DK175855B1 - New proteolytic enzymes - obtd. by mutagenesis of genes encoding wild-type enzymes for improved properties in detergent applications - Google Patents

Abstract

Enzyme prod. is claimed which comprises a mutant proteolytic enzyme, obtd. by expression of a gene encoding the enzyme having an aminoacid sequence which differs in aminoacid(s) from the wild-type enzyme, which mutant enzyme is characterised in that it shows improved properties for application in detergents. The gene may be derived from a wild-type gene of a strain selected from B. subtilis, B. licheniformis, B. amyloliquefaciens and Bacillus nov. spec. PB92. Also claimed is an enzyme prod. which comprises a mutant proteolytic enzyme characterised by showing improved properties for application in detergents as compared to the wild-type enzyme having the aminoacid sequence of PB92 protease and having a mutation at aminoacid(s) 116, 126, 127, 128, 160, 166, 169, 212 and 216. Also claimed are a mutant gene encoding the mutant proteolytic enzyme, an expression vector which comprises the mutant gene and a prokaryotic host strain transformed with the expression vector. Also claimed is a method for selecting a proteolytic enzyme having improved properties for application in detergents which comprises (a) mutagenising a cloned gene encoding a proteolytic enzyme or fragment, (b) isolating the obtd. mutant protease gene(s), (c) introducing the mutant protease gene(s) into a host strain for expression and prodn., (d) recovering the produced mutant protease and (e) identifying those mutant proteases having improved properties for application in detergents.

Description

in DK 175855 B1

The present invention relates to novel proteolytic enzymes with improved properties for use in detergents. These properties include improved ability to remove discolorations in laundry detergent washing compositions, improved stability of laundry detergents during storage, and improved stability in detergent compositions made from the detergents.

Use of enzyme additives, especially proteolytic enzymes, in detergents to allow the removal of 10 protein-based debris has been extensively described. See, e.g. published European patent applications EP-A-0220921 and EP-A-0232269, U.S. Patent No. 4,480,037 and No. Re 30,602 and the article "Production of Microbial Enzymes", Microbial Technology, vol. 1 (1979) 281-311, 15 Academic Press.

Detergents may be in powder form, liquid form or paste form. They contain one or more anionic, nonionic, cationic, zwitterionic or amphoteric compounds as active detergent materials. Such compounds are described extensively in "Surface Active Agents", Vol. II by Schwartz, Perry and Berch, interscience Publishers (1958). Furthermore, they may contain sequestering agents, stabilizing compounds, fragrances and in some cases oxidizing agents, usually called bleaching agents. Detergents are used for cleaning solid surfaces, toilet cleaning, washing up (either by machine or by hand) and laundry.

Laundry detergents are usually divided into two main types, liquid and powdery. Liquid laundry detergents have high levels of surfactants, neutral to moderately alkaline pH and generally do not contain bleach. Washing powder has

I DK 175855 B1 I

I 2 I

In most high alkalinity (washing solution pH 9-11), the I

You contain sequestrants such as sodium tripoly I

In phosphate, and they may, depending on the washing habits of the I

In countries where they are traded, they contain bleach or I

I 5 does not contain bleach. IN

In Enzymes used today in detergents, I-

You are put in the form of liquid suspension, sun or granu-. * I

In lat. In washing powders, the proteolytic enzymes are e.g. IN

You are generally present in an encapsulated form, such as I

In 1 ° in prill form (eg by Maxatas ^ ® and MaxacaJ ^ or in I

In granular form (for example, by Savinas ^ and Alcalas ^). Max I

In the case and Maxacal are negotiated by International Bio-Synthesis

In tics B.v. (Rijswijk, The Netherlands), and Savinase and Alkalase I

You are being negotiated by NOVO Industri A / S (Bagswaard, Denmark). IN

In liquid detergents, the enzymes are most often present in I

In the form of solution. IN

In proteolytic enzymes, in general, water is used

You can combine with detergents. They must be stable

In cars and active in use, e.g. for removal I

20 of protein-containing dyes from textiles during washing I

H I

at temperatures in the range of approx. 10 C to over 60 C

Furthermore, they must be stable for a long time in detergent I

during its storage. Accordingly, the enzymes must be I

stable and active in the presence of sequester I

25 surfactants, surfactants and in some cases I

In bleach and at high alkalinity and temperature. As you

there are neither universal detergents nor universal detergents

sailing washing conditions (pH, temperature, conc. I)

wash mix, hardness of the water), which can i

30 is used worldwide, the requirements for enzymes may vary

on the basis of the type of detergent in which they are to be

H is used and of the washing conditions. IN

The conditions that are crucial for stability I

Generally, the amount of enzymes in washing powders are not I

3 DK 175855 B1 optimal. For example, in the presence of enzyme preparations in detergent powders, oxidizing agents from the detergent can, for example, despite the apparent physical separation of the enzyme from the detergent material, affect the protease and reduce its activity. Another cause of instability of the enzyme in washing powders during storage is autodigestion, especially at high relative humidity.

Furthermore, oxidizing agents often found in washing powders have an important disadvantage in the effectiveness of dye removal when used in laundry or cleaning due to fixation of proteinaceous dyes to the textile material. In addition, these oxidizing agents and other detergent components, such as sequestering agents, also reduce the efficiency of the dye removal process during the washing process.

Liquid detergents have experienced rapid enzyme activation as a major problem, especially at high 20 temperatures. Since the enzymes are found in solution in water | the detergent product, this inactivation already occurs in the detergent under normal storage conditions and significantly reduces the activity of the enzymes before using the product. In particular, anionic surfactants, such as 25 alkyl sulfates, in combination with water and builders tend to irreversibly denature the enzyme and render it inactive or sensitive to proteolytic degradation.

Partial solutions of stability problems in pre-3 ° association with enzymes in liquid detergents have been found in adaptations of the liquid detergent formulations, such as e.g. using stabilizers to reduce the inactivation of the enzymes. See EP-A-

DK 175855 B1! IN

Η

0126505 and EP-A-0199405, U.S. Patent No. 4,318,818 I

and GB Patent Application No. 2178055A. IN

Another method of ensuring stability of one- I

enzymes in liquid detergents are described in EP-A-I

5, physical separation of the enzyme molecules and I

the attacking detergent material is obtained by means of I

formulation technology. In washing powders, alternative I

encapsulation methods have been proposed, see e.g. EP-A-I

0170360. I

10 In the foregoing, the conditions which I

proteolytic detergent enzymes must fulfill in order to:

function optimally and the limitations of the enzymes contained in I

day is available for use in detergents. Uan- I

seen the efforts made to ensure enzyme I

15 stability in detergents, one must still count on one

H

substantially, loss of activity during normal storage

and conditions of use. IN

Identification and isolation of new enzymes for I

a particular intended use, such as use in va- I

20 formulas can be performed in many different ways. And I

way is screening for organisms or microorganisms, I

exhibiting the desired enzymatic activity, isola I

tion and purification of the enzyme from the (micro) organism or I

clay from a culture supernatant of this (micro) organism, I

25 to determine its biochemical properties and to investigate

To determine whether these biochemical properties meet the requirements of I

In the vein of use. If the enzyme identified does not

Ke ke can be obtained from the organism that naturally forms you

In it, recombinant DNA methods can be used for isolation

In 30 tion of the gene encoding the enzyme, expression of I

In the gene of another organism, isolation and purification of I

In the expressed enzyme and testing for its suitability

I for the intended use. IN

5 DK 175855 B1 j

Another method for obtaining new enzymes for intended use is modification of existing enzymes. This can be eg are obtained by chemical modification methods (see I. Svendsen, Carlsberg J 5 Res. Commun. 44 (1976), 237-291). Generally, these methods are unspecific in that all available residues with the same side chains are modified or dependent on the presence of suitable amino acids to be modified, and are often unsuitable for modification of amino acids that are difficult to reach. , unless the enzyme molecule is unfolded. Therefore, the enzyme modification method with mutagenesis of the coding gene is believed to be better.

Mutagenesis can be achieved either by random mutagenesis or by site-directed mutagenesis. Random mutagenesis. of course, when treating an entire microorganism with a chemical mutagen or by imitating radiation can result in modified enzymes. In this case, rigorous selection rules must be available for selecting extremely rare mutants with the desired properties. A greater likelihood of isolation of mutant enzymes by random mutagenesis can be achieved by cloning the coding gene by mutagenesis in vitro or in vivo and expression of the enzyme for which it encodes 25 by cloning the mutated gene into an I

suitable host cell. Also, in this case, appropriate biological selection regulations must be available for selection of the desired mutant enzymes, see International Patent Application WO 87/05050. These biological selection rules do not provide specific selection of enzymes suitable for use in detergents.

The most specific method for obtaining modified enzymes is site-directed mutagenesis,

I DK 175855 B1 I

I I

In allowing specific substitution of one or more 1 I

In amino acids with any other desired amino acid. i

In I EP-A-0130756, examples of the use of this I are given

In a method for generating mutant protease genes capable of

I 5 is expressed to obtain modified proteolysis

In tical enzymes. IN

Recently, the potential for oligonucleotide I

In mediated, site-directed mutagenesis been shown by I

Using mutagenic oligonucleotides synthesized I

I 10 to contain mixtures of bases at several I

In positions in a target sequence. This allows the introduction of I

In a variety of mutations at a specific part of I

a DNA sequence using a single synthetic I

In oligonucleotide preparation as exemplified by (Hui et I

In 15 et al., EMBo J, 2 (1984) 623-629, Matteucci et al., Nucl. IN

Acids Res .. 11 (1983) 3113-3121, Murphy et al., Nucl. IN

In Acids Res 11 (1983) 7695-7700, Wells et al., Gene 34 I

In (1985) 315-323, Hutchingson et al., Proc. Natl. Acad. IN

In Sci. USA 83 (1986) 710-714 and F.M. Ausubel, Current I

In 20 Protocols in Molecular Biology 1987-1988, Greene I

In Publishers Associatin and Wiley, Interscience, 1987). IN

In Stauffer et al., J. Biol. Chem. 244 (1969) I

5333-5338, have already found that the methionine at I

At position 221 in Carlsberg subtilisin is oxidized by Η202 I

I 25 to methionine sulfoxide and is responsible for a violent I

decrease in activity. IN

I As a result of both the random and I method

I with site-directed mutagnesis to form modifi- I

In ready enzymes, mutants are obtained from the serine protease I

30 from Bacillus amylollquefaclens, also referred to as "subti- I

In lisin BPN '"isolated and characterized. In international I

patent application with publication number I

In Wo 87/05050 a mutant subtilisin BPN * with I is described

7 DK 175855 B1 improved thermal stability. EP-A-0130756 discloses that site-directed mutagenesis of methionine at position 222 in subtilisin BPN 'with all the 19 possible amino acids using the so-called "cassette mutagenesis method" can result in enzymes resistant to oxidation by the last one. however, most mutants had low proteolytic activity. The best found mutants were Μ222Ά and M222S, which had specific activities of 53% and 35%, respectively, relative to the natural subtilisin BPN ', see Estell et al., J Biol Chem 260 (1985) 6518-6521.

Previous work to develop modified proteases shows that subtilisin BPN 'mutants with altered stability and kinetic properties can be obtained. See the literature cited above and other references, e.g. Rao et al., Nature 328 (1987) 551-554,

Bryan et al., Protein 1 (1986) 326-334, Cunningham and Wells, Protein Engineering. 1 (1987) 319-325, Russell et al., J. Mol Biol. 193 (1987) 803-819, Katz and Kossia-coff, J. Biol. Chem. 261 (1986) 15480-15485, and the overviews of Shaw, Biochem. J. 246 (1987) 1-17 and Gait et al., Protein Engineering 1 (1987) 267-274. However, none of these references has led to the industrial production of proteolytic enzymes with improved detergent properties and detergent stability. None of the modified proteases have so far been found to have commercial value and be superior to the detergent enzymes used today under the relevant conditions of use.

According to one aspect of the present invention, there is provided a mutant protease as defined in claim 1. These mutant enzymes exhibit improved properties for use in detergents, especially laundry detergents.

I DK 175855 B1 I

I I

In said substitutions in the mutant protease,

In line with [N212D, S160G]. IN

In a preferred embodiment, the enzyme is an I

In PB92 protease mutant. IN

In a further aspect, the invention relates to a process

In tease for use in detergents and with that of claim 4

In the sequence given. IN

In a further aspect, the invention relates to a

In DNA sequence encoding a protease as defined I

In 10 above. IN

The invention also relates to an expression vector, I

In which comprises said DNA sequence. IN

In a further aspect, the invention comprises an I

In prokaryotic host strain transformed with said growth I

For 15 th. IN

The invention also relates to a method for I

preparation of a protease as defined above. IN

Finally, the invention relates to a detergent which I

You include one or more of the proteases and use I

In 20 of one or more of the proteases in a detergent or I

In a washing process. IN

In these and other aspects of the invention you will become

I will describe in more detail in the following detailed description

In relief. IN

I I

Brief Description of the Drawings:

FIG. 1A shows the construction of mutation growth

In the tower containing PB92 protease gene. IN

In FIG. 1B schematically shows the mutagenesis I used

method. IN

In FIG. 1C shows the construction of an expression I

vector containing a mutant PB92 protease gene. IN

9 DK 175855 B1

Pig. Figures 2A, 2B and 3 show the washing ability as a function of specific proteolytic activity for different PB92 protease mutants under different washing conditions.

FIG. 4 shows the nucleotide sequence of the PB92 protease gene and the amino acid sequence of the precursor enzyme it encodes.

By the term "with improved properties" as used in this specification 1 in conjunction with 10 "proteolytic mutant enzymes" is meant proteolytic enzymes with improved washability or enhanced stability with preserved washability over the corresponding wild-type protease.

The term "washability" of proteolytic mutant enzymes is defined herein as the proteolytic one. contribution of the mutant enzyme to the clothing purification in addition to the effect of the detergent without the enzyme under relevant washing conditions.

The term "relevant washing conditions" is used to denote the conditions, especially with respect to the washing temperature, time, mechanical conditions, concentration of detergent in the washing water, the nature of the detergent and the water hardness actually used in the household in a detergent market area.

The term "enhanced washability" is used to indicate that a better end result is obtained in terms of removal of dye under "relevant washing conditions" or that a smaller amount of proteolytic mutant enzyme by weight is needed to achieve the same result as with the corresponding wild type enzyme.

The term "retained washability" is used to indicate that the washability of a proteolytic mutant enzyme by weight is at least 80% of the washability of the additive.

I DK 175855 B1 I

I 10 I

corresponding wild-type protease under "relevant washing conditions

In things. "

The term "improved stability" is used for I

In indicating better stability of proteolytic mutant

In 5 enzymes 1 detergents during storage and / or better I

In stability 1 the wash mixture, which comprises the stability I

Into oxidizing agents, sequestering agents, auto

In bright, surfactant and high alkali, than I

In what you find in the corresponding wild type enzyme. IN

I 10 Biochemical properties determined under well-defined I

In the laboratory conditions are not reliable parameters I

I for predicting the properties of a particular wash- I

In mean protease under the desired and specified uses

In terms of conditions. These parameters include kinetic I

In 15 data measured on well-defined substrates such as proteins I

I such as, for example, casein, dimethylcasein and hemoglobin, or

In substituted oligopeptide substrates, such as IN

In sAAPFpNA (succinyl-L-alanyl-L-alanyl-L-propyl-L-phenyl-I

In alanyl-paranitroanilide). Obviously, other traits I

I 20 at the proteases determine their efficiency in clothing I

In washing or cleaning. IN

The present invention is based on the I

In finding that predicting the impact of specific I

In mutations under actual conditions of use I

I 25 is very difficult or even impossible, although methods I

Introduction to Amino Acid Changes by Which You Can

You induce significant changes in their biochemical I

In properties, are available. IN

According to the invention, one has now far gone far

In 30 research and experiments found a method by which I

In fact, the production of mutant proteases is combined with an I

H effective selection method based on these proteases I

In properties. Surprisingly, relatively large numbers of I-

B1 numbers of enzymes can be efficiently screened for their potency in this way.

This test method is based on the removal of protease-sensitive strains from test samples 1 a laundero-5 meter or tergotometer in which the relevant washing conditions are imitated. Suitable test samples are e.g. the commercially available samples from EMPA (EidgenCssis-check Material Prtifungs und Versuch Anstalt, St. Gallen ,!

Switzerland), which are artificially soiled with protein-containing dyes. Relevant dyes on samples for testing protease include blood, grass, chocolate, dyes and other proteinaceous dyes.

In addition, this test method can control other relevant factors such as detergent composition, concentration in detergent mixture, water hardness, mechanical conditions during washing, time, pH and temperature, in such a way as to mimic the conditions which are typical of household use in a given market area.

The washability of proteases is usually measured by the ability of the residues to remove certain representative dyes under appropriate test conditions. This capability may conveniently be determined by reflectance measurements on test cloths after washing with and without enzymes in a launderometer or 25 tergotometer. The test of the invention for use in the laboratory, when applied to proteolytic enzymes modified by DNA mutagenesis, is representative of household use.

Accordingly, the invention enables testing of quantities of various enzymes and selection of those enzymes which are particularly suitable for use in a specific type of detergent. In this way, tailor-made enzymes for specific conditions of use can easily be selected.

I DK 175855 B1 I

I 12 I

In Some bacterial serine proteases, sub-I is designated

In hindsight. Subtilisins include serine proteases from I

In Bacillus subtilis, Bacillus amyloliquefaclens ("subti- I

In lisin EPN "*) and Bacillus llchenlformls (" subtilisin I

I 5 Carlsberg "). See the overview article by Markland and Smith I

I (1971) in "The Enzymes" (Boyer, ed.) Vol 3, 561-608, I

In Academic Press, New York. Bacillus strains, such as al- I

In calophilic Bacillus strains, other proteases form. IN

In Examples of the latter category, serine protease I

In the 10 series of MaxacalR, hereinafter also referred to as PB92 I

In protease) from Bacillus nov. spec. PB92), and in Savina- I

In seR, as mentioned earlier. IN

In the amino acid sequence of the PB92 protease is shown in I

In FIG. 4. The mature protease consists of 269 amino acids,

In 15 which represents a molecular weight of approx. 27000 D, and have I

At an isoelectric point in the highly alkaline region. IN

In the Activity of PB92 Protease on Protein Substrate Expression I

Check in Alkaline Delft Units (ADU). The activity in ADU is involved

You vote according to the method described in British Patent I

In 20 No. 1,353,317, except that the pH is changed from I

In 8.5 to 10.0. Purified PB92 protease has an activity of I

In AD 21,000 per mg. Turnover number (kcaf) measured on casein I

You are 90 seconds ** 1. IN

The specific activity of purified preparations

In 25 by subtilisin Carlsberg (Delange and Smith, J. Biol. I

In Chem. 243 (1968) 2184) amounts to 10,000 ADU / mg and I

I of subtilisin BPN1 (Matsubara et al., J. Biol. Chem. I

In 240 (1965) 1125) to 7,000 ADU / mg. In addition to the above- I

I te parameters such as specific activity and turnover number I

In 30 ber (kcat), PB92 protease differs from proteases, I

I such as Carlsberg subtilisin, subtilisin BPN 'and others I

In proteases formulated in detergents (e.g., MaxataseR and I

In AlcalaseR) by having a high positive charge that you can

13 DK 175855 B1 is visualized by gel electrophoresis of the natural protein as described later in the experimental section under point 4.

Since PB92 protease is active for removing dye at alkaline pH values, it is commonly used as a detergent additive together with detergent ingredients such as surfactants, builders and oxidizers. The latter agents are most commonly available in powder form. PB92 protease has a high efficiency of dye removal compared to other proteases such as the aforementioned subtilisins. This means that a smaller amount of PB92 protease is required to achieve the same washability.

The sensitivity to oxidation is a major disadvantage of the PB92 protease and all other known serine proteases used in detergents (see also Stauffer et al., J. Biol. Chem. 244 (1969) 5333-5338; Estell et al. J. Biol. Chem. 263 (1985) 6518-6521). Oxidation of PB92 protease with either H2O2 or peracids developed by the activator system containing perborate tetrahydrate and TAED gives an enzyme with a specific activity of 50 and 10% respectively in ADU / mg of the specific activity of the oxidized PB92 protease (see Experimental Section 7 and Example 1).

The method is very suitable for the preparation, screening and selection of proteolytic mutant enzymes obtained from naturally produced bacterial serine proteases. Such mutants are e.g. the mutants encoded by a gene obtained from a wild

Of) u type gene of an alkalophilic Bacillus strain, preferably PB92. Also mutants obtained from the alkalophilic Bacillus serine protease Savinase are suitable. In addition, the method may conveniently be used for selective

I DK 175855 B1 I

I 14 I

I tion of modified proteases obtained from other I

In proteases than serine proteases from alkalophilic Bacillus I

strains PB92. Eg. are the genes encoding serine I

the proteases of Bacillus subtills, Bacillus amylollque- I

In 5 faclens and Bacillus licheniformls known and they can be used

reversed as a target for mutagense. It is clear that either

In oligonucleotide site-directed mutagenesis or region-I

In rectified random mutagenesis or any other I

In suitable method for effective development of mutation I

10 of the protease gene can be used. IN

I The method of selecting proteolytic I

In mutant enzymes, the following steps include: mutagenesis of I

In a cloned gene encoding a proteolytic enzyme of I

interest or a fragment thereof; isolation of the op- I

13 reached the mutant protease gene or the mutant protease obtained

I bless; introduction of this or these mutant protease I

gene (s), preferably on a suitable vector, in a suitable I

In host strain for expression and production; extraction of I

the mutant protease produced and the identification of the

In 20 mutant proteases that have improved properties for an- I

In reverse in detergents. IN

In suitable host strains for the production of mutant

In proteases, transformable microorganisms, I

In which expression of the protease can be obtained. specific

In 25 host strains of the same species or genus; as the protease I

You are obtained from are suitable, such as a Bacillus strain, I

Preferably an alkalophilic Bacillus strain, preferably Bacll-I

In lice nov. spec. PB92 or a mutant thereof with in the tissue

In late the same properties. Also subtilis, B ^ I

In 30 licheniform and amylollquefaclens strains are among I

In the preferred strains. Other suitable and preferred strain I

In more, they include strains which prior to transformation with I

In a mutant gene, it is essentially unable to

DK 175855 B1 15 forms extracellular, proteolytic enzymes. Of particular interest are protease deficient Bacillus host strains, such as a protease deficient derivative of Bacillus nov. spec. PB92. Expression signals include DNA sequences that regulate transcription and translation of the protease genes. The appropriate vectors are capable of replicating with sufficiently high copy numbers in the selected host strains or enabling stable conservation of the protease gene in the host strain by chromosomal integration.

The proteolytic mutant enzymes of the invention are prepared by growing under appropriate fermentation conditions of a transformed host strain comprising the desired proteolytic mutant gene (s) and the desired proteolytic mutant genes and recovery of the 15 enzymes formed.

Preferably, proteases that are expressed are secreted into the culture medium, which facilitates their collection, or, for gram-negative, bacterial host strains, in the pear plasma. For secretion, an appropriate 20 amino-terminal signal sequence, preferably the signal sequence encoded by the original gene, if functional in the selected host strain, is used.

According to one aspect of the invention, suitable washability tests which are representative of all relevant household washing conditions of the market can be developed. Eg. a suitable test was developed for the very demanding European detergent market using powdered detergents which may or may not contain bleach in detergent solution concentrations from 1-10 g of detergent / 1 at 10-20 ° GH (German 0 hardness) and more specifically, a powdered formulated washing agent containing TAED and perborate was used in a washing solution.

I DK 175855 B1 I

I 16 I

concentration concentrate of 4, 7 or 10 g of detergent / 1 l

I at 15 ° GH at 40 ° C. IN

Also representative tests are provided

In tatlve for washing with liquid detergents. Suitable washing I

In 5 ability tests can be developed for other conditions, such as one

In market meetings. Test pieces soiled with protease I

sensitive dyes, especially substances soiled with blood, I

In grass and chocolate and other proteinaceous colors

In Substances, Specifically EMPA Test Topics 116 and 117, I

10 is used in representative wash performance tests. IN

In the properties of the naturally occurring or

In naturally mutated detergent proteases can be improved

I by introducing various mutations into the enzyme. In the I

In most cases, the mutations will be substitutions, I

In either conservative or non-conservative, but part- I

In tions and insertions may also apply. IN

For conservative substitutions, the following may be taken

bells are used:

I 20 Allfatic I

In neutral I

In Unpolar G, A, P I

I L, I, V I

polar C, M, S, T, I

I 25 N, Q I

You charged

anionic D, E I

In cationic K, R I

In Aromatic F, H, W, Y I

I 30 I

In which any amino acid can be substituted by any one

In other amino acid in the same category, especially on the same line. IN

In addition, the polar amino acids N, Q may substitute or I

i DK 175855 B1 i i i 17! is replaced by the charged amino acids. In connection with

* I

The present invention is of substitutions which result in increased anionic character of the protease, especially at sites not directly involved in the active site, of particular interest.

Regions of particular interest for mutations are those amino acids that are within a distance of 4 Å from the inhibitor molecule Eglin C when Eglin C is bound to the active site.

The following numbering is based on PB92 pro tease, but the considerations are relevant to other serine proteases with a substantially homologous structure, especially those proteases having more than ca. 70% homolog !, especially more than 90% homology. These positions will be 32, 33, 48-54, 58-62, 94-107, 116, 123-133, 150, 152-156, 158-161, 164, 169, 175-186, 197, 198 and 203- 216, most of these positions being available for direct interaction with a protein substrate. Positions 32, 62, 153 and 215 will usually not be substituted, as mutations at these sites tend to impair washability.

Substitution sites of particular interest include positions 60, 62, 94, 97-102, 105, 116, 123-128, 150, 152, 153, 160, 183, 203, 211, 212 and 213-216. At some positions, there will be a desire to change an unstable amino acid, e.g. methionine, to an oxidatively more stable amino acid, e.g. threonine, preserving the general conformation and volume of the amino acid at this site. In other situations, it appears that 30 improved results can be obtained by replacing the natural amino acid with almost any other amino acid, this in particular replacing the hydroxylated amino acids, S, T, with polar or non-polar amino acids or even aromatic amino acids.

DK 175855 B1 I

in

Substitutions of particular interest include:

G116 I, V, L I

S126 any amino acid I

P127 any amino acid

S128 an arbitrary amino acid I

S160 anionic or neutral aliphatic or R I

Al66 charged, especially anionic I

M169 neutral, aliphatic, preferably unpolar I

N212 anionic

10 M216 aliphatic polar, especially S, T, N, Q I

While many of the mutations result in a lower one, I

specific activity of the protease with common sub- I

s trater, the washing ability is surprisingly comparable el-

better than the natural enzyme, and in many cases

15 traps will improve storage stability. IN

The washability of some of the PB92 mutant proteases, I

when expressed as the reciprocal value of the rela- I

tive amount of the enzyme needed to obtain I

of the same effect as with the natural protease x I

20 100%, increases to more than 120% and in some cases to I

more than 180%. IN

According to another aspect of the invention, I

are brought to PB92 protease mutants which have a better uptake

durable stability in a powdery detergent

25 retaining bleach than the natural PB92 protease, I

while retaining the washability. Examples of such mutan- I

are mutants M216S and M216Q and mutants having at least one of these

see mutations in addition to mutations elsewhere. IN

According to a further aspect of the invention,

It is surprising that the mutant PB9 proteases M216S, I

M216Q, S160D and N212D retain their activity better than I

PB92 protease in a liquid laundry detergent (not I

contains oxidizing agents). Of these mutants, I retain

19 DK 175855 B1 H216S and M216Q washability, and S160D and N212D have improved washability. The improvement in storage stability in the liquid detergent tested is most pronounced for the S160D and M216Q mutants.

It is also possible to combine a series of different mutations that increase the stability of a protease in detergents. Several mutations that positively affect the washing ability of the same protease can be combined into a single mutant protease gene which allows for the production of possibly even more improved proteases, e.g. [S126M, P127A, S120G, S160D] and [G116V, S126N, P127S, S128A, S160D]. New protease mutants. can be formed by combining the good properties with respect to washability of e.g. N212D and S160D with the stability properties of e.g. M216S or M216Q. The [S160D, M216S] mutant has e.g. improved washability and better storage stability.

Useful mutants can also be obtained by combining any of the mutations or sets of mutations described herein. In addition, it is possible to combine useful mutations as described herein with mutations at other sites which may or may not cause a substantial change in the properties of the enzyme.

Preferred embodiments of the present invention are the following PB92 protease mutants: [N212D] and [S160G, N212D].

To illustrate the importance of the method used according to the present invention to obtain 30 new proteases suitable for use in laundry detergents, i.e. using representative laundry testing as the primary selection criterion, the results of the fluid resistance tests of mutant PB92 proteases are compared

DK 175855 B1 I

with the biochemical parameters usually determined H

by biochemical and enzymological protein studies. H

These results allow the conclusion that no H

is there any relation between parameters that determine H

5 the affinity of defined substrates and the kinetics of I

the proteolytic reaction, and the washing ability (see Table li H

Example 1).

Therefore, of course, it is also possible to

the other two or more mutants with different properties- H

10 creates in one enzyme product or the same washing product H

process. Such a combination can give a synergistic I

effect or not. IN

The invention also includes the use of an electric H

clays several proteolytic mutant enzymes as defined in H

15 above in detergents or during washing process H

look.

Finally, it is clear that the numbering of amino- H

the acids can be altered by deletions or insertions of H

amino acids in the protease polypeptide chain, whether H

20 artificially created or by mutagenesis or occur

naturally in proteases homologous to PB92 pro- H

tease. However, it should be understood that positions that are H

homologous to the amino acid positions of PB92 protease will H

falls under the scope of the claims. H

25 The following examples are given in more detail H

and not to limit the invention. IN

DK 175855 B1 21

Experimental Section

Materials and Methods 5 Construction of PB92 protease mutants j, The basic construct from which the mutagenic work started is designated pM58 and is described in detail in EP-A-0283075.

I The strategy followed included three phases: a. Construction of mutagenesis vector M13M1 b. Mutation c. Construction of pM586Eco and subcloning of the mutated DNA fragment into this vector.

15 l.a. Construction of mutagenesis vector M13M1.

The basic construct, pM58, was digested with the restriction enzymes Hpal and Ball. The 1400 20 bp fragment containing the PB92 protease gene was purified on low melt agarose (Manlatis, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor, 1982).

vector M13MP11 (Messing et al., Nucl. Acid.

Res. 9, (1981) 303-321) was digested with Smal. The relevant 1400 bp DNA fragment was ligated into this vector and transfected into E.coll JM101 following the procedure described by Cohen et al., Prop. Natl. Acad.

SCi. USA 69, (1972) 2110-2114.

After phage propagation in E.coll JM101, 30 ssDNA (Heidecker et al., Gene 10, (1980) 69-73) was isolated, the insert and its orientation examined by DNA sequencing using the method described by Sanger, Proc. Natl. Acad. Sci. USA 74 (1977) 6463.

I DK 175855 B1 I

I 22 I

The vector suitable for mutagenesis was obtained

and was designated M13M1. The procedure described above

You are depicted schematically in Figure ΙΑ. IN

In 5 i.b. Muterinq. IN

I Mutagenesis was performed on M13M1 using I

the ssDNA of this vector and the dsDNA of M13mpl9 (Brass et I

In all. Nucleic Acids Res. 9, (1988) 303-321), the I

The latter vector was digested with restriction enzyme I

EcoRI and HinIII, after which the large fragment is dissolved

I was purified on a low melting agarose. IN

Mutagenesis was performed as described by Kramer et al

In al., Nucleic Acids Res. 12, (1984) 9441-9456 with the I

In modification that E. coll JM105 was used instead of I

In 5 · coli WK-30-3 for selection for the mutants. IN

The length of the oligonucleotides used I

to form the specific mutations, was 22 l

In nucleotides. Region-specific mutation used for dan- I

Of several mutations at a time in a specific I

In DNA sequence, an oligonu was performed

In cleotide preparation having a length of 40 nucleotides with al-I

In le four nucleotides incorporated randomly into sites I

In accordance with the amino acid or amino acids to be

I 25 mutated. IN

After mutagenesis, potential mutant I was investigated

Iter for the relevant mutation by sequence analysis under I

For the use of the dideoxy method according to Sanger, see above. IN

In the entire single stranded piece (see Figure 1B) sequence I

I 30 are prepared for checking the absence of secondary mutation

You down. The process is shown schematically in Figure 1B. IN

The method described is useful for the preparation

Creating DNA fragments with mutations in the 3 'portion of I

In the protease gene (amino acids 154-269). IN

23 DK 175855 B1

It will be appreciated by those skilled in the art that alternative restriction enzymes may be used to generate DNA fragments with mutations in the 5 'portion of the protease gene in a Bacillus vector, and that modified PB92 protease genes may be constructed by a method analogous to the method shown in FIG. 1A.

L. C. Construction of pM58SEco and subcloning of DNA fragments containing the mutations in this vector.

To construct pM586Eco, pM58 was digested with the restriction enzyme EcoRI and ligated with T4 ligase under dilute conditions. The ligation mixture 15 was used to transform B. subtilis 1-A40 (Bacillus Cenetic stock Center, Ohio) following the method of Spizizen et al., J. Bacteriol. 81 (1961) 741-746.

Cells from the transformation mixture were plated 20 on minimal plates containing 20 µg / ml neomycin, as described in Example 1 of EP-A-0283075.

Plasmid DNA of the transformants was isolated following the procedure described by Birnboim and Doly, Nucleic Acids Res. 7 (1979) 1513-1523, and was characterized by restriction enzyme analysis. In this way, pM585Eco was isolated (see Figure 1c).

To produce mutant enzyme, the DNA fragments of M13M1 containing the desired mutation were obtained, subcloned, as described in section l.b. into 30 pM486Eco. dsDNA of M13M1 (described above) was digested with EcoRI and ligated into the EcoRI site of pM586Eco. The ligation mixture was used to transform B. subtilis DB104, Doi, J. Bacteriol. (1984)

I DK 175855 B1 I

I I

160, 442-444, using the method of I

Spizizen at al., See above. IN

Cells from the transformation mixture were plated

I on minimal plates containing 20 µg / ml neomycin and 0.4% I

In casein (EP-A-0283075). DNA of protease-forming trans- I

I formants were isolated according to the method described by I

In Birnboim and Doly, (see above) and characterized by I

In restriction enzyme analysis. IN

2. Preparation of Mutant Proteases. IN

In Transformers of DB104, as had been determined

You contained the vector with the mutated protease gene, Inoku I

I thawed in 10 ml of Trypton Soya Broth (TSB) containing I

H

20 µg / ml neomycin and incubated for 24 hours at 37 ° C.

Samples of the culture (0.1 ml) were inoculated into 500 ml of rum

In step bottles containing 100 ml protease production medium I

I um: 12.5 g / l yeast extract (Difco), 0.97 g / l CaCl 2 x 6 H 2 O, I

I 2.25 g / l MgCl 2 X 6 H 2 O, 20 mg / l MnSO 4 x 4 H 2 O, 1 mg / l

In CoCl2x6H2O, 0.5 g / l citrate, 0.5 ml / l antifoam 5693, 6% I

In w / v maltose, 0.2 M phosphate buffer pH 6.8 and 20 µg / ml I

In neomycin. IN

I After incubation for 65 hours under constant conditions

aeration, the protease activity of the cultures was tested

25 cler use of dimethyl casein as substrate and below I

Using the method described by Lin et al., I

In J. Biol. Chem. 244 (1969) 789-793. For the manufacture of I

In larger representative amounts of wild-type PB92 and mu- I

In Aunt PB92 proteases, these strains were also grown by I

I I

30 37 C in aerated fermentors with a volume of 10 L and

In alleviating the use of essentially the same production I

In medium as used in the shake bottles. IN

The obtained liquid media were used for pro-I

In tease purification. IN

25 Purification and concentration of wild-type PB92 protease and its mutants.

The mutant and wild type PB92 proteases prepared 5 with Bacillus subtills DB104 in shake flasks or 10 L fermenters were purified by cation exchange chromatography using Zeta-PrepR plates or modules (PS type, LKB). Liquid fermentation media was centrifuged (3000 x g, 10 min) and the supernatant was diluted 10 times with 10 mM sodium phosphate buffer, pH 5.5, and then transferred to the cation exchanger. After washing with several module volumes and phosphate buffers, the protease was eluted by adding 0.4 M NaCl to the phosphate buffer. Portions containing protease activity 15 were pooled and concentrated by ultrafiltration using a stirred Amicon cell equipped with a PM-10 filter. The NaCl concentration was reduced to ca.

50 mM by diluting and concentrating the protease solution with phosphate buffer, then storing it at -80 ° C at a protein concentration of between 1 and 10 mg / ml.

Alternatively, to collect PB92 protease mutants from the liquid medium from large-scale fermentation media, CaCl 2 (1 wt%) and acetone (30 wt%) were added. After filtration to remove all cell mass, the protease was precipitated from the filtrate obtained by adding 0.2-2% by weight CaSO4x2H2O and by further adding acetone to a final concentration of> 60% by weight. The precipitate was separated by filtration and rinsed with acetone, then dried to give a crude enzyme powder (CEP).

DK 175855 B1 I

4. Methods of analysis for examining the purity of op- I

purified proteases. IN

Proteases were considered pure when one band or I

5 clays one peak was found by electrophoresis and H respectively

high performance gel electrophoresis (HPLC). IN

Polyacrylamide gel electrophoresis (PAGE) was performed in I

presence of sodium dodecyl sulfate (SDS) after H

The method of Laemmli, Nature, 227 (1970) I

10 680-685. Denaturation of the protein samples by SDS at H

H

However, 100 C must be done in advance by inactivating the

the protease activity to prevent self-degradation. IN

This can be done by emulsion with phenylmethylsulfo-I

nyl fluoride (PMSF) (1 mM, 30 min, room temperature) or I

15 by precipitation with trichloroacetic acid (TCA, 8%, 30 min. I

on ice). Natural PAGE was performed at pH 7.45 (gel buffer I

consisting of 20 mM histidine (His) and 50 mM 3- [N-morpho-I

lino] propanesulfonic acid (MOPS) in 5% polyacrylamide gels I

(ratio of acrylamide to bisacrylamide 20: 1). Pro- I

Twenty sample samples were placed on top of gel disks and sub-I

electrophoresis against the cathode. The same His / MOPS- I

buffer was used as electrophoresis (tank) buffer, but I

at pH 6.3. After electrophoresis (lto-2 hours at 350 V), I

the gel was soaked in 8% acetic acid to fix pro-I

The gel was then stained with Coomassie I

Brilliant blue R250 and decolorized by standard methods. H

The purity study by HPLC was performed under AN-H

reversal of a cation exchange column (MonoS-Pharmacia Fi-I

ne Chemicals) and a gel filtration column (TSK 2000 I

30 sw-LKB). The former was run with 10 mM sodium phosphate

hat buffer, pH 5.5, and elution of the bound protease I

was obtained by a linear gradient from 10-300 mM I

of sodium phosphate, pH 5.5. The gel filtration column is running- I

Test in 0.25M sodium acetate, pH 5.5.

5. Determination of the protease concentrate.

In the TU assessment of the protein concentration in a purified protease solution, i) extinction measurements at 280 nm were used using the calculated extinction coefficient (εΜ), and * 0 ii) active site titration.

The extinction coefficient at 280 nm was calculated from the number of tryptophan units (εΜ = 5,600 M_1xcm "1) and tyrosine units (εΜ = 1.330 M-1xcm_1) per

Enzyme molecules. For PB92 protease, εΜ 26.100 M "1xcm" 1 (3 Trp, 7 Tyr residues) corresponding to E at 280 nm = 9.7 (Mr = 26.729 Da) was used. For mutants with altered numbers of Trp and Tyr residues, corresponding corrections.

A determination of the number of active enzyme molecules was obtained by active site titration, using the widely used method of N-transcinnamylimidazole (ML Bender et al., J. Am. Chem. Soc. 88, (1966) 5890). 5931) was found not to be satisfactory for PB92 protease, a method was developed using PMSF instead, which mixed a protease solution with a calculated concentration (from the absorbance at 280 nm) of 0.25, 0.50 respectively. , 0.75, 1.00 and 1.25 equivalents of PMSF, and the mixture was allowed to react for one hour at room temperature in 10 mM sodium phosphate, pH 6.5. The enzyme concentration must be at least 50 µM.

Residual activity was measured spectrophotometrically using succinyl-L-alanyl-L-alanyl-L-propyl-L

I DK 175855 B1 I

I I

In phenyl-alanyl-paranitroanilide (SAAPFpNA) as substrate I

I (see below). The purity (and thus the concentration) of I

In PMSF was determined by NMR spectroscopy and master data I

1 isopropanol was prepared. The result of the titration I

5 of the active site were found to be consistent with re- I

In the results of the purity study by HPLC. IN

I 6. Determination of kinetic parameters of wild-type and I

In mutant proteases. IN

I 10 I

Activity on protein substrates (casein) target I

is tested at pH 10.0 as described in 1 GB patent no

I 1,353,317 (expressed in units of ADU = Alkaline Delft I

In Units). IN

I 2. Turnover number with casein as substrate target I

in a pH state. The reaction chamber of the pH state I

I (Radiometer, Copenhagen) contained 10 ml of 0.1M KC1 with 50 I

In mg casein (Hammerstein, Merck). Protons Released By I

In casein hydrolysis of PB92 protease was titrated with 10 L

In 20 ιπμ NaOH while maintaining the pH of 10.0 (at 40 "c and I

In under a nitrogen gas stream). IN

3. Activity on synthetic peptides was measured under I

Using sAAPFpNA. It formed (yellow) paranitroa- I

I nilide (pNA) was spectrophotometrically analyzed at 410 nm: I

In 25 eM e M "1xcm" 1, (E.G. Delmar et al. Anal. I

In Biochem. 94 (1979) 316-320) with a UVIKON 860 (CONTRON) I

In spectrophotometer equipped with a thermostated six-I

In position cell switch. The kinetic parameters, kcat I

I and Km, were obtained from measurements of the initial velocity of I

30 different substrate concentrations (for PB92 protease I

I from 0.1-6.0 mM) fitting the data to a hyperbolic I

In function using nonlinear regression with I

In the multivariate secant iteration method. Specificity I

The constant kCat ^ Km was calculated. The measurements were carried out at 25 ° C in a final volume of 1 ml containing 0.1M TRIS-HCl + 0.1M NaCl, pH 8.6. The sodium chloride was necessary as PB92 protease in its absence gave nonlinear Linelier-Burk imaging, which could be due to substrate inhibition. The substrate was first dissolved in DMSO to a concentration of 200 mM and then diluted with 0.1M tris-HCl, pH 8.6 to give a stock solution of 20 mM (determined spectrophotometrically at 315 10 nm; εΜ = 14,000 M ~ 1xcm * '1). No corrections were made for the varying concentrations of DMSO (0.05-3.0 vol%).

7. Oxidation of PB92 protease.

The sensitivity of the PB92 proteases to oxidation with H2 O2 was tested following the method described by Estell et al., J. Biol. Chem. 260 (1985) 6518-6521, with the exception that: 20 i) 20 mM H 2 O 2 was used instead of 10 mM and il) 20 mM sodium perborate combined with 10 mM TAED was used as extra oxidant.

8. Washability Test 25 PB92 protease mutants were tested by a specially developed wash test using cotton and polyester / cotton samples soiled with milk, blood and ink (5.0 x 5.0 cm, obtained from EMPA, St. Gallen, Switzerland and 30 denoted by numbers 116 and 117).

The washing tests were performed in an Atlas Launderometer LEF-FC, equipped with stainless steel test containers, each containing a defined detergent and the protease,

I DK 175855 B1 I

· 30 I

In which to test (PB92 protease mutants or PB92 I

In protease). Except where otherwise indicated, tea was carried out

Stir for 30 minutes at a desired temperature. After I

wash the air-dried samples and reflectance of test cloth

In 5 there was measured at 680 nm with a Photovolt photometer I

I (model 577) equipped with a green filter. Reflectance I

In data obtained by measurement on the test samples washed with va- I

In formulations containing the respective PB92 protease mutants

The aunts were compared with the reflectance data of one to I

In 10 corresponding series of measurements with detergents containing I

In PB92 protease. Washability values of the mutant proteases I

I was calculated by dividing the amount of PB92 protease I

In protein (mg) with the amount of mutant protease protein (mg), I

I necessary to obtain the same reflectance, x I

In 100%. IN

In Example 1 I

A. The washability of various PB92 protease mu- I

Twenty aunts in European washing powders were determined according to progress

In the procedure described above. IN

I In stainless steel test containers, each containing I

You kept a specified amount of washing powder IEC dissolved in 250 ml I

IN

water with a hardness of 15 GH, two cotton and

In 25 two polyester / cotton samples. The composition of the wash I

In the powder IEC was as follows:

31 DK 175855 B1

Component weight%

Linear sodium alkylbenzene sulfonate (average chain length of alkane chain C11, 5) 6.4 5 Ethoxylated tallow alcohol (14 EO) 2.3

Sodium Soap 2.8

Sodium Tripolyphosphate (STPP) 35.0 Sodium Silicate 6.0

Magnesium silicate 1.5

Carboxymethyl cellulose 1.0

Sodium sulphate 16.8

Sodium perborate tetrahydrate 18.5 TAED 1.5

Different + water to 100

To each container was added a selected purified PB92 protease mutant at a concentration of between 0 and 1.74 mg (purified) protease per ml. liters of wash water. A container was used for testing PB92 protease of the same 0 for comparison. The washing tests were performed at 40 ° C. The results are shown in Table 1.

in

32 I

DK 175855 B1 I

--p--

j = g I

+ \ g NOllAnOrlNrllCIB Ot I

djjj. O r> Η H CM CO H

We ιβ W H

+ j u \ QJ X r- e - <0 (0 PU -V> ~

m o) S \. iftor-mr ^ ra ^ 'O' ^ o to I

^^ OH r *> Η H CM O <*) ΙΠ

φ tQ ^ Η H

H * H

-μ

Vi C)

0J C

4- »-H

g «« 0

jj cg OHnr-CMCMCMOlCNP * Η H

3 v H

g 3 HHHHHHHOHHH I

rh

tP

O ----> - C V »Ό * Λί 0) rH (M Q) σ> · Η 4-> O Ot 03 O H-l -Η Λ pQ R)

(U O -W o 04 S * loooh-tor ^ nioovoiAin I

ω QI 4J tw rH 0 ^ oio ^ nn ^ p '^ - or-æw «Di Λ -η µ ri 1-4> - 0) CO (0 -H + J Qi

P

H O

<U Vi -Ω Oi --- Ό ft N - σ> <m>

CQ w 4J

CV O

Ό J. T3 H

tuH'TFic'vooooiniflin n O dl c m tr o co ot η cm in r *>

Λ 01 0)> H H

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&> Ot H C

W 0) CQ g O

tu a ni ή h oooooHineoooeon

-¾ ^ \ ΟΟΟΟΗΙΠΝΗΟίΝΓ'Γ-Η H

® H Dl | J ni HH CM H CM H H

(d · η 14 Vi> 4-1> -Μ -0 ·

Ό Ό H

(0 0) IC N Cl

0) * H CM

Q) 4) fl H

03 O H CM

Id X S5 oi and o>

4-1 ICOCHUKOUQOCOM

o CMtøtOtOvOOOOCMOcMM '

We CPHHHHSOVOVOHtOHO

B OfMfMCMCMHHHCMHCMH

assssww coswaw 33 DK 175855 B1 η o «n h co o n.

CO V> W 01 CO HH

H

«COOOOI ^ W HO <J

vo vo σ \ o in «· o O

CO <M CO CO n N} Q

"tea

ΟΓ ^ ΟΗΗΗΟ CO O S

te »s K K ^ K S K

OHn ^ incoro nn - H o te \ o - - ...------ te

CO

^ 55 + j. **

tf dP

ΜθίθΡ * Ρ '\ θηΗΝ <Λΐη ^, <ΛΐηηΗ in g h • PoHHrOH ^ COlOlOH ^ flOHt ^ CO ON ir £ V-l Η H cH · 03

> £ 2 ΐ B

--- jj: H>> <ur, Ti ° J3 inoooininoooininot ^ ininoo y ti u tf cnr ~ r ~ ®oioomoo »r ^ Hcor ^ oiH» P'S 53 H HHCOCOCO hhh u go a 1¾ g vooincoHoinoooooonotnino ^ 01 (no » <tfioioin \ ooino cocoo \ Hc *) cor «in S - r>

Η H Γ0 H CO CO Γ0 HHCO H> § · J

___fe s | 4

. Η H O I

Ό JJ 4J <H -H ω i

O c S M

Z> <JM fU H EO .C Hl

co co æ coco co cocoj <w p -M

co co ro roes ro ro ro Bi, ¾ 9 S

HHH HH H HH Η Ό «* H

t / 1 V) 10 to to WW w M · TJ ffi te te, te te te te MQ / ^ «ζ σ so uKxwoia ^ -h 3 r · r- c * c * co f ~ r ~ r- r ^ r »mu _u m ro ro ro ro ro ro ro ro -ro ro ro SSe ..

HHHHH HHHHHHH _p r; > p h

0 <P <O * fe l /> fe PU fe fe fe fe fe fe H 4J B

te te te te te te te te te te te «m (D (Q 03 ho waw> pp> <coxzsoifc.o6.« {2 «^ o> io« eiovoio «oioior ^ icu> ioiovovoioio c iH Si.

UJHHHHorrorororoHorrorororororo g J3 .µ g

HorrororHHHHHroHHHHHHH £ O QJ -H

ESSSESWWWW & iEWWWWWWW I ^ + J + j

Q a a p p>>>> S- H>>>>>>> I ro-5 S

ιοοορ> Γ ^ ιοιοιοιο \ οσιιονονονοιοιονο ω on U tn lOVOlOHHHHHHCOVOHHHHHHH nj S) ΐ Q)

HHHHHHHHHHHHHHHHHH ^ M s H

<WWSSUOOOWXOOOUOOU

ft * +

DK 175855 B1 I

B. The washability test was repeated at 25 ° C with the same wash- H

agent containing some of the PB92 protease mutants. IN

PB92 protease was used again for reference. The remaining

test conditions were the same as described above. H

The results are shown in Table 2. H

Table 2 I

^ B

Washability of PB92 protease mutants at 25 ° C in STPP

10 dime of washing powder in relation to the washing ability of PB92 pro- H

tease (%).

Protease Washability (%) H

Detergent concentration in the wash water H

15 _; _ 4 q / 1_7 q / 1_ B

M216S 90 80 B

M216Q 95 80 B

N212D 250 135 B

S160D_185_120_ B

B

C. The washability of the proteases was also determined in a B

European washing powder containing phosphate bleach- B

agent in a Launderometer at 25 ° C and 40 ° C. PB92 pro- B

tease was used again as a reference. The other test B

Twenty-five opinions were the same as described above. Resul- B

the tates are shown in Table 3. B

DK 175855 B1 35

Table 3

P O

Washability of PB92 protease mutants at 25 ° C and 40 ° C in a European washing powder not containing phosphate-5 bleach, 1 relative to the washability of PB92 protease (%).

Protease Washability (%)

Detergent concentration in the wash water 4 g / l 7 g / l 4 g / l 7 g / l

0 O

10 _ temperature 25 C_ temperature 40 C

M216S 80 85 100 90 M216Q 80 80 120 100 N212D 170 105 200 75 S160D_Γ70_105_230_165 15

Example 2 The PB92 protease mutant M216S was tested for stability during storage in the powdery detergent IEC described in Example 1. The stability of storage was tested in climates at 30 ° C and 80% relative humidity (RH). The protease for this experiment is encapsulated as follows:

A mixture was prepared containing (in 25% w / w): 2% purified protease, 50% nonionic surfactant (ethoxylated C14-C18 alcohol at 50-80 E.0 units), 5% TiO2, 3 -10% CaSO4.2H2O and

Na 2 SO 4 to 100%. The mixture was heated to 65-90 ° C and cooled to room temperature. The obtained solid mixture was ground to particles. Particles with a diameter of 0.5 to 1 mm were screened and used for the storage tests in detergents.

3.5 g of wash powder IEC containing mutant M216S at a concentration of 6140 ADU / g detergent

DK 175855 B1 I

preserved in 18 ml vials. In comparison, I

PB92 protease was maintained under the same conditions. After 2,

At 4, 5 and 6 weeks, the residual activity of the proteases was measured. Re- I

the results are shown in Table 4.. IN

Table 4

Residual activity of PB92 protease and its mutant M216S, I

after storage (for weeks) at 30 ° C and 80% RH in the wash.

10 powder

Protease Residual Activity (%) I

_0 weeks 2 weeks 4 weeks 5 weeks 6 weeks I

PB92 protease 100 25 5 3 2 I

M216S_100 68_31_20_11 I

Example 3 I

PB92 protease and various PB92 protease mu- I

aunts tested in terms of storage stability in I

20 a washing powder, in the test for stability during storage.

In the case of the proteases 1, an encapsulated form was used. H

The protease products were prepared by mixture H

of the following components at 80 C.

37 DK 175855 B1

Component Weight% AE1 50

TiO2 2 protease (CEP) 7 5 PVPa 1.5 BHT * 1

Na 2 SO 4 residue 1 AE = C 14 -C 18 alcohol polyethoxylate. The alcohol was ethoxylated with 50-80 ethylene oxide (EO) groups.

* PVP = polyvinylpyrrolidone K17 * BHT = 2,6-bis (t-butyl) -4-methylphenol.

This mixture was encapsulated by prilling, essential as described in British Pat.

1603640. The particle fraction having a particle size of between 0.3 and 0.8 mm was used to determine the storage stability of the proteases. The encapsulated protease (140 mg) was mixed with ALLR base (6.4 g) and 20 sodium perborate tetrahydrate (0.6 g). [ALL is a registered trademark of Unilever N.V.]. The ALL base powder used did not contain enzymes or bleaching agents.

The enzyme / detergent / sodium perborate tetrahydrate mixture was incubated at 30 ° C and 80% relative humidity (RH). Table 5 gives the residual activity after storage for the specified period.

in

I DK 175855 B1 I

IN

In Table 5 I

H Residual activity of PB92 protease and some mutants thereof I

After storage (for weeks) at 30 ° C and 80% RH 1 a diaper

In 5 parts containing detergent powder

Protease Residual Activity (%) I

I _0 weeks 1 week 3 weeks 5 weeks I

PB92 protease 100 51 25 15 I

I M216Q 100 94 84 52 I

I 10 M216S 100 89 83 50 I

I S160D 100 47 20 9 I

I N212D_100 59_31_19 I

In Example 4 I

I 15 · I

In PB92 protease and various PB92 protease mu- I

Aunts were encapsulated according to the procedure described in I

Example 3. In this example, 70 mg of In-I was mixed

In capsule protease having a particle size of between I

However, in 0.3 and 0.9 mm with 3.2 g of ALL and 0.3 g of Na-I

In triumper borate tetrahydrate. The samples were stored at I

lo I

30 C in 18 ml vials in a vacuum desiccator. vacuo

I was applied (25 mm Hg) for the first 3 days of storage I

In the period. IN

I 25 When vacuum was applied, the transport speed increased

In the heat of water vapor, so that the system reached its I

In equilibrium at 80% RH faster than the system does without I

In applying vacuum. The relative humidity of 80% I

I was established with a saturated potassium bromide solution. IN

Table 30 of Table 6 gives the residual activities after the other

During given storage period (for weeks). IN

DK 175855 B1 39

Table 6

Residual activity of PB92 protease and some mutants thereof after storage at 30 ° C and 80% RH in a bleach-containing detergent

Protease Reactivate (%) _0 weeks 1 week 2 weeks 3 weeks PB92 protease 100 27 22 14 M216Q 100 63 53 44 10 M216S 100 55 49 31 S160D_100_23_18_13

Example 5 Det. the following liquid detergent was prepared:

Component Weight% linear C1-C13 alkylbenzene sulfonic acid 12 C13 alcohol polyethoxylate, 8 EO 13 20 lauric acid 8 oleic acid 4 triethanolamine 6 1,2-propanediol 6 ethanol 5 sodium citrate 4 diethylenetriamine pentaacetic acid 0.3 calcium formate 0.12 sodium formate 1 borax 1.9 NaOH, 25% w / w solution to pH 11.2 water the residue PB92 protease and various PB92 protease mutants were added to this mixture in an amount

DK 175855 B1 I

reaching a starting protease concentration of 0.13 wt / H

weight%.

The protease stability (1% residual activity) is H

voted after storage of the protease-containing bian- H

5 at 37 ° C for the specified number of days. The results are I

shown 1 Table 7. I

Table 7 I

Residual activity of PB92 protease and some mutants thereof H

after storage at 37 ° C a liquid detergent

Protease Residual Activity (%) H

_0 days 1 day li days 15 days 21 days H

PB92 protease 100 23 10 5 3 H

15 S160D 100 57 30 14 8 H

M216Q 100 59 32 18 9 B

M216S 100 45 18 10 5 H

N212D_100 38_14_9_4 I

Example 6 H

PB92 protease and some mutants thereof formula H

read as follows. With each protease, an H was prepared

mixing the following components:! H

DK 175855 B1 41

Component weight%

Amylogum CLS 45

Sucrose 23 5 Sorbitol 17

Glycerol 4 paraffin oil 3

NaH 2 PO 4 0.5

Protease (CEP) 5.0 10 PVP K17 1.5

Ti02 1

From these mixtures, granules were prepared, essentially following the procedure described in Example 5 and U.S. Patent No. 4,242,219, except that: 1). the mixture described in this example is replaced by the above mixture; 2) openings having a diameter of 0.7 mm in place of 1.0 mm were used; 3) the granules were not coated.

These granules (140 mg) were mixed with ALL base (6.4 g) and sodium perborate tetrahydrate (0.6 g) and placed in 36 ml vials.

The protease stability (in% residual activity) was then determined after storing the vials at 25 ° C and 80% RH for the indicated number of weeks. The results are shown in Table 8.

I DK 175855 B1 I

I 42 I

In Table 8 I

I Residual activity of granules of PB92 and some mutants I

Therein, after storage at 30 ° C and 80% RH 1, a bleach

In medium detergent____ I

Protease Residual Activity (%) I

I _0 weeks 1 week 3 weeks 5 weeks I

I PB92 protease 100 45 29 17 I

I M216Q 100 93 66 45 I

I 10 M216S_100_90_70_41 I

Example 7 I

Prilled products of PB92 protease and some mu- I

In 15 aunts thereof, were prepared and mixed with detergent

In part and bleach as described in Example 3. I

In the storage stability of these samples (in% I

In residual activity) were determined at 30 ° C and 60/80% RH (Shift I)

In tea 60% RH for 12 hours and 80% RH for 12 hours). These I

In 20 results are shown in Table 9. I

DK 175855 B1 43

Table 9

Residual activity of purified products of PB92 protease and some mutants thereof after storage at 30 ° C and 560/80% RH

Protease Residual activity (%) _0 weeks 1 week 3 weeks 5 weeks PB92 protease 100 70 34 15 M216Q 100 98 88 67 10 M216S 100 93 87 48 SI6OO 100 67 35 10 N212D_100_75_53_26

Example 8 15

Granules containing PB92 protease and some mutants thereof were prepared and mixed with detergent and bleach as described in Example 6.

The storage stability of these samples (in% 20 residual activity) was determined after incubation for the indicated time at 30 ° C and an RH held alternately at 60% for 12 hours and at 80% for 12 hours. The results are shown in Table 10.

DK 175855 B1 I

H 44 I

Table 10 I

Residual activity of granules of PB92 and some mutants I

Η I

thereof after storage at 30 ° C and 60/80% RH

H 5 Protease Residual Activity (%) I

I _0 weeks 1 week 3 weeks 5 weeks I

I PB92 protease 100 54 39 28 I

M216Q 100 93 81 67 I

M216S_100_99_87_72 I

I 10 I

Example 9 I

In PB92 protease and some mutants thereof, I tested

in terms of storage stability in a bleaching agent

In 15 containing washing powder at 30 ° C and 80% RH. For this purpose I

In search, the proteases were used in an encapsulated form. Each I

The protease was encapsulated as follows:

At 80 ° C a mixture was prepared with the following mixture.

In composition:

I 20 I

In Component weight% I

I Nonionic surfactant1 45 I

I Ti02 2 I

I Protease (CEP) 10 I

I 23 Na 2 SO 4 residue I

I 1 Non-ionic surfactant = C 4 -C 4 island alcohol-I

In polyethoxylate (50-80 EO groups) I

The above mixture was allowed to cool to 1

At room temperature. The solidified mixture was ground to I.

In smaller particles. Particles of 0/3 to 0.8 mm were screened

In out and used for the storage experiment. IN

For the storage experiment, 140 mg of each H was mixed

In Encapsulated protease ed 6.4 g ALL-base washing powder and H

0.6 g of sodium perborate tetrahydrate. The ALL base powder contained neither enzyme nor sodium perborate. The ALL-base / protease / sodium perborate / tetrahydrate mixtures were incubated at 30 ° C and 80% RH.

After storage for 0, 1, 2 and 4 weeks, the protease stability was determined as percent residual activity for each protease. The results are shown in Table 11.

Table 11 10

Residual activity of PB92 protease and some mutants thereof after storage at 30 ° C and 80% RH in a bleach-containing detergent powder

Protease Residual Activity (%) 15 _ 0 weeks 1 week 2 weeks 4 weeks PB92 protease 100 61 36 12 [S160D, M216Q] 100 78 58 35 rS160D, M216S1 100_86_68_39 20 Example 10 PB92 protease mutants were tested by a wash test under essentially the same conditions as described in Example 1 except that 0.375 g of liquid water powder of the following composition was added to 250 ml of water at 5 ° GH in the Launderometer vessel.

DK 175855 B1 I

46 I

Component guard% I

Lauric Acid 8 I

Oleic Acid 4 I

Linear C1-C13-alkylbenzene-I

Sulfonic acid 12

C13 alcohol polyethoxylate, 8 EO 13 I

Triethanolamine 6 I

1,2-propanediol 6 I

Ethanol 5 I

Sodium hydroxide, 45% w / w% 4 I

Sodium citrate '4 I

Water to 100 I

(pH of the wash water 7.2)

15 The washability of the various proteases in this I

liquid detergent was determined in a Launderometer at I

25 ° C for 30 minutes. After washing, the reflectance was measured

of the test garments as described in Example 1. Washability I

of the mutant proteases was determined as described in Example I

The results are shown in Table 12. I

---- - - -η DK 175855 B1 47

Table 12

Washability of PB92 protease mutants at 25 ° C in a liquid detergent_5 Protease Washability Specific activity relative to PB92 protease (%) _ 212D + 100 160D + 73 10 S160G, N212D + 76 M216S 0 40 M216Q_0_37_ 0: 100% ± 20 % washability relative to PB92 protease 15 (retained washability).

+:> 120% washability relative to PB92 protease.

<80% washability relative to PB92 prptease.

The washability of the various proteases was determined in the commercially available liquid detergents.

TideR, WiskR and ArielR. The stainless steel containers contained either 0.375 g Tide in 250 ml water at 5 GH, 0.375 g Wisk in 250 ml water at 5 ° G or 1.25 g Ariel in 250 0 0 ml water at 15 GH. Washability was determined at 25 ° C or 25 ° C. The other conditions were the same as described in Example 1. The results are shown in Table 13.

DK 175855 Bl | IN

Table 13 j I

Washability of PB92 protease mutants in Tide, Wisk and H

Ariel at 25 ° C and 40 ° C

5 ______ I

Protease Tide Wisk Ariel Η

25 'C 40 * C 25 * C 40 * C 40 * C I

N212D + + + · + 0 I

10 S160D ++++++

M216Q 0 + 0 + + I

M216S ND 0 0 0 0 I

S160Q ....

S160N - - -. 0 15 S160K .... _

S160A .... I

S160D, M216Q + + + + + H

S160D, H216S 0 - + + 0 I

M117L, M216Q ND - ND - 0 I

20 M117L, M216S ND - ND - I

ND s not determined H

0: 100% ± 20% washability relative to PB92 protease. IN

25 +:> 120% washability 1 relative to PB92 protease. IN

<80% washability relative to PB92 prptease. H

Example 11 H

The washability of PB92 protease mutants was determined H

by a statistical full-scale washer test at TNO, H

Delft, The Netherlands (Cleaning Techniques Research Insti-

tute). The washability of these mutants was compared to H

the washability of PB92 protease. H

---- DK 175855 B1 'i t i

All proteases were dosed in 1 IEC detergent on | based on protein weight (0.007 w / w%). Based on {activity, these doses gave: PB92 protease 1460 ADU / g detergent 5 M216S 679 ADU / g detergent M216Q 479 ADU / g detergent S160D 1080 ADU / g detergent N212D 1455 ADU / g detergent

For each detergent protease mixture, 8 10 tests were performed at 40 ° C in identical AEG Turnamat twinup washers. 170 g of detergent were used in each washing machine. During the tests, the detergents tested were used in such a way that each powder underwent the same number of wash cycles in each machine.

During the tests, tap water was normally used as available in the city of Delft with the following intermediate specifications: alkalinity (M): 2.2 mmol / 1 hardness (H): 1.6 mmol / 1 (9 ° GH)

2 ° temperature: 20 ° C

Removing dirt and dye from test clothes

During each test run, 6 samples of three 25 types of cotton EMPA dirt (Nos. 111, 116 and 117) were washed together with the soiled laundry. The removal of dirt and dye from the artificially soiled test cloth was determined by passing the blue light tristimulus perpendicular to the test cloth. The amount of light returned from the test garment in an amount of 45 was measured, in accordance with I EC Publication 456, the remission value for magnesium oxide was set to 100. The higher the remission value, the better the washing process for a particular kind.

DK 175855 B1 I

dirt. IN

Load of the washing machine I

5 The washing machines were filled to a load of

4.75 kg consisting of test clothes and laundry, I

been dirty in normal use.

the laundry consisted of: I

6 tea towels I

4 pieces of underwear I

4 sheets I

4 pillowcases. IN

If necessary, clean sheets and I

cushion covers to achieve the required load. IN

The laundry was carefully selected for each wash.

process. Each piece of clothing selected for one of the ma- I

the rails had an equally dirty counterpart which was washed

in one of the other washing machines. In this way you were

the dirt load the same during each process. IN

20 I

Parameters of the washing process I

Program_40 C_

25 water flow during main wash 19 1 I

Time to reach the highest temperature 13 min I

Highest temperature 43 C I

Washing time 45 min

Water Intake 71 I

30 Temperature after dilution of wash water 30 C I

Drain 17 1 I

5 Rinses with approx. 17 liters of cold water each 51 B 175

The mean values for the emission (V) and ratio (R) 'were determined after 8 washing tests. The results of two independent experiments are shown in Tables 14A and 14B.

Table 14A

Removing artificial dirt

Protease EMPA Sample No. Total _in · 116_117_

_V RVRVRV R

PB92 protease 49 1.00 44 1.00 56 1.00 149 1.00 M216S_49 1.00 46 1.05 58 1.04 153 1.03

Table 14B

Removing artificial dirt

Protease EMPA Sample No. Total 20 _111_116_U / 7_

_V RVRVRV R

PB92 protease 41 1.00 35 1.00 46 1.00 123 1.00 M216S 40 0.96 33 0.94 42 0.91 115 0.93 M216Q_42 1.01 35 0.99 43 0.94 120 0, 98 25 In Table 15, the ratios (R) obtained from the statistical full-scale washer tests were divided by the corresponding ratio (R ') calculated from the washability values relative to PB92 protease obtained from the Laun derometer washing test under the same conditions (10 g / l IEC , the test clothes EMPA 116 and 117, washing time 30 minutes

ISLAND

ter, temperature 40 C).

DK 175855 B1 I

Table 15 I

Correlation between full-scale washer testing and Launde- I

room cleaning test_ I

5 Protease ____ R / R1_ I

PB92 protease 1,001 I

M216S 1.18 I

M216Q 1.10 I

S160D 1.02 I

N212D_0.98_ I

* Rate. definition I

The values of R / R1 for mutant protease approach I

1.0 shows correlation between full scale machine tests and H

15 Launderometer Tests. IN

Example 12 I

FIG. 2A and 2B show the washability in 4 g of IEC / 1 of I

20 different PB92 protease mutants according to I

with the tests described in Example 1 relative to na-I

turbulent PB92 protease as a function of their spread

get some activity. The figures in the figures refer to those I

the following mutant proteases:

- ^ DK 175855 B1 53

1 PB92 protease 11 K216P

2 H216A 12 M216T

3 M216C 13 M216W

4 K216S 14 M2161 5 5 M216L 15 M2166

6 M216E 16 M117L, H118D

7 M216K 17 M117L, M216Q

8 M216H 18 M117L, H118D, M216Q

9 M216N '19 M117L, M216S

10 101.2160 31S2S9K

21 M169S 32 W235R

22 M216Y 33 H243R

23 M169I, M216S 34 H243R, S259K

15 24 M216-OX '35 D175N

25 N212S 36 E134K

26 N212D 37 W235R, S259K

27 S160G, N2120 38 W235R, H243R

28 L211Y 38 S259K

20 29 L211Y, N212S 40 T207K

30 A166D, M169I

51 S160I

41 S160N 52 S160G, M216S j 42 S160G 53 S160G, M216Q!

25 43 S160P 54 S160L

44 S160T 55 S160Y

45 S160C 56 S160D, M216S

46 S160Q 57 G116V, S126V, P127E, S128K

47 S160D 58 G116V * S126L, P127N, S12BV

30 59 G116V, S126L, P127Q, S128A

48 S160K

60 G116V, S126V, P127K

49 S160R

50 S160A

----- - - _j

I DK 175855 B1 I

I I

I 61 S126M, P127A, S128G I

I 62 G116V, S126Y, P127G, S128L I

I 63 G116V, S126N, P127H, S128I I

I 64 G116V, S126H, P127Y I

I 5 65 G116Vr S126R, P127S, S128P I

I 66 G116V, S126F, P127Q I

I 67 G116V, S126G, P127Q, S1281 I

I 68 G116V, S126F, P127L, S128T I

I 69 G116V, S126Q, P127D I

In 10 Pig. 3 shows the washability in 7 g IEC / 1 for difference I

equal PB92 protease mutants in the tests described in Ex. I

In Example 1 relative to natural PB92 protease as one

In function of their specific activity. The figures in FIG

In pure, refer to the same mutant proteases as the numbers in Figures 2A and 2B.

All publications (including patent applications)

As mentioned in this specification, the prior art discloses

In nod upon which the present invention is based. All pubs

In equations are incorporated herein by reference in the same

To 20 extent, as if each publication specifically and j I

I was individually designated as being incorporated herein

You by reference. IN

Although the invention is described above I

In respect of some details by way of example,

In order to make it clearer and easier to understand, I

It is clear to those skilled in the art that many changes and modi I

Fixations can be performed without going beyond the scope of the invention

the idea and framework. IN

H

Claims (8)

A mutant protease for use in detergents and having at least 70% homology to the amino acid sequence of PB92 serine protease with the sequence 5 H * 1J-A-Q-S-V-P-H-G-I-S-R-V-Q-A-P-A-A-H-N ~ R-G-L-T-G-S-G-. VAVLDTGI-STT-HPDLNIRGGAS ~ ~ GEPSTQDGNG FVP-HGTHVAGTIAALNNSIGVLGV-APNAE-LY-AVKVLGASGSGSVSSIAQGLE WAGNNGMHVANLSLGSPSPSA-TLEQAVNSATSRGVLVVAA-5-GN ~ SGAGSISYPARYANAMAVGAT-D-Orn-NNRAS- fsqygagldivapgvnvosty-pgstyaslng- TSMATPHVAGAAALVKQKNPS-WSNVQIRNHL-10 KNrATSLGSTNLYGS-6-LVHAEAATR-COOH Which differ by at least one amino acid substitution at a selected site corresponding to position 212 in said PB92 protease from asparagine to aspartic acid or glutamic acid; and with improved washing performance and / or improved stability to PB92 protease. The mutant protease of claim 1, wherein said 20 substitutions correspond to [N212D, S160G]. The mutant protease of claim 1, wherein said enzyme is a PB92 protease mutant. 4. Protease for Use in Detergents and with the Sequence 25 fkN-AQSVPGGGVGGGVGGVGGVGGVGGVGGVGGVGGVGGVGGVLGVGGVGGVGGVGVGGVGGVGGAPGVGGVGGVGGAPVGGVGVGGVLGVGGAPLVGGVLGVGGVLGAP - GNSGAGSISYPARYANAMAVG-ATDQNNNRASfsygygagldiv-apgvnvqstypgstyasldgT-SMAT — PHVAGAAALVK — O — KNPSHSNVQIRNHL-30. _K-N-T-A-T-S-I-G-S-R-K-L-Y-G-S-G-L-V-N-A-E-A-A-T-R-COOH. "* '1 _______ ___J -I DK 175855 B1 I A DNA sequence encoding a protease such as H defined in any one of claims 1 to 4. H An expression vector comprising the DNA-H sequence defined in claim 5. H A prokaryotic host strain transformed with an H expression vector according to claim 6. H The transformed prokaryotic host strain of H claim 7, wherein said host strain is Bacillus. H The transformed prokaryotic host strain according to H 10 claim 8, wherein said Bacillus is an alkalophilic Bacil-H loop. H The transformed prokaryotic host cell of H claim 9, wherein said alkalophilic Bacillus is Bacillus H nov. spec. "PB92 or a mutant thereof. H Transformed prokaryotic host cell according to H of claim 10, wherein said host strain prior to transformation was at least substantially unable to produce extracellular proteases. Process for producing a protease H 20 as defined in any one of claims 1 H to 4, characterized in that one cultivates an H microorganism or a host strain transformed with an expression vector comprising a DNA sequence encoding for said protease, thereby forming said protease H 25 and by recovering said protease. H A detergent comprising one or more proteases as defined in any one of claims 1 to 4. Use of one or more proteases such as H 30 defined in any one of claims 1 to 4 in a detergent. H 57 DK 175855 B1 Use of one or more proteases as defined in any one of claims 1 to 4 in a washing process. ------ j I DK 175855 B1 II FIGURE 1A I i — i, H ar I east uj I ® \ I i CC IIU \ CO II LLI - // \ m I f Q> 00 -QI / co in i co I is s'? tei »II \ \ * -« Q. CO / -1- CO _ I \\ ° / x ° ω 2 I v ^> / 5 -S ° - _ II / ω 2 E / lj ^ ----- - \ I * 3 o § / Λ II x “Lii 2 // \ IIS · H ^ 3“ i [S 15 II o «i" e “ife." ®. II o E co \ \ o S00 / Φ ; W / I a BI - 1 * 2 I rt - c EJ ^ XI co E x co c HI ω IX) IEI <I ► I --- ^ DK 175855 B1 FIGURE IB i * Θ EcoRl ^ y / proteaseS ^ EcoR ] Hlndm ^ // ~ YI M13M1 I l 8.6kb J x EcoRI / HIndlll ds DNA ss DNA I__1 ^ Annealing ♦ ollgonucleotld {- * -) EcoRl - Hindmvy ^^ Toteas ^ X ^ EcoRI I (gap-duplex II _I polymerase T4-li gas f EcoRl _ __ Hin din ------- EcoRl EcoRl __mutation // protease '^ s ^ \ ^' '^^ / protease ^ i ^ k ^ II II I M13M1mut I Il il l 8 6kb J ^ »^ xTransfectlon - --- ^ to E.coll BMH7 1-1 BmutL and JM105 ____j DK 175855 B1 I FIGURE 1C EcoRI I / ^ 5roteasem \ I / X ^ EcoRI I / pM58 VI 1 6'3kb II \\ NE ° / \\, ° ^ l // x EcoRI I ligation V EcoRI I EcoRI m srs I I. pM5.8 ^ .Eh ° II M13M1 1 IU 4'6kb I l 8.6kb I x EcoRI .. x EcoRI I, _ligation_, transformation of B. subtllisT In DB 104. selection on fEcoRl In neoR, prot® ___. ,. I ^^^^ mutation / ^ protease ^^ w EcoRI II pM58m \ I It 5r2kb I \ Wo ori // I DK 175855 B1 - - FIGURE 2A AK 5 »i -: - η i» - 2. a N * 200 - 0 3, β) '27 4J S 10-! ... 1 ia- * \ * * 100- 4 1 * - 00- ... 23 24 25' i S "» SI 60 - 20 19 32 I 28 2 3 31 «- O 21 35 31 a- / 5 15 1J11 M-1-YYY I yy I yy] 0 20 40 60 80 100 120 SPECIFIC ACTIVITY relative to PB92 protease {%) ______ _____i I DK 175855 B1 II FIGURE 2B II Λ II k II β 260 -1 --- I li II · 240 - 57 60 III i ^ 66 I II®- 47 II * 200 - 59 61 II h 180- -HI 2 58 II 3 160 - I Æ MIO tf wm - 1A 90 I .H '40 - I In 65 I 0 IS 120- IMI \ 45 I «100-55 41 42; s * 6249 s51 0 III 60 - 45 II 44 II> 40- II 20 - 37 «54 40 II 36 1 I β_ I 0“ i-1-1-1-1-1 IIIIIII 0 20 40 60 80 100 120 II SPECIFIC ACTIVITY relative to PB92 protease (*) H ----- - DK 175855 B1 FIGURE 3 Λ R \ 260 n- »• 2« - [I 22. i (4 i * 200- 59 61 I 0 1 1 1 3 1S> - e i 1i0 '47 «2 .η · 140-0 a. 120- \ 57 67 0 p 100- 1 K 66 23 i ^ 104 I 8. O 19 a „h 56 17 ° 5026 i (ΟΙ 46 I · * 55 20- * 0- | -1-1-1-1--1-1-1-1-1-1-1 0 20 40 60 80 100 120 SPECIFIC ACTIVITY relative to PB92 protease (%) ______ _i I DK 175855 B1 II FIGURE 4 I i-Pre II -100 II ................. .......... ATGAAGAAACCGTTGGCCAAAATTCTCGCAAGC in KKKPLGKIVAS in i-Pro I -90 I ACCGCACTACrcATTTCTGTTGCTTTTAGTTCATCGATCGCATCGGCTGCTGAAGAAGCA in TALLISVAFSSSIASAAE EA II AAAGAAAAATATTTAATTGGCTTTAATGAGCAGGAAGCTGTCAGTGAGTTTGTAGAACAA in KEKYLIGFNEQEAVSEFVEQ I "50 in GTAGAGGCAAATGACGAGGTCGCCATTCTCTCTGAGGAAGAGGAAGTCGAAATTGAATTG VEANDEVAILSEEEEVEIEL II CTTCAIXIAATTTtlAAACGATTCCTGTTTTATCCGTTGACnTAAGCCCAGAAGArGTGGAC in LHEFETIPVLSVELSPEDVD I -10 II GCGCTTGAACTCGATCCAGCGATTTCTTATATTGAAGAGGATGCAGAAGTAACGACAATG ALELDPAISY1EEDAEVTTN I I I-Mature H 1 + 1 10 20 I GCGCAATCAGTGCCATGGGGAATTAGCCGTGTGCAAGCCCCAGCTGCCCATAACCGTGGA HI AQSVPWGISRVQAPAAHNRG I 30 40 II TTGACAGGTTCTGGTGTAAAAGITGCTGTCCTCGATACGGTTGTCCTCGATACGGTTGTCCTCGATACGGTTGTCCTCGATACC I 30 60 II TTAAATATTCGTGGTGGCGCTAGCTTTGTACCAGGGGAACCATCCACTCAAGATGGGAAT LNIRGGASFVPGEPSTQDGN I I 70 80 GGGCATGGCACGCATGTGGCTGGGACGATTGCTGCTTTAAACAATTCGATTGGCGTTCTT HI GHGTHVAGTIAAL NNSIGVL ----------- 1 DK 175855 B1 FIGURE '4 (cont'd) 90 100 110 120 GGCGTAGCACCGAACGCGGAACTATACGCIGfrTAAAGTATTAGGGGCGAGCGCTTCAGGT GVAPNAELYAVKVLGASGSG TCGGTCAGCTCGATTGCCCAAGGATTGGAATGGGCAGGGAACAATGGCATGCACGTTGCT SVSSIAQGLEWAGNNGHHVA 130 1 * 10 AATTTGACTTTAGGAAGCCCTTCGCCAAGTGCCACACTTGAGCAAGCTGTTAATAGCGCG NLSLGSP SPSATLEQAVNSA ISO 160 ACTTCTAGAGGCGTTCTTGTTGTAGCGGCATCTGGGAATTCAGGTGCAGGCTCAATCAGC TSRG.VLVVAASGNSGAGSIS 170180 TATCCGGCCCGTTATGCGAACGCAATGGCAGTTCGGAGCTACTGACCAAAACAACAACCGC 'YPARYANAMAVGATDQNHNR 1 I9O 200 GCCAGCTTTTCACAGTATGGCGCAGGGCTTGACATTGTCGCACCAGGTGTAAACGTGCAG ASFSQYCAGLDIVAPGVNVQ 210220 AGCACATACCCAGGTTCAACGTATGCCAGCTTAAACGGTACATCGATGGCTACTCCTCAT STYPOSTYASLNGTSMATPH 23Ο 2l "0 GTTGCAGGTGCAGCAGCCCTTGTTAAACAAAAGAACCCATCTTGGTCCAATGTACAAATC VAGAAALVKQKNPSWSNVQ I ψ 25Ο 260 CGCAATCATCTAAAGAATACGGCAACGAGCTTGGGAAGCACGAACTTGTATGGAAGCGGA RNHLKNTATSLGSTNLYGSG 270 CTTGTCAATGCAGAAGCGGCAACACGCTAA LVNAEAATR End ___ Å