CUTINASE CLEANING COMPOSITION

Abstract: This invention relates to cleaning compositions and methods for using them. Particularly, the invention relates to compositions comprising a surfactant and a cutinase enzyme.

Description: Cutinase Cleanincr Cαπpositions Badgfround of the Invention a) Field of the Invention The invention relates to enzymatic cleaning ∞πpositions and methods for using them. Particularly the invention relates to cleaning compositions comprising a surfactant and a cutinase enzyme. b) Bac&ground Information A wide variety of enzymes are well known for use in cleaning compositions. The use! of B. subtilisins and B. licheniformis protease in cco_nmercial preparations is common. Other enzymes have also been used in commercial cleaning compositions such as, for example, U.S. Patent No. 4,011,169, and British ' Patnet No. 1,293,613. Also a cα_τp_ * ehensive review article of lipases in cleaning compositions can be found in Journal of Applied Biochemistr y . 2:218-229 (1980) in an article entitled "Lipases as Detergent Ccsπponents". Lipolytic detergent additives are also known frcm e.g., British Patent Specification No. 1,293,613 and Canadian Patent No. 835,343. U.S. Patent No. 3,950,277 and British Patent Specification No. 1,442,418 disclose lipase enzymes combined with an activator and calcium and or magnesium ions, respectively, which are utilized to pre-soak soiled fabrics and to remove triglyceride stains and soils from polyester or polyester/cotton fabric blends, respectively, Suitable micrcbial lipases for use therein (apart from animal and plant derived lipases) are said to be those derived from Pseudσmonas. As p ererillus. Pneu ococcus. Stat-hylococcus. and Staphylococcus toxins. Mvcobacterium tuberculosis, v∞torula lipolvtica. and Sclerotinia. SUBSTITUTESHEET British Patent Specification No. 1,372,034 discloses a detergent ccsπposition comprising a bacterial lipase produced by Pset-dσmonas stutzeri strain ATCC 19154. Furthermore, -" i " is " reeesnmeaded that the ρ__efβ_ςϊed lipolytie enzymes should have a pH cptimum between 6 and 10, and should be active in said range, preferably between 7 and 9. Around 1970, this presumed Pseudcmonas stutzeri strain was reclassified as Pseudcmonas aeruσinosa. as appears for example from the ATCC catalogues. European Patent Application EP-A-0130064 discloses an enzymatic detergent additive comprising a lipase isolated from Fusariυro — oxysporum with an alleged higher lipolytic cleaning efficiency than conventional lipases. In European Patent Application No. 0214761, Enzymatic detergent additives are described as the active component of which is a microbially produced lipase from a strain of Pseudcmonas cepacia. The lipases described therein are claimed to be superior to the lipolytic detergent action of the prior art, especially at lew temperature washing processes (around 60"C and below). I PCT Patent Application No. 87/00859 other novel lipolytic enzymes are described as having an optimal pH in the range of 8 - 10.5 at a ten-perature of 60*C or less frcm bacterial strains selected frcm ]=*seudσmonas pseudoalcaliqenes. P. stutzeri and Acinetobacter calcoaceticus. These enzymes are described as particularly effective at low teπperatures; i.e., 40*C or lower and effective in both liquid and solid detergent compositions. Also in U.S. Patent No. 3,950,277, it is described in general terms that lipases frcm Pseudcmonas are suited as agents for removal of oily stains frcm fabrics, if used together with a special group of lipase activators. The art cited does not, hcwever, cover cutinase enzymes frcm Pseudcmonas or any other micrcbial source. However, prior art enzymes for use in cleaning α_mpositions, while effective on many proteins and lipids, are not αampletely effective against all stains commonly found in laundry and other cleaning applications. SUBSTITUTESHEET Further, many lipases are not stable at pH 8 - 11 where most cleaning compositions are used. Even further, most enzymes for use in cleaning cαπpositions are not very stable, if at all, under oxidative conditions or in the presence of other enzymes such as proteases * SUMMARY OF THE INVENTION Accordingly it has been discovered that combinations of a surfactant and a substantially pure microbial cutinase enzyme are effective expositions for cleaning applications. The cutinase enzyme -- - - preparations possess activity at pH of from about 8 and 11, exhibit- cleaning activity in aqueous solution at concentrations frcm about .05 mg L to about 100 mg/L or more at teπperatures frcm about 20 β C to about 50 β . The enzymes are oxidatively stable and stable in the presence of other enzymes such as proteases. Even __urther, the cutinases show a synergistiσ effect when a plurality of surfactants are used with the cutinase. The invention also relates to the iraproved process for enzymatically cleaning a material with an aqueous solution; the iiiprOvemeπt comprising adding a substantially pure cutinase to the cleaning solution. Efli_TAIIED DESCRIPTION OF THE INVE TTON Applicant has discovered that cutinase enzymes are useful when included in cleaning compositions. These compositions may take on a variety of forms such as for laundry cleaning, household and industrial cleaning, and the like. The cleaning compositions σαπprise combinations of known .surfactants and a micrcbial cutinase enzyme which can be used to clean a wide variety of materials. The composition can be added to aqueous solution or solid powder, or formulated in an aqueous solution or solid powder and used according to conventional cleaning techniques. SUBSTITUTE SHEET Enzyme Cutinases are well known in the art and are available frcm a wide variety ©f souϊeeso S-fiβ Cuti_nas-is f__rsm Fιs_gi and Pollen, P.E. Kolattι_k___ly, pg. 472 ■ = 504, incorporated herein by reference, for discussion of cutinases useful in the practice of the invention. A preferred cutinase is that cutinase isolated in a substantially pure form frcm s * ^-^oιτγτrv.s putida, particularly the P. putida, ATCC 53552, described in ccpending U.S. patent application. Serial No. 932,959 filed November 19, 1986 and incorporated herein by reference, which enzyme therefrom has the following amino acid sequence: SUBSTITUTE SHEET 10 |l* »« • • ι». -t- IIP thr P'l fly ♦*# P'» P*t »r» JO lit Ml |t| tm pht itp • '1 ttr $ ' # P'l tyr thr 30 I ttr ttr ttr f 1« fty f> f * »_» Cyt Irf lit 40 tyr trf pr« «rf UP it* ι»» ι'« ϊ» fi »•' ••'i )0 10 kit f *4. tit 1«t» trp |ly im |'r thr fly ll )0 fly pr* ur thr tyr ill fly It* It* l«r kit trp •0 ill »r kit fly pht v«l *«1 ill l'ι Ill flu thr to ttr ISA ill fly thr fl lj tf% |1« ctt It* ill cyt 100 K itp tyr It* «|1 iff flu lit li; thr pr* tyr 110 120 ,1, .e l»r ur fly ϊy» u l.» Uf fly irf **l 130 fly thr tir fly hit . »*r f<A fly fl; fly fly ter 140 111 Mt 111 fly fl* IIP thr ι * | -»' rf thr thr HO 111 pr» tit fl* pr* X-r thr It. fl - ltd fly .felt 110 l-> ttr ill ttr fl* irf irf fU ft.* fly pr- ~t no 110 Hi Itw *tt ttr fly fly fly *ι$ thr Ut ill pht MO »r» tyr It* it* ill fl pr* *«« tyr irf irf ill ?03 ItA *ιl pr- vil pfct trp fly fll« « irf tyr til 210 tir kit put flu pr* «ιl fly ttr fly fly l-i | yf 270 ff fly pr* ttr thr ill trp pr.t f ht fl* 1 t« no }«0 att itp Itp fl« tp ll rf U thr p t tyr fly ISO ill |1A cyt ttr Itw cyt thr ttr ltw ttu trp Itr fil fly irf |rf fly Itt. SUBSTITUTE SHEET Other sources of bacterial and fungal cutinaises include: Fusarium solani pisi Fusarium roseum saπifcruciπum Fusarium. roseum r_πi * ττr.τ*t * --. Hel-Ci nthosporum sativum Ulocladium cσnsortiale Streptαmyces scabies Oolletotrichum capsici Irhytopthora cactorum Bσtrvtis cineria Colletofbrichum qloeosporioides - ~ - - - The cutinase of the invention should preferably be selected to cause at least about 10%, and preferably 20%, hydrolysis of the given fat under given conditions. Normally the amount would be in a concentration of frcm about .01% to about 5.0% by weight of the surfactant, and preferably frcm about .05% to about 3%, such that upon dilution in wash water it is in a concentration of at least about .05mg L. Further, one skilled in the art could take the preferred cutinase or, for that matter, airy cutinase of the invention or any i_ι_tπuniologically identical cutinase and use random or selective replacement of amino acids to produce other cutinases vfliich. are more or less selective toward given substrates or include modification in activity such, as oxidative stability. Substantially pure cutinase includes the isolated enzyme as well as the broth containing the enzyme in unpurified form but essentially free of other enzymes and enzyme sources. The natural substrate of cutinase is cutin which is a bicpolyester polymer which covers the plant leaves, fruits, etc. , see Structure, Biosynthesis and Biodegradation of Cutin and Suberin. (1981), P.E. Kolatt ikuSy, Ann. Rev. Plant Physiol., 32, pgs. 539-567. Stains comprising lipids which could be hydrolyzed or bound by cutinase on a substrate such as cloth would be similar to the natural substrate cutin. Cutinase, for these types of stains, will be more SUBSTITUTESHEET effective than the prior art lipases. The cutinases will work especially well on gravy, oils and greases, plant or grass, oil based makeup and collar stains. Cutinases are distinguishable from other lipases by methods well known in the art. See R.E. Purdy and P.E. KolatΛukudy, Biochemistry, "Cutinase Assay", 14:2831-2840, (1975). Microbial cutinases frcm both fungal and bacterial sources have very good activity at pH 8 - pH 11 which is an ideal pH condition for detergent use. Because of the specific activity of cutinases, it is a preferred aspect of the invention to combine one or more other cutinases, or one or more other enzymes, such as proteases, amylases or other lipases, along with the cutinase of the invention in the cleaning composition. Further, Applicant shows a synergistic increase in hydrolytic activity of cutinase when two or more surfactants are combined along with the cutinase enzyme. Cutinases then are ideal for cleaning cccposition inclusion. They have stability αxidatively such as in H2θ 2 . They have good stability in a temperature range of from about 20 - 50"C which is ideal frcm a cleaning point of view. They are also stable in the presence of other enzymes; e.g., proteases, and as such, are ideal for mixtures of enzymes. The Surfactant A number of known ccmpc_wr_ds are suitable surfactants useful in the present ccmpcsitiαns. These include noniαnic, anionic, cationic, or zwitteriαnic detergents, as disclosed in U.S. 4,404,128 to Barry J. Anderson and U.S. 4,261,868 to Jiri Hora et al. The art is familiar with the different formulations which can be used as cleaning ccampositions. SU BS TITUTE SHEET -T-hg. (-ip*.*τing Qaπpositions and Method of Use Cutinases can be formulated as a purposefully added ingredient into taown powdered and liquid detergents having pH between 6.1 and 12.0 at levels of about 0.01 to about 5% (preferably .05 to .5%) by weight of the detergent. These detergent cleaning compositions can also include other enzymes such as known proteases and a ylases, as well as bleaches, colorants, builders, and stabilizers. The cutinase of the invention may be added to powdered detergents in the form of granulates or prills, prepared by methods known in the art such as described in British Patent Nos. 1,324,116 and 1,362,365 and U.S. patent Nos. 3,519,570; 4,106,991 and 4,242,219. The cutinase preparations of the invention can be prepared by cultivating the microorganisms defined herein or otherwise cutinase ∞ntaining itύcroorganism under appropriate conditions. In order to obtain reasonable yields of enzyme, media containing readily assimilable carbon and energy scurces as necessary such as a nitrogen source, as well as calcium and magnesium salts and trace elements and ~ cutin, or monc_mers of cutin, or cαpounds resembling cutin or cutin monomers. One could also obtain the gene for cutinan and express in any organism of choice where one may not have to add cutin or cutin monomers into the fermentation. The addition of cutinase to conventional cleaning compositions does not create any special use limitation. In other words, any temperature and pH suitable for detergent ccπpositions cxsπtaining enzymes is also suitable for the present compositions. i_lt_hαugh the preferred form of the invention has been described above, it will be obvious to those skilled in the art to which the ' invention pertains, that, after understanding the invention and in view of the following testing as a whole, various changes and equivalent modifications may be made without parting from the scope of the invention as defined by the claims. SUBSTITUTE SHEET STABILITY CUTINASE AGAINST PROTEASES Reaction conditions: Buffer: 0.1 M NaP, pH 10 Temp: 37'C Lipase: 42 ug ml Approximately 1:1 protease:cutinase aqueous solution were made up with the following results. ENZYME ACTIVITY Protease Incubation Time 0 min 5 min * 10 min 15 min 14 hrs None 1.57 1.60 1.47 1.63 1.61 Maxacal (35ug ml) 1.68 1.58 1.72 1.66 0.134 Esperase 1.73 1.64 1.59 1.51 0.456 (64 ug^ml) Ifcxical is Gist-Brocade's brand of subtilisin enzyme (protease) Esperase is Nσvo's brand of protease enzyme (protease) SUBSTITUTE SHEET TEMPERATURE STABHHY OF BACTERIAL (OTINASE _3AI_F IJFE AT 50 8 C pH Hrs. 7 30 8 25 9 12 10 0.3 Enzyme was incubated at 50 β C in O.IM sodium phosphate buffer at various pH's and activity was measured by hydrolysis of trioctanoin in polyvinyl alcohol emulsions. SUBSTITUTESHEET EFFECT OF DETERGENTS ON HYEROLASE AC IVITY 1 Reaction Conditions: substrate: p-nitro-phenyl butyrate, lma (pnp) pH: 8.0 buffer: 0.1m tris pH 8.0 temperature: 25'C enzyme: bacterial cutinase frcm ATCC 53552 An aqueous solution with the following were made up and the enzyme activity was measured in these soluticns using pnp as a substrate by following absorbance of p-nitrcphenol at 410 mm. Triton xl00% SDS % % Activity 0 0 100 0.2 — 78 0.4 — 60 — 0.05 30 — 0.1 23 — 0.2 14 — 0.4 6 0.4 0.4 78 0.2 0.2 98 0.2 0.05 125 0.2 0.1 138 SUB S TITUTESHEET 6.1 0.1 ~ 130 0.05 0.05 132 1) Non-ionic detergent inhibition is not significant at low concentrations. 2) An ionic detergent inhibitor at high concentrations. 3) Mixture of anionic and non-ionic detergents stimulate activit .- SUBSTITUTESHEET STABILITY TCWARD OXTDANIS Cutinase .5 mg/ml in O.lm sodium phosphate tuffer, was incubated with various levels of hydrogen peroxide at pH 8.4 and 25'C for 2 hours, and hydrolytic activity was measured by a pH-stat using tricx ano:i_mrpolyviπyl alcohol emulsion. [H 2 0 2 ppm] Hydrolytic Activity % Remaining 0 100 100 86 200 86 500 91 1000 • 95 SUBSTITUTE SHEET Act. (mmol/mg.mln) SUBSTITUTE SHEET Act. (mmol/mg.mln) SUBSTITUTE SHEET

Claims: What is Claimed is: 1. An enzymatic cleaning cαπposition ccπprising a substantially pure microbial cutinase in an ameuπt sufficient that ι_q?on dilution in an aqueous solution for use there is sufficient cutinase to achieve a concentration of frcm about .05 mg/L to about 100 mg-/L of cutinase. 2. A composition according to Claim 1 wherein the cutinase is the cutinase present-in an amount of from about 0.01% to about 5% by weight of the surfactant. 3. A composition according to Claim 2 wherein the Pseudomonas putida is ATOC 53552. 4. A composition according to Claim 1 which further ccnprises one or more enzymes selected from lipases, aπylases or proteases. 5. A composition according to Claim 1 wherein the cutinase selected is capable of causing at least 10% hydrolysis of a cutin or cutin-like substrate desirous of being cleaned frcm a given surface. 6. A composition according to Claim 1 wherein the cutinase is a non-naturally occurring cutinase having at least one amino acid randcmly or selectively replaced by an amino acid not naturally found at that position. SUBSTITUTE SHEET 7. A σcπposition according to Claim 1 wherein the cutinase is added in the form of cell fermentation broth containing cutinase tut essentially free of other enzymes or enzyme sources. 8. An enzymatic cleaning additive wherein the active component which is a substantially pure micrcbially produced cutinase. 9. A αarπposition according to Claim 8 v-hich further ccnprises one or more enzymes selected from lipases, amylases or proteases. 10. A ccπposition according to Claim 8 cccprising a plurality of surfactants. 11. A composition according to Claim 10 wherein the surfactants are SDS and Triton X-100. * - 12. An iDproved method for enzymatically cleaning a material having a cutin or cutin-l e stein ccuprising: a) selecting a cutinase enzyme; b) forming an.aqueous solution with said enzyme in a concentration of frcm about 0.05 mg/L to about 100 mg/L; c) contacting the material with the solution of step b); and d) rinsing the material of step c) . 13. A method according to Claim 12 wherein the aqueous solution further comprises a surfactant compatible with the selected cutinase. SUBSTITUTE SHEET 14. A method according to Claim 13 wherein the aqueous solution further comprises a surfactant c πpatible with the selected cutinase. SUBSTITUTE SHEET

Inventors: KOLATTUKUDY PAPPACHAN, POULOSE AYROOKARAN J, KOLATTUKUDY, PAPPACHAN, POULOSE, AYROOKARAN, J.
Applicants: GENENCOR INC, GENENCOR, INC.
Application year: 1988
Main classification: C11D3
IPC codes: C11D3, C12N9