis var, thermoidea Subrahmanyam og Thirumalachar og H. brevispora Subrahmanyam og Thirumalachar. j I en særligt foretrukket udførelsesform for det enzymatiske detergentadditiv ifølge opfindelsen kan lipasen fremstilles | 25 fra H. lanuginosa (Griffon og Maublanc) Bunce, H. brevispora iCooney & Emerson, H. insolens, Cooney & Emerson, Thermomyces ibadanensis, Apinis & Eggins, H. hyalothermophila Moubasher, 20 Mazen and Abdel-Hafez, H. grisea var, Indica Subrahmanyam, H. brevis var, Subrahmanyam thermoidea and Thirumalachar and H. .brispora Subrahmanyam and Thirumalachar. In a particularly preferred embodiment of the enzymatic detergent additive of the invention, the lipase can be prepared | 25 from H. lanuginosa (Griffon and Maublanc) Bunce, H. brevispora i
Subrahmanyam og Thirumalachar, H. brevis var, thermoidea Subrahmanyam og Thirumalachar eller H. insolens Cooney & Emerson.Subrahmanyam and Thirumalachar, H. brevis var, Thermoidea Subrahmanyam and Thirumalachar or H. insolens Cooney & Emerson.
H. lanuginosa er endvidere beskrevet under synonymerne Thermomyces lanuginosus Tsiklinsky, Sepedonium lanuginosum 30 Griffon og Maublanc, Sepedonium thermaphilum cyclosporum og S. thermaphilum ovosporum Velich, Acremoniella sp. Rege,H. lanuginosa is also described under the synonyms Thermomyces lanuginosus Tsiklinsky, Sepedonium lanuginosum 30 Griffon and Maublanc, Sepedonium thermaphilum cyclosporum and S. thermaphilum ovosporum Velich, Acremoniella sp. Rege,
Acremoniella thermophila Curzi og Monotospora lanuginosa (Griffon og Maublanc) Mason.Acremoniella thermophila Curzi and Monotospora lanuginosa (Griffon and Maublanc) Mason.
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Ydermere blev arten Scytalidium thermophilum (Cooney 4 Emerson) Austwich af Hedger (1975, "The Ecology of thermophilic Fungi in Indonesia", i "Biodegradation et Humification", "Rapport du ler colloque International" - Nancy 1974 (ed. G. Kilbertius, 5 0. Reisinger, A. Mourey 4 J. A. Cancela Da Fonseca),Furthermore, the species Scytalidium thermophilum (Cooney 4 Emerson) became Austwich by Hedger (1975, "The Ecology of Thermophilic Fungi in Indonesia", in "Biodegradation et Humification", "Report du ler colloque International" - Nancy 1974 (ed. G. Kilbertius, 5 0. Reisinger, A. Mourey 4 JA Cancela Da Fonseca),
Sarreguemines: Pierron Editeur - 57206) anset for at tilhøre Humicola insolens.Sarreguemines: Pierron Editeur - 57206) considered to belong to Humicola Insolence.
Lipasen som anvendes ifølge opfindelsen kan fremstilles af en af følgende stammer: 10 taxonomisk deponerings- benævnelse internt Nr. deponeringsnr. dato H. lanuginosa A 1231 DSM 3819 13. aug 1986 H. lanuginosa H 126 DSM 4109 4. maj 1987 H. brevispora A 2121 DSM 4110 4. maj 1987 15 H. brevis var.The lipase used according to the invention can be prepared from one of the following strains: Internal taxonomic deposit designation No. Deposit. date H. lanuginosa A 1231 DSM 3819 Aug 13, 1986 H. lanuginosa H 126 DSM 4109 May 4, 1987 H. brevispora A 2121 DSM 4110 May 4, 1987 15 H. brevis var.
thermoidea A 2106 DSM 4111 4. maj 1987 H. insolens C 579 DSM 1800 1. okt 1981 DSM står for Deutsche Sammlung von Mikroorganismen. Stammerne er deponeret under Budapesttraktatens bestemmelser.thermoidea A 2106 DSM 4111 May 4, 1987 H. insolens C 579 DSM 1800 Oct 1, 1981 DSM stands for Deutsche Sammlung von Microorganism. The tribes are deposited under the provisions of the Budapest Treaty.
20 Lipase til brug i opfindelsen kan fremstilles ved aerob dyrkning af en af ovenstående stammer i henhold til de i teknikken kendte retningslinier, f.eks. som i de senere angivne eksempler.Lipase for use in the invention can be prepared by aerobically culturing one of the above strains according to the guidelines known in the art, e.g. as in the examples given later.
pH-afhængigheden af lipaseaktiviteten blev bestemt 25 ved en traditionel metode, hvor tributyrin blev anvendt som substrat ved 30°C i en pH-stat og med gummi arabicum som emulgeringsmiddel. Aktiviteten ved forskellige pH-værdier blev fundet udfra forbruget af alkali overfor tid.The pH dependence of lipase activity was determined by a conventional method using tributyrin as a substrate at 30 ° C in a pH state and with gum arabic as the emulsifier. The activity at various pH values was found based on the consumption of alkali over time.
pH-afhængigheden blev også checket med et mere 30 realistisk substrat, nemlig olivenolie, der var adsorberet på PVC (i-henhold til US patentskrift 4,284,719).The pH dependency was also checked with a more realistic substrate, namely olive oil adsorbed on PVC (according to US Patent 4,284,719).
pH-aktivitetskurverne for lipase fra H. lanuginosa DSM 3819 fremgår af fig. 1 (tributyrin) og fig. 2 (oliven-olie/PVC). Kurverne for DSM 4109, DSM 4110 og DSM 4111 varThe pH activity curves for lipase from H. lanuginosa DSM 3819 are shown in FIG. 1 (tributyrin) and FIG. 2 (olive oil / PVC). The curves for DSM 4109, DSM 4110 and DSM 4111 were
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6 tilsvarende, idet de viste optimum ved pH 10,0 - 10,5 ved begge metoder. pH-aktivitetskurverne for lipase fra H. insolens DSM 1800 fremgår af fig. 3 (tributyrin) og fig. 4 (olivenolie/PVC): 5 Isoelektrisk fokusering blev udført på de fem lipaser, hvorefter et lag med tributyrin blev lagt ovenpå for at påvise lipaseaktiviteten. Det viste sig, at DSM 3819, DSM 4109, DSM 4110 og DSM 4111 alle har lipaseaktiviteter med pi omkring 4,5, mens DSM 1800 har størstedelen af dets lipase-10 aktivitet med pi omkring 9,0 - 9,5 og kun en ubetydelig mængde lipaseaktivitet med pi omkring 4,5.6 similarly, showing optimum at pH 10.0 - 10.5 by both methods. The pH activity curves for lipase from H. insolens DSM 1800 are shown in FIG. 3 (tributyrin) and FIG. 4 (olive oil / PVC): 5 Isoelectric focusing was performed on the five lipases, after which a layer of tributyrin was superimposed to detect lipase activity. It was found that DSM 3819, DSM 4109, DSM 4110 and DSM 4111 all have lipase activities with pi around 4.5, while DSM 1800 has most of its lipase 10 activity with pi around 9.0 - 9.5 and only one negligible amount of lipase activity with p1 about 4.5.
Ved en foretrukket udførelsesform tilvejebringes det enzymatiske detergentadditiv ifølge opfindelsen som et ikke-støvende granulat eller som en væske. Disse er egnede til 15 anvendelse i henholdsvis pulverdetergenter og flydende deter-génter. Granulater kan fremstilles på flere forskellige måder.In a preferred embodiment, the enzymatic detergent additive of the invention is provided as a non-dusting granule or as a liquid. These are suitable for use in powder detergents and liquid detergents, respectively. Granules can be prepared in several different ways.
Der kan henvises til britisk patentskrift nr. 1,362,365, som beskriver fremstillingen af enzymholdigt granulat til anvendelse som detergentadditiver ved hjælp af et apparat omfattende 20 en ekstruder og en sfæronisator (solgt som MAMUMERIZER®), og til US patentskrift nr. 4,106,991, som beskriver fremstillingen af enzymholdigt granulat til anvendelse som detergentaddi-tiver ved hjælp af en tromlegranulator.Reference is made to British Patent Specification No. 1,362,365, which describes the preparation of enzyme-containing granules for use as detergent additives by an apparatus comprising an extruder and a spheronizer (sold as MAMUMERIZER®), and to U.S. Patent No. 4,106,991 which describes the preparation. of enzyme-containing granules for use as detergent additives by means of a drum granulator.
Hvad angår en flydende formulering har lagerstabili-25 teten en tendens til at være utilstrækkelig, og en væske med et enzymstabiliseringsmiddel er derfor foretrukket. Stabiliseringsmidler kan være propylenglycol eller andre midler, der er kendt som stabiliseringsmidler til enzymopløsninger. Som det fremgår senere i denne beskrivelse har en ren vandig op-30 løsning af lipasen som anvendes ifølge opfindelsen en ringe lagerstabilitet, men den kan forbedres betydeligt ved at inkludere stabiliseringsmidler, f.eks. propylenglycol.In the case of a liquid formulation, the storage stability tends to be insufficient and a liquid with an enzyme stabilizer is therefore preferred. Stabilizers may be propylene glycol or other agents known as enzyme solution stabilizers. As will be seen later in this specification, a pure aqueous solution of the lipase used according to the invention has poor storage stability, but it can be greatly improved by including stabilizers, e.g. propylene glycol.
I en særligt foretrukket udførelsesform for det enzymatiske detergentadditiv ifølge opfindelsen er lipase-35 aktiviteten over ca. 10.000 LU/g additiv. Lipaseenhed (LU) bliver defineret senere i denne beskrivelse. På denne måde dannes en passende lipaseaktivitet i vaskeflotten, når deter-In a particularly preferred embodiment of the enzymatic detergent additive according to the invention, the lipase activity is above ca. 10,000 LU / g additive. Lipase unit (LU) is defined later in this specification. In this way, a suitable lipase activity is formed in the wash fleet when
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7 gentadditivet tilsættes til detergentet i en mængde af 0,1 til 5,0 g/100 g detergent, og når detergentet tilsættes til vaske-flotten i en mængde af 0,5 - 20 g detergent/1 vaskeflotte.7, the additive is added to the detergent in an amount of 0.1 to 5.0 g / 100 g detergent, and when the detergent is added to the wash float in an amount of 0.5 - 20 g detergent / 1 wash float.
I en særligt foretrukket udførelsesform indeholder 5 det enzymatiske detergentadditiv ifølge opfindelsen andre detergentenzymer udover lipasen, såsom protease, amylase eller cellulase. Alkaliske Bacillus-proteaser er foretrukket på grund af deres velkendte virkning som detergentproteaser. Som sådanne enzymer kan det proteolytiske enzym ALCALASE® fra Novo 10 Industri A/S, fremstillet mikrobielt ved dyrkning af Bacillus licheniformis, eller de proteolytiske enzymer SAVINASE® og ESPERASE®, også fra Novo Industri A/S, fremstillet i henhold til US patentskrift nr. 3,723,250, anvendes. Det blandede enzymatiske additiv kan fremstilles enten ved at blande et 15 tidligere fremstillet granulat af proteinase med et tidligere fremstillet granulat af lipase, eller ved at blande et koncentrat af proteinase med et koncentrat af lipase og derefter indføre blandingen i et granuleringsapparat, sammen med de sædvanlige granuleringshjælpemidler.In a particularly preferred embodiment, the enzymatic detergent additive of the invention contains other detergent enzymes besides the lipase such as protease, amylase or cellulase. Alkaline Bacillus proteases are preferred because of their well-known action as detergent proteases. As such, the proteolytic enzyme ALCALASE® from Novo 10 Industri A / S, produced microbially by the cultivation of Bacillus licheniformis, or the proteolytic enzymes SAVINASE® and ESPERASE®, also from Novo Industri A / S, can be produced according to US Patent 3,723,250 is used. The mixed enzymatic additive can be prepared either by mixing a previously prepared granule of proteinase with a previously prepared granule of lipase, or by mixing a concentrate of proteinase with a concentrate of lipase and then introducing the mixture into a granulator, together with the usual granulating.
20 Nutildags er protease en almindelig detergent- bestanddel, og som det vil fremgå senere, er lipaser som anvendes ifølge opfindelsen i udstrakt grad kompatible i detergentopløsninger med vigtige detergentproteaser, såsom de ovenfor angivne. Hvis både lipase og protease skal tilsættes 25 til et detergent kan det være passende at anvende dem i form af et blandet additiv.Nowadays, protease is a common detergent component and, as will be seen later, lipases used according to the invention are extensively compatible in detergent solutions with important detergent proteases such as those listed above. If both lipase and protease are to be added to a detergent, it may be appropriate to use them in the form of a mixed additive.
I en særligt foretrukket udførelsesform for det enzymatiske detergentadditiv ifølge opfindelsen er den proteolytiske aktivitet mellem ca. 0,5 og ca. 3,0 Ansonenheder/g 30 additiv. På denne måde tilvejebringes en passende proteolytisk aktivitet i vaskeflotten når detergentadditivet tilsættes til detergentet i en mængde af 0,2 - 2 g/100 g detergent, og når detergentet tilsættes til vaskeflotten i en mængde af 0,5 - 20 g detergent/1 vaskeflotte. Den velkendte Anson hæmoglobin-35 metode-for proteolytisk aktivitet er beskrevet i Journal of General Physiology, 22, 79-89 (1959).In a particularly preferred embodiment of the enzymatic detergent additive of the invention, the proteolytic activity is between ca. 0.5 and approx. 3.0 Ansonic units / g 30 additive. In this way, a suitable proteolytic activity is provided in the wash float when the detergent additive is added to the detergent in an amount of 0.2 - 2 g / 100 g detergent and when the detergent is added to the wash float in an amount of 0.5 - 20 g detergent / 1 wash float . The well-known Anson hemoglobin-35 method for proteolytic activity is described in Journal of General Physiology, 22, 79-89 (1959).
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I overensstemmelse med den tidligere nævnte udførelsesform for additivet ifølge opfindelsen kan detergentet ifølge opfindelsen være et pulver eller et flydende detergent, og kan om ønsket omfatte andre detergentenzymer, såsom protea-5 se, amylase eller cellulase, enten i det samme additiv eller som separate additiver.In accordance with the aforementioned embodiment of the additive according to the invention, the detergent according to the invention may be a powder or a liquid detergent and may, if desired, comprise other detergent enzymes such as protease, amylase or cellulase, either in the same additive or as separate additives. .
I en særligt foretrukket udførelsesform for detergentet ifølge opfindelsen indeholder detergentet det enzymatiske _ detergentadditiv ifølge opfindelsen i en mængde af mel- / 10 lem 0,1 og 5% w/w, mere fortrinsvis i en mængde af 0,2 - 2% w/w. På denne måde tilvejebringes en passende balance mellem énzymvirkningen og virkningen af andre detergentbestanddele.In a particularly preferred embodiment of the detergent of the invention, the detergent contains the enzymatic detergent additive according to the invention in an amount of between 0.1 and 5% w / w, more preferably in an amount of 0.2 - 2% w / w. w. In this way, an appropriate balance is provided between the enzyme action and the action of other detergent ingredients.
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Detergentet anvendes typisk i koncentrationer på 0,5 - 20 g/1 vaskeflotte, og passende lipaseaktivitet i vaskeflot-15 ten er 1.000 - 10.000 LU/l, mere fortrinsvis 1.000 - 5.000 LU/1. Således er lipaseaktiviteten i detergentet i en foretrukket udførelsesform 50 - 20.000 LU/g, fortrinsvis 50 - 10.000 LU/g, mere fortrinsvis 250 - 2.000 LU/g og mest fortrinsvis 500 - 2.000 LU/g detergent.The detergent is typically used at concentrations of 0.5 - 20 g / L wash float and appropriate lipase activity in the wash float is 1,000 - 10,000 LU / L, more preferably 1,000 - 5,000 LU / L. Thus, in a preferred embodiment, the lipase activity of the detergent is 50 - 20,000 LU / g, preferably 50 - 10,000 LU / g, more preferably 250 - 2,000 LU / g and most preferably 500 - 2,000 LU / g detergent.
20 Som ovenfor nævnt er den foretrukne lipase i additi vet over 10.000 LU/g, og denne tilsættes til detergenterne i mængder af fortrinsvis 0,1 - 5% w/w og mere fortrinsvis 0,2 -2% w/w. Følgelig er lipaseaktiviteten i detergenterne ifølge en anden foretrukket udførelsesform 10 -500 LU/g, mere for-25 trinsvis 20 - 200 LU/g detergent.As mentioned above, the preferred lipase in the additive is above 10,000 LU / g and is added to the detergents in amounts of preferably 0.1-5% w / w and more preferably 0.2-2% w / w. Accordingly, in another preferred embodiment, the lipase activity of the detergents is 10 -500 LU / g, more preferably 20 - 200 LU / g detergent.
'I en særligt foretrukket udførelsesform omfatter detergentet ifølge opfindelsen andre detergentenzymer foruden lipasen, mest fortrinsvis en protease. Foretrukne detergent-proteaser er de, der allerede er nævnt. Lipase og protease kan 30 tilsættes^til detergentet enten separat eller i form af et blandet additiv. Som allerede nævnt anvendes proteaser almindeligvis i detergenter, og lipa^er som anvendes ifølge opfindelsen udviser en bemærkelsesværdig stabilitet i detergentopløsning med de kommercielt vigtige proteaser. I 35 overensstemmelse med ovennævnte foretrukne værdier for proteaseaktivitet foretrækkes en i 9In a particularly preferred embodiment, the detergent of the invention comprises other detergent enzymes in addition to the lipase, most preferably a protease. Preferred detergent proteases are those already mentioned. Lipase and protease can be added to the detergent either separately or in the form of a mixed additive. As already mentioned, proteases are commonly used in detergents and the lipids used according to the invention exhibit remarkable stability in detergent solution with the commercially important proteases. In accordance with the above preferred values for protease activity, one in 9 is preferred
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proteaseaktivitet i detergentet på 0,0005 - 0,15 AU/g, mere fortrinsvis 0,001 - 0,060 AU/g, endnu mere fortrinsvis 0,003 -0,025 AU/g og mest fortrinsvis 0,006 - 0,010 AU/g detergent.protease activity in the detergent of 0.0005 - 0.15 AU / g, more preferably 0.001 - 0.060 AU / g, even more preferably 0.003 -0.025 AU / g and most preferably 0.006 - 0.010 AU / g detergent.
I en særligt foretrukket udførelsesform for deter-5 gentet ifølge opfindelsen omfatter det overfladeaktive materiale 30-100% anionisk og 0-70% ikke-ionisk overfladeaktivt stof, mest fortrinsvis 50-100% anionisk og 0-50% ikke-ionisk overfladeaktivt stof. Vaskevirkning af lipase som anvendes ifølge opfindelsen er særlig udtalt i detergenter med et højt^ 10 indhold af anioniske midler, såsom LAS (lineær i alkylbenzensulfonat).In a particularly preferred embodiment of the detergent according to the invention, the surfactant comprises 30-100% anionic and 0-70% nonionic surfactant, most preferably 50-100% anionic and 0-50% nonionic surfactant. The washing action of lipase used according to the invention is particularly pronounced in detergents with a high content of anionic agents such as LAS (linear in alkylbenzene sulfonate).
I en særligt foretrukket udførelsesform i vaske-processen ifølge opfindelsen indeholder vaskeflotten detergentet ifølge opfindelsen i en mænge af mellem 0,5 og 20 g/1 15 vaskeflotte. På denne måde tilvejebringes en passende lipase-aktivitet i vaskeflotten, nemlig typisk mellem 1.000 og 10.000 LU/1 vaskeflotte, fortrinsvis mellem 1.000 og 5.000 LU/1 vaskeflotte.In a particularly preferred embodiment of the washing process of the invention, the washing float contains the detergent of the invention in an amount of between 0.5 and 20 g / l of washing float. In this way, a suitable lipase activity is provided in the wash float, namely typically between 1,000 and 10,000 LU / 1 wash float, preferably between 1,000 and 5,000 LU / 1 wash float.
EKSEMPLEREXAMPLES
20 LipaseaktivitetLipase activity
Fremgangsmåden er baseret på hydrolyse af tributyrin i en pH-stat. 1 LU (Lipase Unit - lipaseenhed) er mængden .af enzym, som frigør 1 μπιοί titrerbar smørsyre pr. minut ved 30°C, pH 7.0 med gummi arabicum som et emulgeringsmiddel.The process is based on the hydrolysis of tributyrin in a pH state. 1 LU (Lipase Unit - lipase unit) is the amount of enzyme which releases 1 μπιοί titratable butyric acid per liter. per minute at 30 ° C, pH 7.0 with gum arabic as an emulsifier.
25 Yderligere detaljer gives i Novo Analytical Method AF 95/5, som efter anmodning kan rekvireres.25 Further details are provided in Novo Analytical Method AF 95/5, which can be obtained upon request.
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EKSEMPEL 1 ' Lipase fra H. lanuginosa DSM 3819EXAMPLE 1 Lipase from H. lanuginosa DSM 3819
Fyrre 500 ml rystekolber hver med 200 ml PL-lc medium (sammensætning følger) blev hver podet med 0,2 ml af en 5 opslemning af sporer, fremstillet ud fra skrårør med H. lanuginosa DSM 3819, voksende på YPG-agar (sammensætning følger) i 5 dage ved 45°C. De således podede rystekolber blev rystet i 3 dage ved 45°C med en hastighed på 240 omdrejninger pr. minut.Forty 500 ml shake flasks each with 200 ml PL-1c medium (composition follows) were each seeded with 0.2 ml of a 5 slurry of spores made from H. lanuginosa DSM 3819 swab, growing on YPG agar (composition follows ) for 5 days at 45 ° C. The thus inoculated shake flasks were shaken for 3 days at 45 ° C at a rate of 240 rpm. minute.
På dette tidspunkt var lipaseaktiviteten i den opsamlede væske 10 (6,7 liter).104 LU/ml. Cellerne blev fjernet ved centrifugering ved en hastighed på 4000 omdrejninger pr. minut i 25 minutter. Derved fik man 5,9 liter supernatant. Supernatanten blev filtreret gennem et 10 μ nylonfilterstof og derefter koncentreret 8 gange ved ultrafiltrering på Pellicon UF Cassette system 15 (membraner med en nominel molekylevægtsgrænse på 10.000).At this time, the lipase activity in the collected fluid was 10 (6.7 liters) .104 LU / ml. The cells were removed by centrifugation at a rate of 4000 rpm. 25 minutes. This yielded 5.9 liters of supernatant. The supernatant was filtered through a 10 μ nylon filter fabric and then concentrated 8 times by ultrafiltration on Pellicon UF Cassette system 15 (membranes with a nominal molecular weight limit of 10,000).
UF-koncentratet (endeligt volumen på 740 ml) blev omdannet til råpulver ved frysetørring. Dette råpulver udviste en lipaseaktivitet på 13.310 LU/g.The UF concentrate (final volume of 740 ml) was converted to crude powder by freeze-drying. This crude powder exhibited a lipase activity of 13,310 LU / g.
Sammensætningen af YPG-agar var som følger: 20 Gærekstrakt, Difco 4 g/1The composition of YPG agar was as follows: 20 Yeast extract, Difco 4 g / l
Glucose 15 g/1 ’ K2HP04 1 g//1Glucose 15 g / l 'K2HPO4 1 g // 1
MgS04, 7H20 0,5 g/1MgSO 4, 7H 2 O 0.5 g / l
Agar 20 g/1 r 25 Autoklavering ved 121°C i 40 minutterAgar 20 g / l 25 Autoclaving at 121 ° C for 40 minutes
Sammensætningen af PL-lc medium var som følger:The composition of PL-1c medium was as follows:
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Pepton 15 g/1Peptone 15 g / l
Tween-80 18 g/1Tween-80 18 g / l
MgSC>4, 7H O 2 g/1MgSC> 4, 7H O 2 g / l
CaCl2, 2H20 0,1 g/1 5 Nalco-10 2 g/1 pH før autoklavering 6,0CaCl 2, 2H 2 O 0.1 g / l Nalco-2 2 g / l pH before autoclaving 6.0
Autoklavering ved 121°C i 40 minutter EKSEMPEL 2Autoclaving at 121 ° C for 40 minutes Example 2
Lipase fra andre Humicola-stammer 10 Stamme DSM 4111 blev gæret på medium PL-lc, stamme DSM 4109 på medium GT, stamme DSM 4110 på medium GTS-1, og Stamme DSM 1800 på medium LR-8ST, hovedsageligt som beskrevet i eksempel 1.Lipase from Other Humicola Strains 10 Strain DSM 4111 was fermented on medium PL-1c, strain DSM 4109 on medium GT, strain DSM 4110 on medium GTS-1, and Strain DSM 1800 on medium LR-8ST, mainly as described in Example 1 .
Sammensætning af gæringsmediumComposition of fermentation medium
15 I GT GTS-1 LR-8ST15 I GT GTS-1 LR-8ST
Gærekstrakt g/1 | 22,5 15 0Yeast extract g / 1 | 22.5 15 0
(65% tørstof) I(65% solids) I
20 Pharmamedia | 0 0 5020 Pharmamedia | 0 0 50
Tween 80 — | 18 5 5Tween 80 - | 18 5 5
Span 80 - I 0 5 5 25 " jSpan 80 - I 0 5 5 25 ”j
MgS04,7H20 - I 2 2 0,5MgSO4.7H2O - I 2 2 0.5
CaCl2,2H20 - I 0,1 0,1 0 30 K2HP04 - I 0 0 5CaCl2.2H20 - I 0.1 0.1 0 30 K2HPO4 - I 0 0 5
NaNO^ - I 0 0 1NaNO ^ - I 0 0 1
Nalco-10 I 5 2 0Nalco-10 I 5 2 0
35 I35 I
pH før · autoklavering j ca. 6,5 ca. 6,0_ ca. 7,0pH before · autoclaving j approx. 6.5 approx. 6.0_ ca. 7.0
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Udvinding fra kulturvæskerne blev hovedsageligt udført som beskrevet for DSM 3819 i eksempel 1. I tilfældet med lipase fra DSM 4109 blev et ekstra oprensningstrin udført forud for frysetørring, idet UF-concentratet blev udfældet med acetone, 5 hvorefter det udfældede blev opløst igen i vand og frysetørret.Recovery from the culture liquids was carried out mainly as described for DSM 3819 in Example 1. In the case of lipase from DSM 4109, an additional purification step was performed prior to freeze drying, the UF concentrate being precipitated with acetone, then the precipitated being dissolved again in water and freeze-dried.
De endelige frysetørrede pulvere udviste følgende lipaseaktivi-teter:The final lyophilized powders exhibited the following lipase activities:
Stamme DSM 4111 DSM 4109_DSM 4110 DSM 1800 LU/g 32.000 211.000 6.600 1.800 10 VaskemetodeStrain DSM 4111 DSM 4109_DSM 4110 DSM 1800 LU / g 32,000 211,000 6,600 1,800 10 Washing method
Det prøvemateriale, der blev anvendt i vaskeforsøg, var bomuldsstof (med en overfladevægt svarende til ca. 1,2 g/50 2 cm ) imprægneret med olivenolie (Sigma 0—1500). Lapperne blev fremstillet ved at påføre 50 eller 85 μΐ (som angivet) 15 olivenolie, opvarmet til 50-60°C, i midten af hver prøvelap (7 x 7 cm) ved hjælp af en mikropipette. Efter oliepåføringen blev lapperne ældet ved stuetemperatur i 2 dage.The test material used in washing experiments was cotton fabric (with a surface weight corresponding to about 1.2 g / 50 2 cm) impregnated with olive oil (Sigma 0-1500). The patches were prepared by applying 50 or 85 μΐ (as indicated) of 15 olive oil, heated to 50-60 ° C, in the center of each sample patch (7 x 7 cm) using a micropipette. After the oil application, the patches were aged at room temperature for 2 days.
Lipasepræparaterne fra eksempel 1 og 2 blev anvendt og identificeret ved stammenumrene.The lipase preparations of Examples 1 and 2 were used and identified by the stem numbers.
20 Til sammenligning anvendtes endvidere i dette eksem pel et lipolytisk pulver på basis af Fusarium oxysporum, fremkommet som beskrevet i eksempel 23 i europæisk patentansøgning med publikationsnummer 0 130 064, som repræsenterende det mest effektive kendte lipolytiske detergentadditiv. Fusarium oxy-25 sporum lipasepræparatets aktivitet var 90.000 LU/g.In comparison, in this example, a lipolytic powder based on Fusarium oxysporum, as described in Example 23 of European Patent Application Publication No. 0 130 064, as the most effective known lipolytic detergent additive, was also used. The activity of the Fusarium oxy-sporum lipase preparation was 90,000 LU / g.
Lipaserne blev bedømt ved vaskeforsøg i en Terg-0-Tometer forsøgsvaskemaskine. Terg-O-Tometer forsøgsvaske-maskinen er beskrevet hos Jay C. Harris, Detergency evaluation and testing, Interscience Publishers Ltd., 1954, side 60 - 61.The lipases were assessed by washing experiments in a Terg-0-Tometer test washer. The Terg-O-Tometer test washer is described by Jay C. Harris, Detergency evaluation and testing, Interscience Publishers Ltd., 1954, pages 60 - 61.
30 Vaskeforsøgene udførtes under følgende betingelser:The washing experiments were performed under the following conditions:
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Omrøring 100 omdrejninger pr. minutStirring 100 rpm. minute
Vandets hårdhedsgrad 18°dH (fra hanen), hvis andet ikke er angivet stof/væske forhold 7 lapper/1000 ml 5 skylning 15 min under løbende hane Mængden af olie, der var indeholdt i 7 lapper med 85 μΐ hver er ca. 535 m.j (vægtfylde 0,90).Water hardness 18 ° dH (from tap), unless otherwise stated substance / liquid ratio 7 patches / 1000 ml 5 rinses 15 min under running tap The amount of oil contained in 7 patches with 85 μΐ each is approx. 535 m.j (density 0.90).
Efter skylning blev lapperne lufttørret. Restindholdet af olie i lapperne blev bestemt ved Soxhlet ekstration 10 med n-hexan i 5 timer efterfulgt af gravimetrisk bestemmelse af restmængden.After rinsing, the patches were air dried. The residual oil content of the patches was determined by Soxhlet Extraction 10 with n-hexane for 5 hours followed by gravimetric determination of the residual amount.
Sammensætningen af restolien blev analyseret ved hjælp af TLC-FID (TLC-FID er en forkortelse af "thin layer chromatography/flame ionization detector", og fremgangsmåden er 15 beskrevet i Lipids, vol. 18, nr. 10 (1983), side 732) metoden, hvor der anvendes en Iatroscan TH-10 (Iatron Lab. Inc., Tokyo) kombineret med en Chromatocorder II (System Instruments Co.,The composition of the residual oil was analyzed by TLC-FID (TLC-FID is an abbreviation of "thin layer chromatography / flame ionization detector" and the procedure is described in Lipids, vol. 18, no. 10 (1983), page 732 ) method using an Iatroscan TH-10 (Iatron Lab. Inc., Tokyo) combined with a Chromatocorder II (System Instruments Co.,
Ltd., Tokyo) integrator ved følgende betingelser: fast fase Chromarod S-II (Iatron) 20 mobil fase Hexan/ethylæter/eddikesyre (60:50:2 v/v/v) hydrogentilførsel 160 ml/min lufttilførsel 2000 ml/min scanningshastighed 30 sek. pr. scanning 25 Prøver til TLC-FID analysen blev fremstillet som følger. Efter gravimetrisk bestemmelse af restmængden blev det tørrede ekstrakt opløst igen i 20 ml hexan, og 5 ml intern standard (lithocholsyre, 12,5 mg/ml) opløst i ethanol blev tilsat. Til hver analyse brugtes 1 μΐ af prøven.Ltd., Tokyo) integrator under the following conditions: solid phase Chromarod S-II (Iatron) 20 mobile phase Hexane / ethyl ether / acetic acid (60: 50: 2 v / v / v) hydrogen supply 160 ml / min air supply 2000 ml / min scan rate 30 sec. per. scan 25 Samples for the TLC-FID assay were prepared as follows. After gravimetric determination of the residual amount, the dried extract was redissolved in 20 ml of hexane and 5 ml of internal standard (lithocholic acid, 12.5 mg / ml) dissolved in ethanol was added. For each assay, 1 μΐ of the sample was used.
30 Baseret på standardkurver for triolein, diolein, monoolein og olies'yre blev restoliens relative sammensætning (% w/w) beregnet.Based on standard curves for triolein, diolein, monoolein and oleic acid, the relative composition (% w / w) of the residual oil was calculated.
Antallet af uhydrolyserede glyceridbindinger i restolien blev beregnet ved følgende formel:The number of unhydrolyzed glyceride bonds in the residual oil was calculated by the following formula:
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14 3 2 1 n = 10 x M (885 X Xm_ + 621 x X^ + 357 x X ) (μ mol)14 3 2 1 n = 10 x M (885 X Xm + 621 x X ^ + 357 x X) (µmol)
1G UG MG1G UG MG
hvor X = procent triglycerid (% w/w) i Gwhere X = percent triglyceride (% w / w) in G
Xn_ = procent diglycerid (% w/w)Xn_ = percent diglyceride (% w / w)
UGUG
5 X = procent monoglycerid (% w/w)5 X = percent monoglyceride (% w / w)
MGMG
M = restmængden af olie (mg) 885, 621 og 357 er molekylevægten for henholdsvis triolein, diolein og monolein.M = residual amount of oil (mg) 885, 621 and 357 is the molecular weight of triolein, diolein and monolein respectively.
EKSEMPEL 3 10 Virkningen af vasketemperaturEXAMPLE 3 The effect of washing temperature
Dette eksempel demonstrerer virkningen af Humicola lanuginosa lipasen (DSM 3819) i et anionisk detergent ved forskellige vasketemperaturer.This example demonstrates the effect of the Humicola lanuginosa lipase (DSM 3819) in an anionic detergent at different washing temperatures.
Detergentsammensætning: LAS (0,5 g/1), Na2C0^ (1/0 g/1) 15 Vasketid: 20 minutterDetergent composition: LAS (0.5 g / l), Na 2 CO 2 (1/0 g / l) Washing time: 20 minutes
Lipasedosering: 3000 LU/1 pH: 9,5Lipase dosing: 3000 LU / 1 pH: 9.5
Besmudsning: 85 μΐ olivenolie LAS er lineær alkylbenzensulfonat (Nansa HS80/S, Albright & 20 Wilson), et anionisk overfladeaktivt stof.Contamination: 85 μΐ olive oil LAS is linear alkyl benzene sulfonate (Nansa HS80 / S, Albright & Wilson), an anionic surfactant.
Uden Fusarium HumicolaWithout Fusarium Humicola
Temp._lipase_oxysporum lanuginosaTemp._lipase_oxysporum lanuginosa
Restolie (mg) 185 187 165 30°C η (μ mole) 590__571__360_ 25 Restolie (mg) 187 192 157 50°C η (μ mol)_606_628_432_ 15Residual oil (mg) 185 187 165 30 ° C η (μmole) 590__571__360_ 25 Residual oil (mg) 187 192 157 50 ° C η (μmol) _606_628_432_ 15
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EKSEMPEL 4EXAMPLE 4
Virkning af vasketid I dette eksempel vises virkningen af Humicola lanuginosa lipasen (DSM 3819) ved at anvende forskellige vasketider.Effect of washing time In this example, the effect of the Humicola lanuginosa lipase (DSM 3819) is shown by using different washing times.
5 Detergentsammensætning: LAS (0,5 g/1), Na^CO^ (1,0 g/1)Detergent composition: LAS (0.5 g / l), Na 2 CO 2 (1.0 g / l)
Temperatur: 30°CTemperature: 30 ° C
Lipasedosering: 3000 LU/1 pH (initial): 9,5Lipase dosing: 3000 LU / 1 pH (initial): 9.5
Besmudsning: 85 μΐ olivenolie 10 Vasketid Uden Fusarium Humicola (min. )_lipase_oxysporum lanuginosa 20 Restolie (mg) 185 187 165 η (μ mol) 590 571 360 40 Restolie (mg) 177 167 128 15 η (μ mol) 568 526 246 60 Restolie (mg) 141 147 93 η (μ mol) 465 454 153 90 Restolie (mg) 139 135 78 η (μ mol) 431 419 111 20 EKSEMPEL 5Contamination: 85 μΐ olive oil 10 Washing time Without Fusarium Humicola (min) _lipase_oxysporum lanuginosa 20 Residual oil (mg) 185 187 165 η (μ mol) 590 571 360 40 Residual oil (mg) 177 167 128 15 η (μ mol) 568 526 246 60 Residual oil (mg) 141 147 93 η (μmol) 465 454 153 90 Residual oil (mg) 139 135 78 η (μmol) 431 419 111 20 EXAMPLE 5
Virkning af vandets hårdhedsgrad ved vaskEffect of water hardness on washing
Dette eksempel viser indflydelsen af vandets hårdhedsgrad på vaskevirkningen af Humicola lanuginosa lipase (DSM 3819). Hårdhedsgraden (°dH = tysk hårdhed) blev indstillet ved 25 at blande vand fra hanen med destilleret vand.This example shows the influence of water hardness on the washing effect of Humicola lanuginosa lipase (DSM 3819). The hardness (° dH = German hardness) was set by mixing water from the tap with distilled water.
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Detergentsammensætning:. LAS (0,5 g/1), Na2C03 (1,0 g/1)Detergent :. LAS (0.5 g / l), Na 2 CO 3 (1.0 g / l)
Temperatur: 30°CTemperature: 30 ° C
Vasketid: 20 minutterWashing time: 20 minutes
Lipasedosering: 3000 LU/1 5 pH (initial): 9,5Lipase dosing: 3000 LU / 1 pH (initial): 9.5
Besmudsning: 85 μΐ olivenolieDirt: 85 μΐ olive oil
Hardhedsgrad Uden Fusarium Humicola °dH lipase oxysporum lanuginosa 0 Restolie (mg) 254 244 242 10 η (μ mol) 820 752 627 6 Restolie (mg) 210 192 173 η (μ mol) 670 595 415 12 Restolie (mg) 182 179 170 η (μ mol) 579 548 405 15 18 Restolie (mg) 185 187 165 η (μ mol) 590 571 360 EKSEMPEL 6Hardness Without Fusarium Humicola ° dH lipase oxysporum lanuginosa 0 Residual oil (mg) 254 244 242 10 η (μ mol) 820 752 627 6 Residual oil (mg) 210 192 173 η (μ mol) 670 595 415 12 Residual oil (mg) 182 179 170 η (μ mol) 579 548 405 15 18 Residual oil (mg) 185 187 165 η (μ mol) 590 571 360 EXAMPLE 6
Virkning af lipasedosering ved vaskEffect of lipase dosing on washing
Dette eksempel viser, hvilken betydning dosering af H.This example demonstrates the importance of dosing of H.
20 lanuginosa lipase har ved vaskeudførelser under anvendelse af acetonefraktioneret lipasepulver fra DSM 3819.20 lanuginosa lipase has in wash designs using acetone fractionated lipase powder from DSM 3819.
Detergentsammensætning: LAS (0,5 g/1), Na2C03 (1,0 g/1)Detergent composition: LAS (0.5 g / l), Na 2 CO 3 (1.0 g / l)
Vasketid ‘ 20 minutterWashing time '20 minutes
Temperatur 30°CTemperature 30 ° C
25 pH (initial): 9,5PH (initial): 9.5
Besmudsning: 85 μΐ olivenolieDirt: 85 μΐ olive oil
De besmudsede lapper, der anvendtes, var fra en anden portion end i eksemplerne 2-5, så resultaterne kan ikke sammenlignes direkte.The soiled patches used were from a different portion than in Examples 2-5, so the results cannot be directly compared.
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Lipasedosering, LU/ml 0 500 1500 3000 6000 10,000Lipase dosing, LU / ml 0 500 1500 3000 6000 10,000
Restolie (mg) 232 209 202 202 194 176 η (μ mol) 761 558 521 471 437 363 EKSEMPEL 7 5 Sammenligning af Humicola lipaser ved vaskResidual oil (mg) 232 209 202 202 194 176 η (μmol) 761 558 521 471 437 363 EXAMPLE 7 5 Comparison of Humicola lipases by washing
Dette eksempel sammenligner den vaskevirkning, der blev opnået med lipase fra H. lanuginosa (DSM 4109), H. brevis var, thermoidea (DSM 4111), H. brevispora (DSM 4110) og H. insolens (DSM 1800).This example compares the washing effect obtained with lipase from H. lanuginosa (DSM 4109), H. brevis var, thermoidea (DSM 4111), H. brevispora (DSM 4110) and H. insolens (DSM 1800).
10 Detergentsammensætning: LAS 0,50 g/1Detergent composition: LAS 0.50 g / l
Talgsæbe 0,05 -Soap Soap 0.05 -
Alkoholethoxylat (C^.^r^EO) 0,10 -Alcohol ethoxylate (C ^ ^ r ^ EO) 0.10 -
Alkoholethoxylat (C^g_^g,30EO) 0,02 -Alcohol ethoxylate (C ^ g_ g, 30EO) 0.02 -
Zeolite 1,20 - 15 Na2C03 . 0,50 -Zeolite 1.20 - Na2 CO3. 0.50 -
Natriummetasilicat 0,10 - EDTA (titriplex III) 0,01 -Sodium metasilicate 0.10 - EDTA (titriplex III) 0.01 -
Na2S04 2,00 -Na2 SO4 2.00 -
Temperatur: 30°CTemperature: 30 ° C
20 Vasketid: 20 minutter20 Washing time: 20 minutes
Lipasedosering: 6000 LU/1 pH: 9,5Lipase dosing: 6000 LU / 1 pH: 9.5
Besmudsning: 50μ1 olivenolieDirt: 50μ1 olive oil
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Lipasepræparat_η (μ mol) intet 546 H. lanuginosa 454 H. brevis var. thermoidea 468 5 H. brevispora 484 H. insolens 350 EKSEMPEL 8Lipase preparation_η (μ mol) nothing 546 H. lanuginosa 454 H. brevis var. thermoidea 468 5 H. brevispora 484 H. insolens 350 EXAMPLE 8
Stabilitet af Humicola lipase overfor proteaseStability of Humicola lipase to protease
Den udmærkede stabilitet af Humicola lipaser i deter-10 gentopløsninger indeholdende proteolytiske enzymer vises i nedenstående.The excellent stability of Humicola lipases in detergent solutions containing proteolytic enzymes is shown below.
Et Humicola lanuginosa præparat (DSM 4109) sammenlignes med den Fusarium oxysporum lipase, der blev anvendt i de foregående eksempler og med det kommercielle lipasepræparat, 15 Amano P (Amano Pharmaceutical co. Ltd., Nagoya, Japan), som er fremstillet af Pseudomonas fluorescens.A Humicola lanuginosa preparation (DSM 4109) is compared with the Fusarium oxysporum lipase used in the previous examples and with the commercial lipase preparation, 15 Amano P (Amano Pharmaceutical co. Ltd., Nagoya, Japan), produced by Pseudomonas fluorescens .
De alkaliske Bacillus proteaser ALCALASE®, SAVINASE® og ESPERASE® anvendtes. Disse er kommercielle detergentproteaser fra Novo Industri A/S, Danmark.The alkaline Bacillus proteases ALCALASE®, SAVINASE® and ESPERASE® were used. These are commercial detergent proteases from Novo Industri A / S, Denmark.
20 Den proteolytiske aktivitet blev bestemt med kasein som substratet. En Kasein proteaseenhed (CPU) blev defineret som mængden af enzym, der frigør 1 mM af primære aminogrupper (bestemt ved sammenligning med en serinstandard) pr. minut under standardbetingelser, dvs inkubation i 30 minutter ved 25°C og pH 25 9,5. En 2% (w/v) opløsning af kasein (Hammersten, solgt af Merck A.G., Vesttyskland) blev fremstillet med Universalpufferen, der er beskrevet i Britton og Robinson (Journ.Chem.Soc. 1931, s.The proteolytic activity was determined with casein as the substrate. A casein protease unit (CPU) was defined as the amount of enzyme that liberates 1 mM of primary amino groups (determined by comparison with a serine standard) per day. per minute under standard conditions, i.e. incubation for 30 minutes at 25 ° C and pH 25 9.5. A 2% (w / v) solution of casein (Hammersten, sold by Merck A.G., West Germany) was prepared with the Universal Buffer described in Britton and Robinson (Journ.Chem.Soc. 1931, p.
1451) indstillet til pH 9,5.1451) adjusted to pH 9.5.
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Detergent: 1,3 g/1 af et fosfat-frit pulver indeholdende 25% overfladeaktivt stof (alfa-olefin sulfonate (AOS) og lineær alkylbenzensulfonat 5 (LAS)), natriumsulfat, zeolite, natriumsilicat og optisk hvidt.Detergent: 1.3 g / l of a phosphate-free powder containing 25% surfactant (alpha-olefin sulfonate (AOS) and linear alkylbenzenesulfonate 5 (LAS)), sodium sulfate, zeolite, sodium silicate and optical white.
Vandets hårdhedsgrad: 4,5°dHWater hardness: 4.5 ° dH
pH: 10,0 (indstillet)pH: 10.0 (adjusted)
Temperatur: 25°CTemperature: 25 ° C
10 Lipaseaktivitet (initial): 3000 LU/1Lipase activity (initial): 3000 LU / 1
Proteaseaktivitet: 0 eller 0,05 CPU/1Protease activity: 0 or 0.05 CPU / 1
Resterende lipaseaktivitet (%): 1) Protease: SAVINASE® [ Inkubationstid (min)Residual lipase activity (%): 1) Protease: SAVINASE® [Incubation time (min)
Lipd.se 15 I 0 15 I 30 I 60 90 , I I -Lipd.se 15 I 0 15 I 30 I 60 90, I I -
Humicola lanuginosa | 100 99 | 94 | 91 89Humicola lanuginosa | 100 99 | 94 | 91 89
Fusarium oxysporum j 100 32 | 14 j 3 -Fusarium oxysporum j 100 32 | 14 j 3 -
Pseudomonas j j | 20 fluorescens_| 100_1 j_0 |_-_ - 2) Protease: Ingen T . I Inkubationstid (min)Pseudomonas j j | Fluorescence | 100_1 j_0 | _-_ - 2) Protease: None T. In Incubation Time (min)
Lipase ' I 0 I 15 I 30 I 60 I 90 -i-1-1-1-1- 25 Humicola lanuginosa | 100 | 101 j 99 | 102 | 96Lipase 'I 0 I 15 I 30 I 60 I 90 -i-1-1-1-1- 25 Humicola lanuginosa | 100 | 101 j 99 | 102 | 96
Fusarium oxysporum | 100 1 57 | 42 | 18 ( 6Fusarium oxysporum | 100 1 57 | 42 | 18 (6)
Pseudomonas ί I I I IPseudomonas ί I I I I
fluorescens_j 100 j 94 j 94 j 88 j 90 20fluorescence_j 100 j 94 j 94 j 88 j 90 20
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Detergent: LAS 0,40 g/1Detergent: LAS 0.40 g / l
Alkoholethoxylat (Berol 065) 0,15 g/1Alcohol ethoxylate (Berol 065) 0.15 g / l
Talgsæbe 0,15 g/1Tallow soap 0.15 g / l
Natriumtripolyfosfat 1,50 g/1 5 Natriummetasilicat 0,40 g/1 CMC 0,05 g/1Sodium tripolyphosphate 1.50 g / l Sodium metasilicate 0.40 g / l CMC 0.05 g / l
Na2S04 2,10 g/1Na 2 SO 4 2.10 g / l
Vandets hårdhedsgrad: 18°dH pH: 9,5Water hardness: 18 ° dH pH: 9.5
10 Temperatur: 30°CTemperature: 30 ° C
Lipaseaktivitet: 3000 LU/1Lipase activity: 3000 LU / 1
Proteaseaktivitet: 0 eller 0,05 CPU/1 1) Protease: SAVINASE® I Inkubationstid (min)Protease Activity: 0 or 0.05 CPU / 1 1) Protease: SAVINASE® In Incubation Time (min)
Lipase 15 ( 0 I 15 ( 30 I 60 ( 90 j | i-1-1-Lipase 15 (0 I 15) 30 I 60 (90 µl
Humicola lanuginosa | 100 | 99 | 98 | 97 | 97Humicola lanuginosa | 100 | 99 | 98 | 97 | 97
Fusarium oxysporum | 100 j 5 j 0 j |Fusarium oxysporum | 100 j 5 j 0 j |
Pseudomonas I I I I IPseudomonas I I I I I I
20 fluorescens_j 100 j_0 |_ j_ j_- 2) Protease: ALCALASE® _. I Inkubationstid (min)20 fluorescence_j 100 j_0 | _ j_ j_- 2) Protease: ALCALASE® _. In Incubation Time (min)
Lipase ' ( 0 I 15 I 30 I 60 J 90 -1-1-1-1-1- 25 Humicola lanuginosa | 100 [ 98 | 97 | 95 j 95Lipase '(0 I 15 I 30 I 60 J 90 -1-1-1-1-1- 25 Humicola lanuginosa | 100 [98 | 97 | 95 j 95
Fusarium oxysporum | 100 | 24 j 4 j 0 jFusarium oxysporum | 100 | 24 j 4 j 0 j
Pseudomonas I j I I IPseudomonas I j I I I
fluorescens_| 100 j 18 j_0 j_ (_-fluorescens_ | 100 j 18 j_0 j_ (_-
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21 3) Protease: ESPERASE® I Inkubationstid (min)21 3) Protease: ESPERASE® I Incubation Time (min)
Lipase ' _0 15 30 60 90 5 Humicola lanuginosa 100 97 96 96 98Lipase '0 15 30 60 90 5 Humicola lanuginosa 100 97 96 96 98
Fusarium oxysporum 100 20 0Fusarium oxysporum 100 20 0
Pseudomonas fluorescens_100_0_-_"_~ 4) Protease: Ingen 10 I Inkubationstid (min)Pseudomonas fluorescence_100_0 _-_ "_ ~ 4) Protease: None 10 In Incubation Time (min)
Lipase (0 I 15 I 30 I 60 I 90Lipase (0 I 15 I 30 I 60 I 90
1 I 1 I I1 I 1 I I
Humicola lanuginosa | 100 j 96 | 95 | 96 | 94Humicola lanuginosa | 100 j 96 | 95 | 96 | 94
Fusarium oxysporum j 100 [ 30 | 10 j 0 jFusarium oxysporum j 100 [30 | 10 j 0 j
15 Pseudomonas j | III15 Pseudomonas j | III
fluorescens_j 100 j 101 j 102 | 102 j 99fluorescence_j 100 j 101 j 102 | 102 j 99
Det ses, at Humicola lipasen ifølge opfindelsen er meget stabil i detergentopløsninger med protease i modsætningen til kendte détergentlipaser (Fusarium og Pseudomonas).It is seen that the Humicola lipase according to the invention is very stable in detergent solutions with protease in contrast to known detergent lipases (Fusarium and Pseudomonas).
20 EKSEMPEL 9EXAMPLE 9
Stabiliseret flydende Humicola lipasepræparatStabilized liquid Humicola lipase preparation
Lipasestabilitet i opløsninger med forskellige stabiliseringmidler blev undersøgt.Lipase stability in solutions with various stabilizers was investigated.
Lipase: Humicola lanuginosa (DSM 4109)Lipase: Humicola lanuginosa (DSM 4109)
25 Lagringstemp: 30°CStorage temperature: 30 ° C
pH: 7,0pH: 7.0
Rodalon blev tilsat til alle præparaterne som konserveringsmiddel (0,2 mg aktivt stof pr. ml).Rodalon was added to all the preparations as a preservative (0.2 mg of active substance per ml).
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Resultater: I III INITIAL || Restaktivitet (%), I 1,2-propandiol ,Sorbitol|C^l2.2H20|AmOTIET|, ^ I (ml/ml) I (cx/ml) I (mq/ml) I (LU/ml) II 0 I 2 I 13 I 29 I 49Results: I III INITIAL || Residual activity (%), I 1,2-propanediol, Sorbitol | C12.2H20 | AmOTi |, I (ml / ml) I (cx / ml) I (mq / ml) I (LU / ml) II I 2 I 13 I 29 I 49
5 I || I II I I I I5 I || I II I I I I
1 I 0_I 0 I 0 I 4520 || 100 I 17 I 2 | 1 | 11 I 0_I 0 I 0 I 4520 || 100 I 17 I 2 | 1 | 1
I II I II I I I II II I II I I I I
2 I 0,50_I 0 I 0 I 4520 11 100 | 93 | 63 | 35 | 342 I 0.50_I 0 I 0 I 4520 11 100 | 93 | 63 | 35 | 34
I II I II I I I II II I II I I I I
10 3 I 0,50_I 0 I 3 | 4720 11 100 | 86 | 76 | 54 | 4810 3 I 0.50_I 0 I 3 | 4720 11 100 | 86 | 76 | 54 | 48
I II I II I I I II II I II I I I I
4 I 0_1 0.30 I 0 I 4880 11 100 | 57 | 10 | 1 [ 14 I 0_1 0.30 I 0 I 4880 11 100 | 57 | 10 | 1 [1
Disse resultater viser, at 1.2-propandiol (MPG = mono-propylenglycol) er et udmærket stabiliseringsmiddel for Humicola 15 lipase. Lagerstabiliteten blev yderligere forbedret ved tilsætning af Ca salt. Sorbitol har en svagt stabiliserende virkning.These results show that 1,2-propanediol (MPG = monopropylene glycol) is an excellent stabilizer for Humicola lipase. The storage stability was further improved by the addition of Ca salt. Sorbitol has a slightly stabilizing effect.
Et vaskeforsøg blev udført med stabiliseret flydende lipasepræparat med følgende sammensætning:A wash experiment was performed with stabilized liquid lipase composition having the following composition:
Humicola lanuginosa DSM 4109 lipase 5000 LU/ml 20 1.2-propandiol 50% v/vHumicola lanuginosa DSM 4109 lipase 5000 LU / ml 20-propanediol 50% v / v
Deioniseret vand 50% v/vDeionized water 50% v / v
CaC^^^O 3 mg/mlCaCl 3 O 3 mg / ml
Detergentsammensætning: LAS (0,5 g/1), Na2CC>2 (1,0 g/1)Detergent composition: LAS (0.5 g / L), Na 2 CC> 2 (1.0 g / L)
Temperatur: 30°CTemperature: 30 ° C
25 Vasketid: 20 minutter pH: 9,5Washing time: 20 minutes pH: 9.5
Besmudsning: 50 μΐ olivenolieDirt: 50 μΐ olive oil
Lipasedosering: 1 ml stabiliseret flydende præparat pr.Lipase dosing: 1 ml of stabilized liquid preparation per day.
liter vaskeflotte.liters of washing floats.
23 DK 157560 B23 DK 157560 B
Resultater:________Results: ________
Lipasedosering Restolie (mg) η (μπιοί) LU/1___ 0 157 507 5 5000 145 418 EKSEMPEL 10Lipase Dosage Residual Oil (mg) η (μπιοί) LO / 1 ___ 0 157 507 5 5000 145 418 EXAMPLE 10
Humicola lipase som støvfrit granulatHumicola lipase as dust-free granules
En granulatbærer uden enzym blev hovedsageligt fremstillet i henhold til US patentskrift nr. 4,106,991 med følgende 10 sammensætning: 15% cellulosefibre 4% titaniumdioxid 5% gult dextrin 10% saccharose 15 64% natriumsulfatA granule carrier without enzyme was prepared mainly according to US Patent No. 4,106,991 having the following composition: 15% cellulose fiber 4% titanium dioxide 5% yellow dextrin 10% sucrose 15 64% sodium sulfate
Dette granulat blev sigtet for at opnå en partikelstørrelse på 300-710 μπι.This granulate was screened to obtain a particle size of 300-710 μπι.
30,8 g of dette blev befugtet med 6,2 g af en 30% opløsning i ethanol af polyvinylpyrrolidon (PVP K30, produkt af 20 GAF Corp., USA). Efter grundig blanding blev 12,3 frysetørret H. lanuginosa DSM 4109 lipase (92.700 LU/g, hovedsageligt fremstillet som i eksempel 2) tilsat, og blev grundigt blandet. Granulatet blev tørret (fordampning af ethanol) ved gennemblæsning (fluidisering) ved ca. 50°C. Partikler med partikelstørrelser på 25 300-850 μπι blev opsamlet ved sigtning.30.8 g of this was wetted with 6.2 g of a 30% solution in ethanol of polyvinylpyrrolidone (PVP K30, product of 20 GAF Corp., USA). After thorough mixing, 12.3 lyophilized H. lanuginosa DSM 4109 lipase (92,700 LU / g, mainly prepared as in Example 2) was added and thoroughly mixed. The granulate was dried (evaporation of ethanol) by purging (fluidization) at ca. 50 ° C. Particles with particle sizes of 25 300-850 μπι were collected by sieving.
Granulatet blev derefter overtrukket i tre trin som følger:The granulate was then coated in three steps as follows:
DK 157560 BDK 157560 B
24 - 5% polyethylenglycol (MW 600) - 11,25% TiC^/Mg silicat (4:1) - 4% polyethylenglycol (MW 600)24 - 5% polyethylene glycol (MW 600) - 11.25% TiC 2 / Mg silicate (4: 1) - 4% polyethylene glycol (MW 600)
Det overtrukkede granulat blev gennemblæst ved 0,8 5 m/sekund i 10 minutter for at fjerne fine partikler af overtræksmateriale. Endelig blev materialet igen sigtet, og partikler med partikelstørrelser fra 300 til 850 pm blev opsamlet. Et støvfrit, offwhite granulat blev opnået.The coated granulate was purged at 0.8 5 m / second for 10 minutes to remove fine particles of coating material. Finally, the material was screened again and particles with particle sizes from 300 to 850 µm were collected. A dust-free, off-white granule was obtained.
Udbytte og aktivitet blev som følger: 10 frysetørret pulver 92.700 LU/g 12,3 g uovertrukket granulat 21.100- 45,0 - overtrukket granulat 18.200 -Yield and activity were as follows: 10 freeze-dried powder 92,700 LU / g 12.3 g uncoated granules 21,100- 45,0 - coated granules 18,200 -
Et vaskeforsøg blev udført med det frysetørrede pulver og granulatet som følger: 15 Detergentsammensætning: LAS (0,5 g/1), Na^CO^ (1/0 g/1)A wash experiment was performed with the freeze-dried powder and the granulate as follows: Detergent composition: LAS (0.5 g / l), Na 2 CO 2 (1/0 g / l)
Temperatur: 30°CTemperature: 30 ° C
Vasketid: 20 minutterWashing time: 20 minutes
Lipasedosering: 6000 LU/1 pH: 10,0 20 Besmudsning: 50 μΐ olivenolieLipase dosage: 6000 LU / 1 pH: 10.0 20 Contamination: 50 μΐ olive oil
Resultater:_Results: _
Lipasepræparat_η (μ mol)_ intet 515 frysetørret pulver 386 25 granulat 415Lipase Preparation_η (μmol) _ No 515 Freeze-dried Powder 386 25 Granules 415
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DK449987ADK157560C (en) | 1986-08-29 | 1987-08-28 | ENZYMATIC DETERGENT ADDITIVE, DETERGENT CONTAINING THE ENZYMATIC DETERGENT ADDITIVE AND PROCEDURE FOR WASHING TEXTILES. |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DK411786 | 1986-08-29 | ||
| DK411786ADK411786D0 (en) | 1986-08-29 | 1986-08-29 | ENZYMATIC DETERGENT ADDITIVE, DETERGENT AND WASHING METHOD |
| DK481686ADK481686D0 (en) | 1986-10-09 | 1986-10-09 | ENZYMATIC DETERGENT ADDITIVE, DETERGENT AND WASHING METHOD |
| DK481686 | 1986-10-09 | ||
| DK449987ADK157560C (en) | 1986-08-29 | 1987-08-28 | ENZYMATIC DETERGENT ADDITIVE, DETERGENT CONTAINING THE ENZYMATIC DETERGENT ADDITIVE AND PROCEDURE FOR WASHING TEXTILES. |
| DK449987 | 1987-08-28 |
| Publication Number | Publication Date |
|---|---|
| DK449987D0 DK449987D0 (en) | 1987-08-28 |
| DK449987A DK449987A (en) | 1988-03-01 |
| DK157560Btrue DK157560B (en) | 1990-01-22 |
| DK157560C DK157560C (en) | 1990-06-11 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| DK449987ADK157560C (en) | 1986-08-29 | 1987-08-28 | ENZYMATIC DETERGENT ADDITIVE, DETERGENT CONTAINING THE ENZYMATIC DETERGENT ADDITIVE AND PROCEDURE FOR WASHING TEXTILES. |
| Country | Link |
|---|---|
| DK (1) | DK157560C (en) |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| USRE43341E1 (en) | 1995-06-07 | 2012-05-01 | Danisco A/S | Method of improving the properties of a flour dough, a flour dough improving composition and improved food products |
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| ATE220502T1 (en) | 1997-04-09 | 2002-08-15 | Danisco | IMPROVED METHOD FOR PRODUCING FLOUR PASTA AND PRODUCTS MADE FROM SUCH PASTA USING GLYCERINE OXIDASE |
| WO2000005396A1 (en) | 1998-07-21 | 2000-02-03 | Danisco A/S | Foodstuff |
| MXPA03010511A (en) | 2001-05-18 | 2004-03-02 | Danisco | Method of improving dough and bread quality. |
| US20050196766A1 (en) | 2003-12-24 | 2005-09-08 | Soe Jorn B. | Proteins |
| US7955814B2 (en) | 2003-01-17 | 2011-06-07 | Danisco A/S | Method |
| MXPA05007653A (en) | 2003-01-17 | 2005-09-30 | Danisco | Method. |
| US7718408B2 (en) | 2003-12-24 | 2010-05-18 | Danisco A/S | Method |
| US7906307B2 (en) | 2003-12-24 | 2011-03-15 | Danisco A/S | Variant lipid acyltransferases and methods of making |
| GB0405637D0 (en) | 2004-03-12 | 2004-04-21 | Danisco | Protein |
| EP1776455B1 (en) | 2004-07-16 | 2015-03-18 | DuPont Nutrition Biosciences ApS | Lipolytic enzyme, uses thereof in the food industry |
| EP2405007B1 (en) | 2007-01-25 | 2013-12-04 | DuPont Nutrition Biosciences ApS | Production of a lipid acyltransferase from transformed Bacillus licheniformis cells |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| USRE43341E1 (en) | 1995-06-07 | 2012-05-01 | Danisco A/S | Method of improving the properties of a flour dough, a flour dough improving composition and improved food products |
| Publication number | Publication date |
|---|---|
| DK157560C (en) | 1990-06-11 |
| DK449987A (en) | 1988-03-01 |
| DK449987D0 (en) | 1987-08-28 |
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| Date | Code | Title | Description |
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| PUP | Patent expired |