CN1088256A - Enzymatic detergent compositions - Google Patents

Abstract

The invention provides a kind of enzymatic detergent compositions, it comprises the surfactant system of (a) 0.1-50% (weight), and this system comprises one or more anion surfactants of (a1) 0-95% (weight) and (a2) one or more nonionogenic tensides of 5-100% (weight); (b) 10-20000LU enzyme/gram detergent composition, this enzyme can show tangible lipolytic activity in the major cycle of washing process.

Description

Enzymatic detergent compositions

The present invention relates generally to enzyme-containing detergent and sanitising agent field.More particularly, the present invention relates to contain enzymatic detergent compositions with lipolytic activity enzyme.

Known various types of enzyme is as the additive of detergent composition.For example, describe the detergent composition that contains proteolytic enzyme, cellulase, amylase, lipase and various mixtures thereof in the literature, and occurred several such products on the market.The present invention relates to contain the detergent composition of lipolytic enzyme or lipase.Such enzyme can help to remove fatty dirt by the one or more ester bonds in the hydrolysis triglyceride from fabric.

EP-A-214761(Novo Nordisk) lipase that obtains from Pseudomonas cepacia class organism, EP-A-258068(Novo Nordisk are disclosed) disclose that title is Humicola before the Thermomyces() belong to the lipase that organism obtains.The application of these lipase as detergent additives also described in these two pieces of patent applications.

EP-A-205208 and EP-A-206390(are Unilever for two pieces) example of the other detergent composition that contains lipase is provided, they disclose the lipase of a class according to their immune contextual definition, and have described in detergent composition and textile washing their application.The preferred lipase lipase that to be those obtain from Pseudomonas fluorescens, Pseudomonas gladioli and Chromobacter species.

EP-A-331376(Amano) describe some lipase, their application and the production method of their use recombinant DNA (rDNA) technology, and comprised a kind of aminoacid sequence of the lipase that obtains from Pseudomonas cepacia.At WO-A-89/09263 and EP-A-218272(all is Gist-Brocades for two pieces) in provided the other example of the lipase of producing with recombinant DNA technology.

Although disclose the improvement of a large amount of lipase and they,, found so far only to obtain from Humicola lanuginosa and be widely used as the additive of fabric washing product at the lipase that Aspergillus Oryzae produces as the host.It can be with trade mark Lipolase(TM) bought from Novo-Nordisk.

At the chemical industrie magazine, in the article of 1990, the 183-186 page or leaf Henrik Malmos, Henrik Malmos points out that people know that the activity of lipase in washing process generally is low, and is Lipolase(TM) also unexceptional.In drying process, when the water-content of fabric reduced, this enzyme had recovered its activity, fat spot hydrolysis alive.In wash(ing)cycle thereafter, remove the material of this hydrolysis.What action of lipase after first wash(ing)cycle this also is interpreted as is low, but is tangible in the cycle of back.(J.Chem Tech.Biotechnol.50 has also described these discoveries in 321-330) to people such as Aaslyng (1991) at " Mechanistic studies of proteases and Lipases for the Detergent Industry ".

The present inventor is its shortcoming as the existing Betengent product that contains lipase, and promptly when with these fabrics that product washing does not have before this with this Betengent product contacts, can estimate does not have obvious superiority with lipolytic enzyme.

In addition, in the Europe and the U.S., in the middle of the user of automatic washing machine, use the dry fabric that washs of roller dryer that the trend of growth is arranged.In these machines, wet clothes is promptly dry by contacting with warm air.Use roller dryer to reduce to the drying process that at room temperature needs dry 6-48 hour usually and be less than 1 hour.As mentioned above, in drying process, when the water-content of fabric reduced, lipase recovered its activity.When using roller dryer, shorten greatly for cycle of lipase optimum water concentration.Therefore, when using in the washing process that comprises drying step in roller dryer when containing the Betengent product of common lipase, the benefit that the human consumer obtains from such product seldom.

Therefore, an object of the present invention is to provide a kind of enzymatic detergent compositions, demonstrate lipolytic activity significantly in the major cycle of said composition washing process in automatic washing machine, therefore, it will demonstrate lipolytic activity when it is used to wash before this not with this Betengent product contacts fabric.It also is one object of the present invention that the enzymatic detergent compositions that is specially adapted to be used in combination with roller dryer is provided.

Another object of the present invention provides in major cycle of the washing process of a kind of production in automatic washing machine and shows the method for such enzyme of lipolytic activity significantly.

There is the lipolytic enzyme that shows the detergent composition of tangible lipolytic activity in the major cycle that can be used for being formulated in washing process in our surprised discovery now.In addition, we have found in the major cycle of washing process to show the ability of such lipolytic activity and between their the inactivation performance a kind of good relation have been arranged during with fluoro di(2-ethylhexyl)phosphate isopropyl esters (DFP).So their inactivation performances were selected when suitable lipolytic enzyme usually can be according to DFP.The at of particularly finding the eucaryon source is the suitable enzyme that shows a kind of like this lipolysis in the major cycle of washing process.

WO-A-88/09367(Genencoy) propose tensio-active agent mixed with the at of pure basically microorganism and prepare the effective cleaning agent.The detergent composition that contains the at that obtains from (prokaryotic organism) Pseudomonas putida ATCC 53552 is disclosed.But, in more recent European patent application EP-A-476915(Clorox), same enzyme (being called lipase at that time) is disclosed, when using with usual method, this enzyme is more effective unlike other lipase removing from fabric aspect the grease.In other words, do not think that such enzyme shows washing effect.

WO-A-90/09446(Plant Genetics Systems) described from Fusarium solani pisi and intestinal bacteria (E.Coli), cloned and produced the eucaryon at, and mention this at especially and can be used to produce sanitising agent, as the preparation of cloth-washing detergent and other special lipolyses, as cosmetic composition and shampoo.Do not have to disclose or propose special enzymatic detergent compositions, therefore the people who is skilled in technique can not be subjected to inspiring the detergent composition of selecting this specific enzymes to be used for fabric washing with preparation.

According to a first aspect of the invention, provide an enzymatic detergent compositions, comprising:

(a) 0.1-50%(weight) reactive systems, this system contain (a1) 0-95%(weight) one or more anion surfactants and (a2) 5-100%(weight) one or more nonionogenic tensides; (b) every gram detergent composition 10-20000LU enzyme, this endonuclease capable demonstrates tangible lipolytic activity in the major cycle of washing process.

According to a second aspect of the invention, provide a kind of according to differentiating and be chosen in the method that in the major cycle of washing process, shows the enzyme of tangible lipolytic activity in the automatic washing machine with the performance of fluoro di(2-ethylhexyl)phosphate isopropyl esters (DFP) enzyme deactivation.

According to another aspect of the present invention, provide a kind of method of producing such enzyme.

(a) surfactant system

One aspect of the present invention provides a kind of enzymatic detergent compositions, contain 0.1-50%(weight) reactive systems, it contains 0-95%(weight successively) one or more anion surfactants and 5-100%(weight) one or more nonionogenic tensides.This surfactant system can contain both sexes or zwitierionic detergent compound in addition, still, because their expense is than higher, so this is unwanted usually.

Generally, the nonionic of this surfactant system and anion surfactant can be selected from the tensio-active agent of describing in the following document: Schwartz and Perry, " Surface Active Agent " Vol.1, Schwartz, Perry and Berch, Interscience, vol.2; Interscience, 1958; " McCutcheon ' s Emulsifiers and Detergents " of the recent release of publishing by Manufacturing Confectioners Company, or H.Stache, Carl Hauser Verlag, 1981, " the Tenside Tanschenbuch " of second edition.

Operable suitable non-ionic compound particularly comprises the compound that has hydrophobic group and the reaction product of active hydrogen atom, for example Fatty Alcohol(C12-C14 and C12-C18), acid, acid amides or alkylphenol and alkylene oxide, particularly independent oxyethane or with the reaction product of propylene oxide.Specific nonionic detergent compounds is C ₆-C ₂₂Alkylphenol-ethylene oxide condensate generally is 5-25EO, i.e. each molecule 5-25 ethylene oxide unit, and aliphatic C ₈-C ₁₈The condensation product of uncle or secondary straight or branched alcohol and oxyethane, general 5-40EO.

Operable suitable anionic detergent compound normally has an alkali metal salt of the water soluble of organic sulfuric acid of the alkyl that contains about 8-22 carbon atom and sulfonic acid, and used term alkyl comprises the moieties of senior acyl group.The example of suitable synthetic anionic washing compound is sodium alkyl sulfate and alkylsurfuric acid potassium, particularly the senior C that is produced by Tallow, beef or Oleum Cocois by sulfation ₈-C ₁₈Those that alcohol obtains, C ₉-C ₂₀Sodium alkyl benzene sulfonate and potassium, particularly linear secondary C ₁₀-C ₁₅The sodium sulfate of those ethers of sodium alkyl benzene sulfonate and alkyl glycerol ether sodium sulfate, the particularly higher alcohols that obtains from Tallow, beef or Oleum Cocois and the synthol that obtains by oil.The preferred anionic surfactants washing compound is C ₁-C ₁₅Sodium alkyl benzene sulfonate and C ₁₂-C ₁₈Sodium alkyl sulfate.

Also can be tensio-active agent, as at EP-A-328177(Unilever) in those tensio-active agents of describing, their abilities are saltoutd, alkylpolyglycosides tensio-active agent of describing in EP-A-070074 and alkyl list glycosides.

The preferred surfactants system is the mixture of negatively charged ion and nonionic laundry active, particularly at EP-A-346995(Unilever) in the negatively charged ion pointed out and the kind and the example of nonionogenic tenside.Particularly preferred surfactant system is C ₁₆-C ₁₈Primary alconol sulfuric acid an alkali metal salt and C ₁₂-C ₁₅The mixture of primary alconol 3-7EO ethoxylate.

The amount of nonionic detergent is preferably greater than surfactant system 10%, for example 25-90%(weight).The amount of anion surfactant for example can be about the 5%-40%(weight of surfactant system).

(b). enzyme

Enzymatic detergent compositions of the present invention also contains every gram detergent composition 10-20000LU, preferred 50-2000LU enzyme, and this enzyme can show significant lipolytic activity in the major cycle of washing process.In this manual, LU or lipase unit are by EP-A-258068(Novo Nordisk) unit definition.

Because the effect of significant lipolytic activity or washing in the major cycle in washing process, just mean that the detergent composition that contains enzyme can be in the automatic washing machine of Europe class, in single washing process, with regard to concentration, the water hardness, temperature, use common wash conditions, can from the fabric that pollutes, remove a large amount of greases.Should remember, under same condition, the lipolytic enzyme Lipolase(TM that can buy from Novo Nordisk on the common market) as if without any effective washing effect to grease.

Enzyme can be evaluated with following analytical procedure the washing effect of grease.Betengent product (as given below) pre-wash with no enzyme contains cotton 33% new polyester/cotton test fabric, then rinsing up hill and dale.Then so untainted fabric is besmirched with sweet oil or other suitable hydrolyzable greases.Being incubated in the 30ml washing lotion of each test fabric (heavily about 1g) in 100ml polystyrene bottle.Washing lotion contains the Betengent product of every liter 1 gram dosage given below.These bottles are placed in the Miele TMT washing machine of adorning water also with normal 30 ℃ of main washing procedures stirrings 30 minutes.The enzyme that lipolytic activity is arranged is added in the washing lotion in advance with 3LU/ml.Control group does not contain any enzyme.This washes powder by following the composition (weight %):

The pure nonionogenic tenside 9.5 of ethoxylation

Sodium sulfate 38.6

Yellow soda ash 40.4

Water glass (Na ₂O: Si ₂O=2.4) 7.3

Water 4.2

As nonionogenic tenside, we use the C of 10.5-13EO ethoxylation ₁₂-C ₁₅Alcohol, but find that the character of the pure nonionogenic tenside of ethoxylation can change in wide range.

After the washing,, and in roller dryer, use the freezing air drying with these fabrics of the thorough rinsing of cold water, and the amount of the fat of assessment remnants.This can carry out with several method, and usual method is to test fabric with petroleum ether extraction in the Soxhlet extraction equipment, steams solvent, and measures the percentage ratio (weight) of remaining fatty substance as initial fat quantity on the fabric.

According to second more responsive method, with the bromination sweet oil make dirty the test fabric (Richards, S., Morris, M.A. and Arklay, T.H.1968), Textile Research Journal 38,105-107).Then, each test fabric is placed in the 30ml washing lotion in the 100ml polystyrene bottle is incubated.Then these bottles are placed in the washing machine of adorning water and with normal 30 ℃ of main washing procedures and stir.After main the washing, carefully the test fabric with 5 seconds of cold rinse.After the rinsing, in drying machine, use freezing air drying test fabric immediately.After the drying, can determine the amount of residual fat with the bromine content of x-ray fluorescence spectrometry method mensuration fabric.Can determine the amount of removing of fat, the percentage ratio that it is present in the amount on the test fabric to start with is calculated as follows:

The dirt %=that removes (bromine bw-bromine aw)/(bromine bw) * 100%

Wherein, the percentage ratio of the bromine before bromine bw represents to wash on the fabric, the percentage ratio of bromine after bromine aw represents to wash.

The active another kind of method of assessment enzyme is to measure reflectivity according to the method for standard at 460nm.

According to the present invention, this detergent composition comprises a kind of enzyme, and said composition is compared with the same detergent composition that does not have enzyme, can remove at least more than 5%, preferred at least more than 10% greasy dirt (by initial greasy dirt metering), it is with the determination of test method of introducing in this specification sheets.

The another kind of method of determining enzymic activity is itself and commercially available Lipolase(TM), promptly the activity of the lipase that can obtain from Novo/Nordisk compares.According to the present invention, the test method of introducing with this place, the enzyme grease of removing by enzyme with by Lipolase(TM) ratio at least 3 of the enzyme grease removed, preferably at least 5.The meaning of the enzyme grease of removing is the dirt that can remove owing to the existence of enzyme, promptly deducts the observed dirt of removing when not having enzyme as background.

Since this application has can be prepared the detergent composition with obvious washing effect through disclosing, how the people who is skilled in technique can very clearly select to be used for suitable enzyme of the present invention.But, we have found that a kind of method very easily selects the suitable lipolytic enzyme of the present composition, this method be the lipolytic enzyme found according to us washing effect closely with fluoro di(2-ethylhexyl)phosphate isopropyl esters (DFP) time their inactivation performance relevant.Know all that generally the reactive site serine residue in this compound and the lipolytic enzyme reacts rapidly, so it just loses their enzymic activity.We find by the lipolytic enzyme that promptly has operable reactive site serine residue of the rapid inactivation of DFP good washing effect is arranged generally.On the other hand, promptly to be had a lipolytic enzyme of serine residue of the reactive site that cannot use by the DFP inactivation general not or seldom washing effect arranged more slowly.We find that at room temperature pH after 10 minutes, demonstrated residual activity less than 50% with the DFP insulation at 10 o'clock, preferably have good washing effect less than 40% lipolytic enzyme.Find with having residual activity less than the washing effect of 25% lipolytic enzyme even better, best with having residual activity less than the washing effect of 15% lipolytic enzyme.The details of DFP inactivation test can find in an embodiment.

The enzyme that composition of the present invention is suitable can find (EC3.1.1. in esterase and lipase ^*, wherein * represents any number).

According to the present invention, show that the enzyme of the more preferably type of washing effect is the eucaryon at.At is the group (EC3.1.1.50) of enzyme, wax-ester hydrolase.These endonuclease capables reduce cutin, i.e. the longer chain fatty acid of esterification and in plant pure the reticulation as the Fatty Alcohol(C12-C14 and C12-C18) of the supercoat of leaf and stem.In addition, they have some lipolytic activities, and promptly they can the hydrolysis triglyceride.Therefore, the lipase that can regard them as particular variety.

The feature of lipase is that they present the interface activation effect.This just means in the enzymic activity of activity ratio on consoluet substrate that forms on interface or the micellar substrate much higher.When concentration of substrate was increased to the micelle-forming concentration (CMC) that is higher than substrate and forms, the interface activation effect just made that lipolytic activity increases suddenly.By experiment, can observe this phenomenon, it is the discontinuous graph of relation of enzymic activity and concentration of substrate.But opposite with lipase, at does not show any tangible interface activation effect.

Because promptly there is not the interface activation effect in this feature, concerning present patent application, we stipulate at as lipolytic enzyme, and it is display interface activation not basically.Therefore, at is different from classic lipase, and they do not have the spiral lid at topped catalyzed combination position.

Owing to their fat acid decomposition performance, the component of at effect enzymatic detergent compositions is proposed generally.For example, WO-A-88/09367(Genencor) propose tensio-active agent and pure basically microorganism at are hybridly prepared into the effective cleaning agent.Disclosed is the detergent composition that contains a kind of (protokaryon is given birth to) at that obtains from Gram negative bacteria Pseudomonas Putida ATCC 53552.But, as mentioned above, in early European patent application EP-A-476915(Clorox), disclose same enzyme (being called lipase afterwards), when using, removed from fabric aspect the grease with ordinary method, compare with other lipase, it is better effect not.

From Fusarium solani pisi clone the at gene and mensuration order (Biochemistry 26,7883-7892) for Ettinger etc., (1987).WO-A-90/09446(Plant Genetics Systems) clone and produce this gene has been described in intestinal bacteria (E.Coli.).Still, there is not and exists the interface in the hydrolysis of catalysis ester and synthetic effectively of this between at and substrate in moisture and non-aqueous media.According to its general stability, propose this at and can be used to produce sanitising agent, as the preparation of cloth-washing detergent and other special lipolyses, as skin moisten composition and shampoo.The method of producing this kind of enzyme in economically viable mode is not open, and the two is not the special enzymatic detergent compositions that contains this at.

Can be from many sources such as plant (for example pollen), bacterium and fungi obtain at.Be used at of the present invention and be selected from eucaryon cutin enzyme.Such eucaryon at can be from various sources such as plant (for example pollen) or fungi obtain.

(eucaryon) fungi cutin enzyme is as comprising having two families that different qualities is leaf characteristic and stem characteristic.At with leaf characteristic often has acidity or neutral pH optimum value, and the at stem characteristic often has the alkaline pH optimum value.At with alkaline pH optimum value more is applicable to the detergent composition of alkaline assistant, washes powder and washing lotion as heavily using as a servant fabric.There is acid to be more suitable for, but also is suitable for industrial cleaning product in light labour product or rinse conditioner to the neutral pH optimum value.

In the table I below, list four kinds of different stem characteristic at, listed file names with their pH optimum value.

The table I

Example pH optimum value with at of stem characteristic

Fusarium solani pisi 9

Fusarium roseum culmorum 10

Rhizoctonia solani 8.5

Alternaria brassicicola（PNBase Ⅰ） 9

Particularly preferably being in the present invention can be from wild Fusarium Solani Pisi(Ettinger etc., 1987) at that obtains.When being used for suitable detergent composition, this at shows tangible washing effect.

The present invention preferably has the at of the aminoacid sequence of height homology than what obtain from Fusarium solani pisi to it in addition.Example is from Colletotrichum capsici, the at that Colletotrichum gloeosporiodes and Magnaporthe grisea obtain.

Be unsuitable for the Humicola of being lanuginosa lipase of the present invention, it is at EP-A-305216(Novo Nordisk) in be described, it is with Lipolase(TM) can sell for from market.But, can contemplate that this kind of enzyme can be used the modification of rDNA technology, like this, it also demonstrates tangible lipolytic activity in the major cycle of washing process.Such modification meeting influences the structure of lipase.Therefore, need experimentize, with the balance between the advantage of the inevitable damage of the structure that finds this enzyme and active aspect.General preferred modification is that electric charge was not too many around it did not influence reactive site.

Lipolytic enzyme of the present invention can be with any suitable form, promptly with the form of the slurries of the form of particulate composition, enzyme or with carrier substance bonded form (for example in EP-A-258068 and the Savinase(TM of Novo Nordisk) and lipolase(TM) product) join in the detergent composition effectively.A kind of good mode that enzyme is added in the detergent product is to contain 0.5-50%(weight in the pure nonionogenic tenside of ethoxylation) the form of slurries of enzyme, for example at EP-A-450702(Vnilever) described.

The enzyme that is used for detergent composition of the present invention can become a kind of suitable generation organism by the gene of clone's enzyme, as Bacilli or Pseudomonaceae, yeast such as Saccharomyces, Kluyveromyces, Hansenula or Pichia or fungi such as Aspergillus produce.

The at of the natural generation of generation microorganism is phytopathogen normally, these microorganisms are not the host cells that is suitable as very much the at gene, therefore, (former) at encoding gene just is incorporated on the rDNA carrier, can be transferred in the preferred host microorganism of rDNA technology.For this reason, can be with the such rDNA carrier of rDNA carrier that is similar to substantially described in the WO-A-90/09446.

In order to improve the productive rate of fermenting process, the gene of coding at should be transferred in the microorganism that grows fast in cheap substratum, and can synthesize and secrete a large amount of enzymes.According to the present invention, the rDNA(host microorganism of suitable improvement like this) be bacterium, comprising Bacilli, Corynebacteria, Staphylococci, and Streptomyces, or rudimentary eukaryote such as Saccharomyces cerevisiae and relevant species, Kluyveromyces marxianus and relevant species, Hansenula polymorphagn and the relevant species and the species of Aspergillus genus.Preferred host microorganism is rudimentary eukaryote, because these microorganisms can well produce the justacrine enzyme during the fermentation, and can glycolysis-(glycolysate) at molecule.Glycosylation may belong to the stability of the enzyme of matter again in the detergent system.

The present invention also provides genetic material, genetic material is for example obtained to clone's rDNA carrier by the gene that Fusarium solani pisi obtains from introducing eucaryon at gene, uses the gene that they change new host cell and express the in the new host cell.

The present invention also provides the polynucleotide that made or improved by the rDNA technology, and the such at of its coding contains the rDNA carrier of such polynucleotide and contains such polynucleotide and/or microorganism that the rDNA of such rDNA carrier improves.The present invention also provides the polynucleotide of respective coding eucaryon at, the polynucleotide of base sequence that for example have the at of encoding mature, a then pause codon and exactly at random have encode in the upstream of the nucleotide sequence of encoding mature the at preceding former sequence of this at or the nucleotide sequence of former sequence of the codon of last transfer in the polynucleotide.

In such polynucleotide, the at coding nucleotide sequence that obtains from initial organism can be improved in such a way, promptly make at least one codon codon preferably as much as possible, make to change quietly and form coding equivalent amino-acid residue and by the new preferred codon of host, the messenger RNA(mRNA) of the induced gene that improves stability is provided when therefore, using in this host cell.

The upstream of the nucleotide sequence of former at or the ripe at of encoding to determine the to encode host's that is suitable for selecting the signal or the nucleotide sequence of secretion order.Therefore, one embodiment of the invention relate to a kind of rDNA carrier, have inserted a coding at or its precursor nucleotide sequence in this rDNA carrier.

This nucleotide sequence for example can obtain from following several sequences:

(a) naturally occurring nucleotide sequence (for example, coding by Fusarium solani pisp produce former before the initial aminoacid sequence of at or former at);

(b) chemosynthesis by the molecular nucleotide sequence of password, these codons are preferentially selected by new host, and are forming nucleotide sequence in stable messenger RNA(mRNA), this initial aminoacid sequence of still encoding in new host;

(c) coding of being mentioned by superincumbent a or b section has the genetic engineering nucleotide sequence different aminoacids sequence and that a kind of nucleotide sequence that excellent stability and/or active Fusarium solani pisp at are arranged obtains in detergent system.

Put it briefly, the rDNA carrier can directly be expressed the nucleotide sequence of aforesaid coding at gene in a kind of host who preferably preferably comprises following component:

(a) at of encoding mature or preceding at or corresponding before two strands (ds) DNA of at, wherein exactly be removed to the small part presequence in the downstream of secretion signal (host cell of selection institute is preferably).The gene that should be transferred in part is not that the ATG codon should be placed on the place ahead under the situation about beginning from codon ATG.The portion gene of this transfer always should finish with the suitable codon that stops;

(b) be positioned at the expression regulators (the host's organism that is suitable for selecting) of upstream of the ds DNA normal chain of this at of coding (component (a));

(c) be positioned at coding at (the termination codon subsequence (the host's organism that is suitable for selecting) in the ds DNA normal chain downstream of component (a);

(d1) impel ds DNA to integrate or the host's that selects genomic nucleotide sequence, or

(d2) be suitable for the host's that selects original copy product;

(e1) at random a kind of (auxotroph) selectable marker.This nutrient defect type mark thing can be formed with the coding region of nutrient defect type mark thing and the promotor of defective;

(e2) the ds dna sequence dna of the coded protein in the at of the maturation among at random a kind of host who is included in selection and/or a kind of parent form of excretory.

A kind of like this rDNA carrier also can move to upstream and/or the downstream as former defined polynucleotide, and other sequence is convenient to the functional expression of at.This nutrient defect type mark thing can be made up of the coding region and the defective promoter region of nutrient defect type mark thing.

The present invention also provides a kind of production can show the method for the lipolytic enzyme of cleaning function, it comprises fermentation culture artificial modification's some steps that had the microorganism of the gene that the rDNA technology of the lipolytic enzyme of detergent active makes by coding that contain, by enzyme that separates the microorganisms producing that obtains by fermentation culture or the enzyme that passes through separate microorganism cells produce from fermentation culture, the division isolated cells, with physics or chemistry concentrate or method of purification is partly purified, the enzyme that is obtained by described nutrient solution or described cell.Such fermentation can be common intermittent type fermentation, charging-intermittent type fermentation or continuously ferment.The selection of the method for using depends on that the host plants system, preferred dirty working method (currently known methods).

The optimum condition of selecting is such, promptly becomes fermentation culture by this enzyme of microorganism secretion, removes cell by filtration or centrifugation and reclaim this enzyme afterwards from nutrient solution.Then, at random can concentrate and purify this enzyme to needed degree.

Except the microorganism of specified property, fermenting process can be according to the fermentation and the dirty processing units of known fermentation process and common usefulness.

On the other hand, the invention provides containing of artificial modification has the enzyme gene of defined lipolytic activity herein and can produce microorganism by the enzyme of described genes encoding.

The present invention also provides the recombinant DNA carrier of the nucleotide sequence that has the lipolytic enzyme of introducing in this place of coding with detergent active.

(c) other components

Enzymatic detergent compositions of the present invention can contain the preferred 20-50%(weight of 5-60% in addition) detergent builder compound.This washing auxiliary detergent can be anyly can reduce in the washings free calcium ion content and preferably provide said composition to have other useful performances as producing alkaline pH, removing the material of suspended substance of the carclazyte material of the suspended substance of dirt and softening fabrics from fabric.

The example of detergent builder compound comprises the precipitation washing assistant, as alkaline carbonate, supercarbonate, orthophosphoric acid salt; The chelating washing assistant is as alkali metal tripolyphosphates or nitrilotriacetic acid(NTA) salt; Or the ion-exchange washing assistant, as amorphous alkali metal aluminosilicate or zeolite.

Be reduced to less than 1mM if detergent composition of the present invention contains the concentration that builder material makes free ca, find that the lipolytic activity to detergent composition of the present invention is particularly advantageous.

In other embodiments, enzymatic detergent compositions of the present invention can comprise that also enzyme and other are generally used for the mixture of other components in the detergent system, and these components comprise the additive of detergent composition.These other components can be any of a lot of known materials of being introduced in for example following document, these documents are GB-A-1372034(Unilever), US-A-3950277, US-A-4011169, EP-A-179533(Procter and Gamble), EP-A-205208 and EP-A-206390(Unilever), JP-A-63-078000(1988) and the Research Disclosure 29056 in June, 1988, and each of several specification sheetss described here.The preparation of detergent composition of the present invention also can be by with reference to EP-A-407225(Unilever) routine D ₁-D ₁₄Be illustrated.

Also exist such detergent composition of proteolytic ferment or proteolytic enzyme can obtain special advantage therein.EP-A-271154(Unilever) describe many suitable PI that have and be lower than 10 proteolytic enzyme.The proteolytic enzyme of reinstating with lipase one can comprise in the document the disclosed for example BPN type or a lot of subtilisins of its alloytype in some cases, proposed that wherein some can be used for washing composition, disclosed mutant proteases in following document for example, these documents are EP-A-130756 or EP-A-251446(Genentech), US-A-4760025(Genencor), EP-A-214435(Henkel), WO-A-87/04661(Amgen), WO-A-87/05050(Genex), Thomas etc., (1986), Nature 5,316 and 5,375-376 and J.Mol.Biol.(1987) 193,803-813.Russel Deng (1987), Nature 328,496-500 and other.

The present invention will further be illustrated with the following examples now.Accompanying drawing has:

Figure 1A

The nucleotide sequence of the box 1 of synthetic Fusarium Solani pisi at gene and specified oligonucleotide.In this box sequence, show the oligonucleotide conversion.The box information of bottom refers to open translating and reads the outside Nucleotide position of sign indicating number (reading frame).

Figure 1B

The nucleotide sequence of the box 2 of synthetic Fusarium.solani pisi at gene and specified oligonucleotide.In this box sequence, show the oligonucleotide conversion.

Fig. 1 C

The nucleotide sequence of the box 3 of synthetic Fusarium solani pisi at gene and specified oligonucleotide shows the oligonucleotide conversion in this box sequence.The box information of bottom refers to open translating and reads the outside Nucleotide position of sign indicating number.

Fig. 1 D

The nucleotide sequence of the synthetic at gene of former at before the coding Fusarium solani pisi.This at presequence, former sequence and mature sequence have been shown.Represented that also the position and the oligonucleotide that are used to clone shift.The box information of bottom refers to open translating and reads the outside Nucleotide position of sign indicating number.

Fig. 2

Connect the former at encoding sequence of Fusarium solani pisi and the synthetic nucleotide sequence of the dna fragmentation of the sequence of the derivative of intestinal bacteria (E.Coli) the PhoA presequence of encoding.Show the ribosomal combining site (RBS) and the restriction enzyme position that are used to clone.Also show the PhoA signal sequence of coding and the aminoacid sequence of part at gene with an information password.

Fig. 3

The nucleotide sequence of box 8, SacI-BclI fragment, the juncture of the encoding sequence of its coding saccharase presequence and ripe Fusarium solani pisi at.

Fig. 4

By lacking the 0.2kb Sal plasmid PUR 2741 that I-the Nru I obtains from PUR2740 are a kind of E.Coli-S.cerevisiae shuttle vectorss, the original copy product in yeast cell that it comprises part PBR322, obtained by 2 μ m plasmids, yeast leu2D gene and under control yeast ga17 promotor have the yeast invertase signal sequence of the joint at plant d-galactosidase gene coding position.

Fig. 5

Plasmid PUR7219 is the E.Coli-S.Cerevisiae shuttle vectors, the yeast invertase signal sequence of the joint at the original copy product in yeast cell, yeast Leu2D gene that it comprises part PBR322, obtained by 2 μ m plasmids and the Fusarium Solani Pisi coding position that has encoding mature under control yeast ga17 promotor.

Fig. 6

Plasmid PUR2740 is the E.Coli-S.cerevisiae shuttle vectors, and it comprises part PBR322, the original copy product yeast cell, the yeast Leu2D gene that obtains from 2 μ m plasmids and the yeast invertase signal sequence that has the joint at plant-d-galactosidase gene coding position under control yeast ga17 promotor.

Fig. 7

Box 5,6 and 7 nucleotide sequences comprise dissimilar encoding sequences of exla presequence and being connected of ripe Fusarium solani pisi at.

Fig. 8

The plasmid PAW14B that obtains by the 5.3kb Sal I fragment of inserting the genomic DNA of Aspergillus niger Var.awamori at the Sal of PUC19 I position.

Fig. 9

Translate the plasmid PUR 7280 that the BSPHI-Af1 II fragment of reading sign indicating number obtains by the exlA that contains with the BSP HI-Af1 II fragment representative that contains former at encoding sequence before the Fusarium solani pisi is open in PAW14B.So, former at gene before plasmid PUR7280 contains Fusarium solani pisi under control A.niger Var.awamori promotor and terminator.

Figure 10

By in PUR7280, bringing out the plasmid PUR7281 that A.nidulans amds and A.niger var.awamori pyrG selectable marker obtain.

Figure 11

The relation that residual activity and various lipolytic enzyme are removed the percentage ratio of dirt behind the DFP-inactivation.With following abbreviation:

Lipolase（TM，ex Novo Nordisk） lip

Fusarium Solani Pisi Cutinase (example 2) CT

Candida cylindracea lipase (sigma) candida

Chromobacterium viscosum lipase (sigma) Chrom

Pancreas Sus domestica lipase (sigma) pancr

Genencor lipase (Pseudomonas putida

Mutation ATCC 53552 Gen

AKG lipase 30B(Amano) AKG

SDL 195 lipase (Showa Denko) SDL

PS.pseudoalcaligines CBS 473.85 lipase PS.ALK

Figure 12

The comparison of the at that obtains by Fusarium solani pisi and the lipolytic activity of lipolytic enzyme (TM).

Figure 13

Fusarium Solani Pisi at and lipolytic enzyme (TM) are singly washed effect to dissimilar dirts.

Figure 14 A

What measure after several wash(ing)cycles is under the 1LU/ml in the enzyme amount, the at that is obtained by Fusarium Solani Pisi and the effect of lipolytic enzyme (TM).

Figure 14 B is the same, at enzyme content 3LU/ml.

Figure 14 C is the same, under enzyme content 5LU/ml.

Figure 14 D is the same, under enzyme amount 10LU/ml.

Figure 15

In PAS/ non-ionic type preparation, use Lipolase(TM), the multicycle washing test of Pseudomonas gladioli lipase and Fusarium solani pisi at.

Figure 16

In the non-release preparation of other PAS/, use Lipolase(TM), the multicycle washing test of Pseudemonas gladioli lipase and Fusarium solani pisi at.

Figure 17

Same Figure 16, but each all after date is heavily made dirty.

Reference

Sambrook，J.，Fritsch，E.F.and Maniatis，T.（1989）.Molecular Cloning：a laboratory manual（2nd ed）.Cold Spring Harbor Laboratory Press，Cold Spring Harbor，New York.ISNB 0-87969-309-6.

Fürste，J.P.，Pansegrau，W.，Frank，R.，Bloecker，H.，Scholz，P.Bagdasarian，M.and Lanka，E.（1986）.Molecular cloning of the plasmid RP4 primase region in a multi-host-range tacp expression vector.Gene 48：119-131.

Michaelis et al.（1983）.J.Bacteriol 154：366-

Tartof and Hobbs（1988）.Gene 67：169-182.

Soliday，C.L.，Flurkey，W.H，Okita，T.W.and Kolattukudy，

P.E.（1984）.Cloning and structure determination of cDNA for cutinase，an enzyme involved in fungal penetration of plants.Proc.Natl.Acad.Sci.USA 81：3939-3943.

Noqi，Y.and Fukasawa，T.（1983）.Nucleotide sequence of the transcriptional initiation region of the yeast GAL7 gene.

Nucleic Acids Res.11：8555-8568.

Taussiq，R.and Carlsson，M.（1983）.Nucleotide sequence of the yeast SUC2 gene for invertase.Nucleic Acids Res.11：1943-1954.

Erhart，E and Hollenberg，C.P.（1981）Curing of Saccharomyces cerevisiae 2-μm DNA by transformation.Curr.Genet.3：83-89.

Verbakel，J.A.M.V.（1991）Heterologous gene expression in the yeast Saccharomyces cerevisiae.Ph.D.thesis.Rijks

Universiteit Utrecht，The Netherlands.

Maat，J.，Roza，M.，Verbakel，J.，Stam，J.，Santos da Silva，M.J.，Bosse，M.，Egmond，M.R.，Hagemans，M.L.D.，v.Gorcom，R.F.M.，Hessing，J.G.M.，v.d.Hondel，C.A.M.J.J.and v.Rotterdam，C.（1992）.Xylanases and their application in bakery.In：Visser，J.，Beldman，G.，Kusters-van Someren，M.A.and Voragen，A.G.J.（Eds），Xylans and Xylanases.

Progress in Biotechnology Volume 7，Elsevier Science

Publishers，Amsterdam.ISBN 0-444-89477-2.

de Graaff，L.H.，van den Broek，H.C.，van Ooijen，A.J.J. and Visser，J.（1992）.Structure and regulation of an Asperqillus xylanase gene.In：Visser，J.，Beldman，G.，Kusters-van Someren，M.A. and Voragen，A.G.J.（Eds），Xylans and Xylanases.Progress in Biotechnology Volume 7，Elsevier Science Publishers，Amsterdam.ISBN 0-444-89477-2.

Hankin，L.and Kolattukudy，P.E.（1968）.J.Gen.Microbiol.51：457-463.

Ettinger，W.F.，Thukral，S.K.and Kolattukudy，P.E.（1987）.

Structure of cutinase gene，cDNA，and the derived amino acid sequence from phytopathogenic fungi.Biochemistry 26：7883-7892.

Huse，W.D.and Hansen，C.（1988）.Strategies 1：1-3.

De Graaff，L.H.，H.W.J. van den Broek and J.Visser（1988）.

Isolation and expression of the Asperqillus nidulans pyruvate kinase gene.Curr.Genet.13：315-321.

Embodiment 1

The construction of the synthetic gene of former at before the coding Fusarium solani pisi.

Basically according to EP-A-407225(Unilever) described method builds the synthetic gene of former at before the coding Fusarium solani pisi.Nucleotide sequence (Soliday etc. according to disclosed Fusarium solani pisi gene, (1984) and WD-A-90/09446, Plant Genetic Systems), design complete synthetic DNA fragment, it comprises the zone of a preceding former at polypeptide of coding Fusarium solani pisi.The nucleotide sequence of primary Fusarium solani pisi gene relatively, this synthetic at gene comprises several mutated nucleotides, introduces restriction enzyme recognition site at position easily by it in the gene that does not influence amino acid sequence coded.The nucleotide sequence of complete synthetic nucleosides acid gene is shown in Fig. 1 D.

Finish the construction of synthesizing the at gene by the isolating box that originates in the synthetic DNA oligonucleotide in conjunction with three.Each synthetic DNA box has an Eco R I position in starting point, has a Hin d III site at terminal point.With a kind of living things system 380A DNA synthesizer synthetic oligonucleotide of application, and purify with polyacrylamide gel electrophoresis.In order to build each box, provide its general method below.The oligonucleotide that equimolar amount (50pmol) is constituted given box mixes, in their 5 ' one end phosphorylations, according to standard method annealing be connected.The mixture of the double chain DNA molecule that produces with the cutting of Eco R I and Hin d III with agarose gel electrophoresis classification fractionation, reclaims from gel by electrolysis.2.7kb Eco R I-Hin d III fragment with PUC9 connects the synthetic DNA box that generates, and converts Escherichia coli to.Arrange Eco R I-Hin III inset sequence of some clones fully with the sequence of the suitable synthetic box of Oligonucleolide primers discriminating at both direction.Build PUR7207(in this way and comprise box 1, Figure 1A), PUR7208(comprises box 2, Figure 1B) and PUR7209(comprise box 3, Fig. 1 C).At last, 0.3kb Nhe I-Hin d III fragment of and PUR7209 segmental with 0.2kb APa I-Nhe I of PUR7208 by the 2.9kb Eco R I-APa I fragment of mixing PUR7207 produces PUR7210, synthesizes the at gene in conjunction with being somebody's turn to do.This plasmid comprises a kind of coding Fusarium solani pisi(Figure 10) the opening of complete preceding former at translate and read sign indicating number.

Embodiment 2

Fusarium solani pisi(is former) expression of in Escherichia coli.

About this synthetic at gene, build a kind of colibacillary expression vector, its effect is similar to WO-A-90/09446(Plant Genetic Systems) situation about being introduced.Design a kind of structure, the partial synthesis gene of the former at of Fusarium solani pisi of wherein encoding is before closing the escherichia coli expression signal of knowing, the escherichia coli expression signal i.e. (ⅰ) a kind of inducible promoter, (ⅱ) ribosome bind site and (ⅲ) signal sequence, this sequence provides the transfer initiation codon, and provides the leap cytoplasmic membrane to export the needed information of former at.

Design a kind of synthetic connexon (see figure 2), become the former sequence of synthetic at gene with the derivative (Michaelis etc., 1983) that engages E.Coli phoA signal sequence.For the cracking of optimizing signal peptide and the secretion of former at, the nucleotide sequence of this connexon is such, the three C terminal amino-acid residues that are PhoA signal sequence (Thr-Lys-Ala) become Ala-Asn-Ala, the-terminal amino acid residue of this cutin proenzyme sequence (Leu 1, sees Fig. 1 D) becomes Ala.The secretion of this structure assurance at becomes encloses film space (seeing WO-A-90/09446, Plaxt Genetic Systems).

In order to obtain a kind of like this structure, the 69bp Eco R I-Spe I fragment that will contain at presequence and the former sequence of part is removed from PUR7210, and replaces with the synthetic DNA connexon sequence (Eco R I-SPe I fragment) of the cutin proenzyme sequence of N-terminal amino acid residue (Fig. 2) of derivative that E.Coli PhoA presequence is provided and change.Plasmid called after PUR 7250 that generates and the 0.7kb Bam H I-segmental segregation of Hin d III that is used to contain ribosome bind site and joins the former at coding region in PhoA signal sequence encoding zone to.This fragment is connected (Furste etc., 1986), obtains PUR7220 with 8.9kb Bam H I-Hin d III fragment of PMMB67EH.In this plasmid, the synthetic gene of the former at of encoding joins in the changing form of PhoA signal sequence, and places inducible promoter control down.

Shake in the bottle at 2 liters that 0.5 liter of IXTB substratum (Tartor and Hobbs 1988) is housed and to cultivate the coli strain WK6 that comprises PUR 7220, the consisting of of this substratum:

0.017M KH ₂PO ₄

0.017M K ₂HPO ₄

12g/l Bacto-tryptones

24g/l Bacto-yeast extract

0.4% glycerine (v/v)

At 25 ℃-30 ℃, in the presence of 100 μ g/ml ampicillins, cultivate down one night of culture acutely shaking (150rpm), optical density(OD) (OD) 10-12 to the 610nm.Add IPTG(sec.-propyl-β-D-thio-galactose pyran-glucoside then) to the excessive 10 μ M of ultimate density, and continue insulation 12-16 hour again.When judging according to the sample analysis that takes out from nutrient solution, when the amount that can observe the lipolytic activity that is produced no longer obviously increases, by the centrifuging collecting cell, and in 0 ℃ of initial volume nutrient solution that is re-suspended into the damping fluid that contains 20% sucrose.By the centrifuging collecting cell and be resuspended in the ice cold water of original culture volume, make by this cell of infiltration vibrations dissolving.By the centrifugal cell debris of removing, cell-free extract is acidified to pH4.8 with acetate, spends the night 4 ℃ of placements, and removes the precipitation of generation.In this step,, obtain the at preparation of purity greater than 75% essentially no endogenous lipase with ultrafiltration and lyophilize cell-free extract.In addition, can be by the acidifying cell-free extract be downloaded on the SP-sephadex, at pH8.0 with this enzyme of buffer solution elution, spissated basic solution is by the DEAE Mierocrystalline cellulose (Whatman DE-52) of suitable volumes, and directly pass through Q-agarose HP(Pharmacia with DEAE) post, the at of purifying is to (being that purity is greater than 95%) of homogeneity.Use the salt gradient wash-out, obtain general overall yield greater than 75% homogeneous phase at preparation.

Embodiment 3

Fusarium solani pisi separates the expression of matter enzyme in Saccharomyces cerevisiae.

In order to be expressed in the synthetic Fusarium solani pisi cutin gene among the Saccharomyces cerevisiae, build a kind of expression vector, wherein, the synthetic gene of encoding mature at is at S.cerevisiae saccharase (Taussig and Carlsson, 1983) presequence and strong derivable ga17 promotor (Nogi and Fukasawa, 1983) afterwards, in order to prepare a kind of like this synthetic at gene of joiner, synthetic a kind of conjugant fragment, wherein the encoding sequence of saccharase presequence joins on the sequence of N-end of encoding mature at.Basically as (box 8 as described in the embodiment 1, see Fig. 3), engage this segment as the Eco R I-Hin d III box in PUC9, produce PUR7217, plasmid PUR7210 and PUR7217 change into E.Coli JM110(and lack the active bacterium pearl of dam methylase for), and 2.8kb Bcl I-Hin d III fragment of PUR721 is connected with 0.6kb Bcl I-Hin d III fragment of PUR7210, generate PUR7218, wherein the nucleotide sequence of encoding mature at polypeptide engages with part S.cerevisiae saccharase presequence coding region.

Be filled in Sal I porous by the 8.9kb Nru I-Sal I fragment of separating PUR2740 with the Klenow polysaccharase, and this fragment of recirculation,, 1991, see Fig. 6 just from PUR 2740(Verbakel) obtain expression vector PUR2741(and see Fig. 4).The 7.3kb Sac I of PUR2741-Hin d III fragment engages with the 0.7kb Sac I Hin d III fragment of PUR7218, produces PUR7219(and sees Fig. 5).At random S.cerevisiae pol II terminator is placed on the back of at gene, in Hin d III site, it was not the effectively expressing of at gene originally substantially.E.Coli-S.cerevisiae shuttle plasmid PUR7219 is containing 2 μ plasmid (Cir ⁺Bacterial strain) contain a kind of original copy product in the S.cerevisiae bacterium pearl, the shortage promotor form of S.cerevisiae Leu2 gene allows at S.cerevisiae Leu2 ^-Select the massive duplication transformant in the bacterial strain, the synthetic gene of the Fusarium solani pisi at of encoding mature part can be linked on the S.cerevisiae saccharase presequence under strictness control, can induce S.cerevisiae ga17 promotor.

With bacterial strain YT6-2-1L(Erhart and Hollenberg, 1987) identical S.cerevisiae bacterial strain SUSO(a, Ciro, Leu2, his4, Canl) with 2 μ S.cerevisiae plasmids and PUR7219 etc. molar mixture use scheme to transform jointly to the standard of yeast cell galvanic deposit (electroporation).Transformant is selected in the leucine prototropy, and from many transformant, separated all DNA.All transformant as if contain 2 μ plasmid and PUR7219, for instance, owing to have 2 μ yeast plasmids simultaneously, the promotor defect form that is contained in the Leu2 gene on the PUR7219 only can functional additional Leu2 defective bacterial strain when duplicating several the existence with height.By in perfect medium, cultivating generation more than 40, then duplicate with complete solid medium middle plateform and cultivate a kind of transformant of handling the PUR7219 plasmid at the selection substratum, produce S.cerevisiae bacterial strain SU51(a, Cir ⁺, Leu2, His4, Can1).

Contain shaking in the bottle of 0.2 liter of MM substratum at 1 liter and cultivate the S.cerevisiae bacterial strain SU51 that contains PUR7219, the consisting of of this substratum:

There is not amino acid whose yeast nitrogen base (yNB) 6.7g/l

Histidine 20mg/l

Glucose 20g/l

Cultivate one night of culture at strenuous vibration (150rpm) with at 30 ℃, to optical density(OD) (OD) 2-4 under 601nm.In 2 liters of 1 liter of yPGAL substratum that shakes in the bottle, this substratum consists of by centrifuging collecting cell and resuspending:

Yeast extract 10g/l

Bacto-peptone 20g/l

Semi-lactosi 50g/l

Continue again to cultivate 12-16 hour.Take a sample from culture in the interval that connects regulation, and the centrifugal biomass of removing.With sweet oil as substrate, with the volumetry at activity of clear liquid analytically.Each sample of filtrate between the 100 and 200 μ l is joined 5.0ml lipase substrate (sigma contains the substrate of sweet oil as lipase) and 25.0ml damping fluid (5mM Tris-HCl pH9.0,40mD NaCl, 20mM CaCl ₂) stirred mixture in.Test at 30 ℃, be titrated to the lipid acid that pH9.0 measures release automatically with 0.05M NaOH with Mettler DL25 titrimeter.Obtain the curve of the amount of titrating solution to the time.The amount of contained lipase activity from the maximum slope calculation sample of this curve.Unit enzymic activity is defined as the amount that discharges the enzyme of 1 μ mol lipid acid in following 1 minute of the defined terms in the above from sweet oil.Such measuring method person skilled in the art knows.

When the at activity that produces no longer increases, remove cell with centrifuging,, press embodiment 1 described method and reclaim to pH4.8 with acetate acidifying cell-free extract.

Embodiment 4

The expression of Fusarium solani pisi in Aspergilli.

In order to be expressed in the synthetic Fusarium at gene among the Aspergillus niger var.awamori, build a kind of expression vector, wherein under the situation of control A.niger var.awamori strong derivable exlA promotor, place the synthetic gene (Maat etc. of former at before the coding Fusarium solani pisi, 1992, de Graaff etc., 1992).

Make up preceding cutin expression of enzymes plasmid (PUR7280) by plasmid PAW14B, it is deposited in (Barrn in the E.coli bacterial strain of JM190 in Gentra-albureau voor schimmel nutrient solution, The Netherlands, N ° of CBS 237.90 times, May 31 nineteen ninety), and contain the 5.3kb Sal I fragment of having an appointment, location 0.7kb endoxylanase II (exlA) gene and 5 ' one flanking sequences of 2.5kb and 3 ' one flanking sequences (Fig. 8) of 2.0kb on this fragment.In PAW14B, the exlA coding region is replaced by preceding former at coding region.The Afl II site (5 '-CTTAAG-3 ') of containing the BSPHI site (5 '-TCATGA-3 ') of first codon (ATG) of exlA gene and containing the terminator codon (TAA) of exlA gene promotes the construction of PUR7280.

The as described below construction: with BSPHI partial cut PAW14B(7.9kb), from sepharose, separate linear plasmid (7.9kb).Thereafter, with the 7.9kb fragment of BSM I cutting and separating, it cuts a small amount of Nucleotide in the downstream in valuable BSPH I site, removes the linear plasmid in other BSPH I sites.On sepharose, separate this fragment, and separate 7.9kb BSPH I-BSM I fragment.This is with Afl II partial cut, and separates the 7.2kb BSPH I-Afl II fragment that forms.

To contain the complete opening of former at before the coding Fuserium Solani Pisi translates 0.7kb BSPH I-Afl II fragment of the PUR7210 that reads sign indicating number and engages generation PUR7280 with 7.2kb BSPH I-Afl II fragment of PAW14B.Can change into mould (moulds) (Aspergillus niger for example to the carrier of building (PUR7280) with conventional common method for transformation then, Aspergillus niger var.awamori etc.), then by introducing the preceding former at gene of endoxylanase II promoter expression.Also can obtain the rDNA carrier of this construction with conventional selectable marker (for example amds or pyrG, Totomycin etc.), can transform mould with the rDNA carrier that generates and produce needed protein.As an example, in this expression vector, introduce amds and pyrG selectable marker, generate PUR 7281(Figure 10).For this reason, by becoming Not I site, produce PUR 7282 with synthetic oligonucleotide (5 '-AATTGCGGCCGC-3 ') Transformed E co R I site (there is 1.2kb in the upstream of the AFG codon of preceding former at gene).The suitable dna fragmentation that contains complete A.nidulans ands gene and A.niger var.awamori pyrG gene is furnished with Not I site, side with their promotor and terminator, and the Not I site of introducing PUR 7282, produce PUR7281(Figure 10).

In Aspergillus niger var.awsamori, express the method for this synthetic Fusarium solani pisi at gene as another kind, build expression vector, the synthetic gene of this maturation at of wherein encoding is not after its preceding former sequence, and after the presequence of A.niger var.awamori exlA.

In order to prepare the synthetic at gene that is used for this joint, synthetic several accepting agent fragments, wherein the encoding sequence of exlA presequence is linked on the sequence of N-end of encoding mature at by different way.In box 5, by carrying out this connection on the former sequence that the exlA presequence is engaged at.In box 6, the exla presequence engages with the N-end residue of ripe at.Box 7 is identical with box 6, but the N-of the ripe at polypeptide of coding end base becomes serine residue from initial glycine here, so that better meet signal peptide cracked needs.Basically it is described to press embodiment 1, is combined into box 5.6 and 7(sees Fig. 7 from the synthetic oligonucleotide).Replace PUR7210 0.1kb Eco R I-spe I fragment with box 5, generate PUR7287.0.1kb Eco R I-Bel I fragment with box 6 and 7 replacement PUR generates PUR7288 and PUR 7289 respectively.For each plasmid PUR 7287, PUR 7288 and PUR 7289,0.7kb BSP H I-Afl II fragment engages with 7.2kb BSPH I-Af II fragment of PAW14B, generates PUR 7290, PUR 7291 and PUR 7292 respectively.

Change method with conventional corotation then, the rDNA carrier of building is converted to mould (moulds) (Aspergillus niger, Aspergillus niger var.awamori) also by (former) at gene before the introducing endoxylanase II promoter expression.With the automatic control mark thing of routine (for example amds or pyrG, the rDNA carrier that Totomycin) also can obtain building, by transforming this mould to PUR 7280 described (seeing above-mentioned) with the rDNA carrier that generates among this embodiment, produce needed protein.

Cultivating the Asperigllus bacterial strain (under the situation of control A.niger var.awamori exlA promotor or terminator, containing the ripe at coding region of the Fusarium solani pisi that is with or without corresponding former sequence and at signal sequence or exlA signal sequence) that transforms with expression vector PUR7280, PUR7281, PUR7290, PUR7291, PUR7292 under the following condition; There is a plurality of 1 liter of 400ml synthetic medium (pH6.5) to shake bottle (final concn 10E6/ml) with spore inoculating.This substratum is by following the composition (AW substratum):

Sucrose 10g/l

NaNO ₃6.0g/l

KCl 0.52g/l

KH ₂PO ₄1.52g/l

MgSO ₄·7H ₂O 0.49g/l

The female extract 1.0g/l of enzyme

ZnSO ₄·7H ₂O 22mg/l

H ₃BO ₃11mg/l

MnCl ₂·4H ₂O 5mg/l

FeSO ₄·7H ₂O 5mg/l

CaCl ₂·6H ₂O 1.7mg/l

CuSO ₄·5H ₂O 1.6mg/l

NaH ₂MoO ₄·2H ₂O 1.5mg/l

Na ₂EDTA 50mg/l

At 30 ℃, under the 200rpm condition, insulation is 24 hours in MKx insulation electromagnetic shaker.Collect the cell of growth with filtering (0.45 μ m strainer), with the AW substratum washed twice that does not have sucrose and yeast extract (salts solution), be resuspended in the 50ml salts solution and transfer to the 300ml that contains the 50ml salts solution and shake in the bottle, in this bottle, add wood sugar and bring out substratum) to ultimate density 10g/l(.Under above-mentioned same condition, continue incubated overnight.Filter the at that generates with Mi Laibu, remove biomass, reclaim at according to embodiment 2 described methods substantially.

Embodiment 5

Differentiate and the gene that separates about Fusarium solani pisi at gene.

From different fungies, separate coding at gene about Fusarium solani pisi at different homology degree.Contain 200ml by Hankin and Kolattukudy(1968 at 500ml) substratum introduced and additionally have shaking of 0.25% sucrose to cultivate fungal cultures in the bottle, and in MKx insulation vibrator (100rpm), be incubated 4 days at 28 ℃.At this moment consume sucrose, induced to produce according to the described adding cutin of people such as Ettinger hydrolyzate basically.At specific time sample thief from nutrient solution at interval, and analyze the lipolytic activity (seeing embodiment 4) that exists according to standard method.Usually bring out the back and lipolytic activity can be described in about two days, filter collecting cell with standard method at this moment.With standard method wash mycelia, freezing and lyophilize in liquid nitrogen.Basically separate the RNA preparation of whole cells according to the described method of people such as Sambrook (1989) with thiocyanic acid metal plate salt method, and purify with cesium chloride density gradient centrifugation.Separate kit(promga with polyATtract mRNA) separation polyA(+) the mRNA fraction.According to standard method, use the cDNA fragment that obtains from Fusarium solani pisi at gene as detecting thing with polyA(t) the mRNA fraction is used for the analysis of Northern hydridization, with check about the at expression of gene.Explanation according to the supplier, with ZAP cDNA synthetic kit(Stratagene, La Jolla) will contain and to be used for synthetic cDNA with the preparation of mRNA of the material that detect thing hydridization, generate but in the Xho I of the lateral sticky end in poly-A zone and the cDNA fragment that engages in the EcoR of the other end I.The cDNA fragment that obtains is used to build expression library by the clone of meaningful orientation in Lambda 2AP II carrier (Stratagene La Jolla), expresses the protein (Huse etc., 1988) that beta-galactosidase enzymes engages.The antiserum(antisera) screening that these storehouses produce with anti-Fusarium solani pisi at.

In addition, with the special primer of at this synthetic cDNA fraction is carried out pcR-screening (seeing Table 2).These primers relatively obtain (Ettinger etc., 1987) by the aminoacid sequence of several fungi at genes.Use the cDNA that obtains by Fusarium solani pisi to compare, each group primer is all optimized the PCR reaction conditions.Preparation with these cDNA may produce special P CR fragment, this segmental length is similar to the segmental length of PCR that (or greater than) uses the cDNA that obtains from Fusarium solani pisi to produce under the same conditions, with purify this PCR fragment and separating from gel of gel electrophoresis.

As another kind of method, also be directly used in the genomic dna of some fungal bacterial strain with the PCR material sieving technology of the special primer of at, with the genomic dna of Fusarium solani pisi as positive contrast.Preparation with this fungal gene group DNA, can produce special P CR fragment, its length is similar to the segmental length of PCR that (or greater than) uses the cDNA that obtains from Fusarium solani pisi to produce under the same conditions, with gel electrophoresis this PCR fragment of purifying, and from gel, separate.

For being positive bacterial strain and many other bacterial strains, the genomic dna of separation of high molecular weight in the method for expression library or in the PCR method for sieving (or with cDNA or use genomic dna).Basically press (1987) described method cultivation bacterial strains such as Ettinger, press (1988) described method isolation of genomic DNA such as cle Graaff.With different restriction enzyme digested genomic dnas, and similarly cDNA introducing thing (expression library method) or PCR fragment (PCR method for sieving) or Fusarium solani pisi at gene (other bacterial strains) are analyzed by Southern hydridization as detecting thing, and build the sterogram of at gene.The genomic dna of suitable absorption is with gel electrophoresis classification fractionation, separates the fragment of suitable size and clone again in pUC19 from gel.Sieve these genomic libraries with corresponding cDNA introducing thing (expression library method) or PCR fragment (PCR method for sieving), generate the clone who contains the at genetic replica.These genes are at the both direction sequencing.Differentiate Introns by the corresponding cDNA of sequencing or by comparing (Ettinger etc., 1987) with other at sequences.By a kind of like this N-end of more also deriving ripe at polypeptide.PCR method with standard, remove introns, build Hind III site in the open downstream of reading sign indicating number of translating immediately, (Sac I site formerly for the encoding sequence of the presequence of Saccharomyces cerevisiae invertase gene, box 8 relatively Fig. 3) joins on the sequence of N-end of encoding mature at.The resulting Sac I-Hin d III fragment of the at gene on the sequence that can be connected to coding S.cerevisiae saccharase presequence that contains changes into S.cereviiae bacterial strain SU51 with the 7.3kb Sac I of PUR7241-Hin d III fragment (see figure 4) merging.Express this fungi at and basically by embodiment 4 described methods from nutrient solution with its recovery.

Embodiment 6

Inactivation test with fluoro di(2-ethylhexyl)phosphate sec.-propyl (DFP).

In this test, the lipolytic enzyme of different sources is carried out inactivation with the DFP of various concentration test cycle regular time that reaches.Inactivation is after the cycle, with the percentage ratio of pH-Stat test determination remaining activity.Test conditions is: the concentration of the test lipolytic enzyme of use is to contain 20mM CaCl at 10mM ₂2H ₂O(Merck) every ml protein 0.5mg in the tris buffer of pH10.0.20 μ lDFP-inhibitor solutions are added in the 0.5ml this kind of enzyme solution (sample), and 209 μ l ether are added in the other 0.5ml enzyme solution (blank).This DFP-inhibitor solution is a 0.5MDFP(fluoro di(2-ethylhexyl)phosphate isopropyl esters, Fluka) in ether (Baker).Two samples room temperature insulation 10 minutes, after the insulation, are used Eppendorf whizzer full speed (14000rpm) centrifugal this solution 2 minutes.Use supernatant liquor.In sample and blank test, at pH10.0, (Sigma, numbering 800-1) measures lipase activity with the lipase substrate in the pH-stat test.Calculate residual activity (RA) then as follows:

RA=lipase activity (sample)/lipase activity (blank) * 100%

This residual activity percentage ratio is as follows:

Lipolytic enzyme residual activity (%)

Lipolase（TM，Novo Nordisk） 86

Fusarium solani pisi at (embodiment 2) 5

Candida cylindracea lipase (sigma) 83

Chromobacterium viscosum lipase (sigma) 65

Porcine pancreatic lipase (sigma) 60

(the mutation of pseudomonas putida of Genencor lipase

ATCC 53552，“Lumafast”） 65

AKG lipase 30B(Amano) 52

SDL 195 lipase (showa Demko) 20

PS.pseudoalcaligines CBS 473.85 lipase 60

Embodiment 7

Measure the lipolytic activity of the enzyme of being tested among the embodiment of front with above-mentioned Br-olive oil process.Wash temperature is 30 ℃, and the water hardness is 27 H.The percentage ratio of the Br-sweet oil of removing in washing test is as follows:

The grease that lipolytic enzyme is removed (%)

Lipolase （TM，Novo Nordisk） 0

Fusarium solani pisi at (embodiment 2) 19

Candida cylindracea lipase (sigma) 0

Chromobacterium viscosum lipase (sigma) 1.6

Porcine pancreatic lipase (sigma) 10

(the mutation of pseudomonas putida of Genencor lipase

ATCC 53552，“Lumafast”） 11

AKG lipase 30B(Amano) 10

SDL 195 lipase (showa Demko) 20

PS.pseudoalcaligines CBS 473.85 lipase 15

Related each other by means of the data (with the residual activity behind the DFP inactivation) that linear regression analysis obtains embodiment 6 with the data that obtain in this embodiment.In Figure 11, use this mutual relationship of diagrammatic representation.Between the scourability of the percentage ratio of residual activity and given lipolytic enzyme, there is a kind of good mutual relationship as can be seen by this figure.Therefore, our conclusion is according to the be in the suds simple prediction experiment of performance of the lipolytic enzyme that the percentage ratio that embodiment 6 usefulness DFP cultivate the back residual activity can be used as any source.

Embodiment 8

Fusarium solani pisi at and lLipolase(TM) the comparison of lipolytic activity.

According to embodiment 2, by separating, the Lipolase(TM that is obtained by Novo Nordisk A/S on the lipolytic activity of the at that will obtain from Fusarium solani pisi and the market), a kind of lipolytic activity of Humicola Lanuginosa lipase compares.

The test fabric with the cotton volume that cotton is knitted is polluted by Pure Olive Oil.To cultivate in the 30ml wash water solution of each test fabric in 100ml polystyrene bottle then.In the Miele TMT washing machine of dress water, use and stir these bottles under 30 ℃ of common main washing procedures.This washing soln is made up of the powder of washing of 2g/ liter (6 H), and this is washed powder and has following composition (by %(weight)):

Nonionogenic tenside C ₁₂C ₁₅Alcohol 10.5-13EO 9.5

Sodium sulfate 38.6

Yellow soda ash 40.4

Water glass (Na ₂O: Si ₂O=2.47 7.3

Water 4.2

After washing for the first time, at room temperature after dry this test fabric of roller dryer, measure residual fat immediately.In the Soxhlet device, test fabric with petroleum ether extraction.Measure the amount of fatty substance then by weighing and be expressed as percentage ratio fatty on fabric, the result provides in Figure 12.

As can be seen from this figure, removing aspect the sweet oil, with controlled trial relatively, Lipolase(TM) do not have anything to improve, shown in the test, this effect shows slightly negative, but does not think significantly.On the other hand, at shows the effect that significantly is in the suds.

Embodiment 9

Fusarium solani pisi at and Lipolase(TM) dissimilar pollutions singly washed effect.

Test fabric peanut oil, the oleate of cotton system mixture oily and these two kinds of fat 50/50 is polluted.Washing powder and wash conditions are with embodiment 8.Pass through mensuration reflectance analysis washing effect after singly washing at the 460nm place.The result provides in Figure 13.

As can be seen from this figure, the detergent composition that contains to the washing effect of the grease of several types all the time than containing Lipolase(TM) the washing effect of composition good.This effect is the most tangible to pure peanut oil.

Embodiment 10

After each wash(ing)cycle, measure Fusarium solani pisi at and Lipolase(TM) effect when different content.

Basically repeat embodiment 9, its test fabric pollutes with the LS-3 grease.This is the emulsion of a kind of 75g peanut oil, 40g emulsifying agent, 125g AS-8 and 125g milk powder.After the washing, will test fabric recontaminate and washing more for the first time, repeat altogether 4 times.Enzyme content be 1,3,5 and the situation of 10LU/ milliliter washing lotion under, after each wash(ing)cycle, measure at and Lipolase(TM) effect, the result provides in Figure 14 A-D.

By these tests, can draw several conclusions.Obviously, under all content of at, all show and significantly singly wash effect.And Lipolase(TM) under high-content 10LU/ml, only show and slightly singly wash effect.To all four wash(ing)cycles, at is all than Lipolase(TM after washing for the first time) good.

Embodiment 11

Fusarium solani pisi at, lipase ex pseudomonas gladioli and Lipolase(TM) effect.

With the test fabric of 67% polyester and 33% cotton with the destarch of following 5g/l Betengent product, and thoroughly rinsing.It is cut sheet of 7.5cm * 7.5cm and beat and scoop up.10 so untainted fabrics (the about 6.5g of total mass) are placed on 75ml contain Betengent product given below, dosage is to wash under 6 H in the washings of 5g/l.Lipase ex Pseudomonas gladioli, Lipolase or Fusarium solani pisi at are added in the wash water solution of 1LU/ml in advance.By EP-A-205208 and EP-A-206390(all is Unilever) the described lipase ex pseudomonas gladioli that obtains.Wash water solution and fabric are placed in the bottle, and this bottle is placed in the Lavamat AEGX washing machine, is stirred 30 minutes at 30 ℃.Washing powder is by following forming (% heavy):

Coconut primary alkyl sulphates 5.2

Nonionogenic tenside C ₁₃-C ₁₅Alcohol 7EO 5.2

Nonionogenic tenside C ₁₃-C ₁₅Alcohol 3EO 6.6

Zeolite 4A 32.00

Yellow soda ash 11.52

Water glass 0.45

Sclerosis tallow soap 2.00

Initial in all cases pH is about 10.After washing finishes this fabric is extracted and about half a minute of rinsing in the 300ml tap water.This process triplicate.And then this fabric extracted and airing at ambient temperature.

Half of exsiccant fabric polluted with sweet oil and placed three days.Measuring by weighing initially approximately is that fabric 5%(is heavy) smeary weight.The result provides in Figure 15.

Obviously, Lipolase(TM after the single wash) smeary is removed inoperative.Lipolase(TM) effect only is just can reach by after the repeated washing.After the washing, P.gladioli lipase obtains few still tangible washing effect, after wash(ing)cycle thereafter positive effect is arranged for the first time.After wash(ing)cycle for the first time, at is removed dirt, and the whole cycle thereafter all keeps this advantage.

Embodiment 12

Fusarium solani pisi at, lipase ex pseudomonas gladioli and Llipolase(TM) effect.

With following Betengent product, with the concentration repetition embodiment 11 of 5g/l.

Coconut primary alkyl sulphates 1.7

Nonionogenic tenside C ₁₂-C ₁₅Alcohol 7EO 6.8

Nonionogenic tenside C ₁₂-C ₁₅Alcohol 3EO 8.5

Zeolite MAP 1) 32.00

Yellow soda ash 11.52

Water glass 0.45

Sclerosis tallow soap 2.00

1) zeolite MAP representative " maximum aluminium zeolite P "; It is at EP-A-384070(Unilever) in the class zeolite described.

The result provides in Figure 16.As can be seen, no matter two kinds of lipase after first washing, still all remove degreasing after wash(ing)cycle thereafter.But at is existing very big washing effect after first wash(ing)cycle.

Embodiment 13

Fusarium solani pisi at, lipase ex pseudomonas gladioli and Lipolase(TM) effect.

Repeat embodiment 12, still recontamination between wash(ing)cycle.The result provides in Figure 17.Observe after second wash(ing)cycle, Lipolase(TM) have some effects with P.gladioli lipase.In single wash, matter angle enzyme is reduced to the smeary amount minimum.Thereafter after wash(ing)cycle even recontamination, grease all further reduces.

Claims (16)

1, a kind of enzymatic detergent compositions comprises:

(a) surfactant system of 0.1-50% (weight), this system contains

(a1) one or more anion surfactants of 0-95% (weight) and

(a2) one or more nonionogenic tensides of 5-100% (weight);

With

(b) 10-20000LU enzyme/every gram detergent composition, this endonuclease capable presents tangible lipolytic activity in the major cycle of washing process.

2, according to the detergent composition of claim 1, wherein,, be benchmark with initial grease amount by the methods described herein analysis, to compare with other same detergent composition that do not contain enzyme, this enzyme can be removed at least more than 5%, and is preferred at least more than 10% grease.

3, according to the detergent composition of claim 1, wherein, by the methods described herein analysis, the enzymatic grease of removing by this enzyme with by Lipolase(TM) ratio of the enzymatic grease removed is at least 3, preferably is at least 5.

4, the detergent composition that requires according to arbitrary aforesaid right, the enzyme that wherein presents lipolytic activity is at room temperature to cultivate by the fluoro di(2-ethylhexyl)phosphate isopropyl esters (DFP) with pH10 described herein to have residual activity less than 50% after 10 minutes, preferably, be more preferably less than 25% lipolytic enzyme less than 40%.

5, the detergent composition that requires according to arbitrary aforesaid right, wherein this enzyme is esterase or lipase.

6, the detergent composition that requires according to arbitrary aforesaid right, wherein this enzyme is the eucaryon at.

7, according to the detergent composition of claim 6, wherein this enzyme is the fungi at.

8, according to the detergent composition of claim 7, wherein this enzyme is the fungi at that the stem characteristic is arranged.

9, detergent composition according to Claim 8, wherein this enzyme is to be selected from Fusarium solani pisi, Fusarium roseum culmorum, Rhizoctonia solani and Alternaria brassicicola(PNBI) at.

10, according to the detergent composition of claim 7, wherein this enzyme is the at that can obtain from the Fusarium organism.

11, according to the detergent composition of claim 7, wherein this enzyme is the at that obtains from Fusarium solani pisi.

12, according to the detergent composition of claim 7, wherein this enzyme and the antibody generation immunological cross-reaction that suppresses to produce from Fusarium solani pisi deutero-at.

13, according to the detergent composition of above-mentioned claim, wherein do not contain anion surfactant.

14, according to the detergent composition of claim 1-12, wherein anion surfactant is a primary alcohol sulfate.

15, according to the detergent composition of claim 14, wherein anion surfactant is C ₁₂-C ₁₅Primary alcohol sulfate.

16, the method for contamination with wash fabric, the step that comprises: (a) use fabric that the solution washing according to the detergent composition of above-mentioned arbitrary claim pollutes and (b) use the hot-air dry fabric.

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