FIELD OF INVENTION

The present invention relates to amylases having improved dishwashing and/or washing performance.

5BACKGROUND OF THE INVENTION

For a number of years α-amylase enzymes have been used for a variety of different purposes, the most important of which are starch liquefaction, textile desizing, starch modifi¬ cation in the paper and pulp industry, and for brewing and 0baking. A further use of or-amylases, which is becoming in¬ creasingly important is the removal of starchy stains during washing and dishwashing.

Examples of commercial αf-amylase products are Termamyl ^® , BAN ^® and Fungamyl ^® , all available from Novo Nordisk 5A/S, Denmark. These and similar products from other commercial sources have an acidic to a neutral pH optimum, typically in the range of from pH 5 to pH 7.5, which means that they do not display optimal activity in detergent solutions owing to the alkaline character of the detergents. 0 It is an object of the present invention to provide novel cv-amylases with improved performance in alkaline solu¬ tions, especially in alkaline detergent solutions.

SUMMARY OF THE INVENTION

The present invention provides α-amylases with a very 5 high specific activity at pH 8-10 and at temperatures of from 30°C to around 60°C, conditions normal in detergent solutions.

Accordingly, the present invention relates to an α- amylase having a specific activity at least 25% higher than the specific activity of Termamyl ^® at a temperature in the range of θ25°C to 55°C and at a pH value in the range of pH 8 to pH 10, measured by the o;-amylase activity assay as described herein.

BRIEF DESCRIPTION OF DRAWINGS

The present invention is further illustrated with reference to the accompanying drawings, in which

Fig. 1 shows the relation between pH and the ot- 5 amylase activity of a novel amylase (obtained from Bacillus strain NCIB 12289) , determined as described in Example 2.

Fig. 2 shows the pH profile of an α-amylase obtained from Bacillus strain NCIB 12512 (I) , of an α-amylase obtained from Bacillus strain NCIB 12513 (II) and of Termamyl ^® (III) 10determined at 55°C in the pH interval of from 4 to 10.5, the test being performed as described in Example 3.

Fig. 3 shows the temperature profile of an α-amylase obtained from Bacillus strain NCIB 12512 (I) , of an α-amylase obtained from Bacillus strain NCIB 12513 (II) and of Termamyl ^® 15 (III) determined at pH 10.0 in the temperature interval of from 25°C to 95°C, the test being performed as described in Example 3.

Fig. 4 shows the RSF-rating - removal of starch film from dish- and glassware, as a function of the dosage of a 20novel α-amylase (obtained from Bacillus strain NCIB 12289) at 55°C, the test being performed as described in Example 4.

Fig. 5 shows the RSF-rating - removal of starch film from dish- and glassware, as a function of the dosage of a novel ce-amylase (obtained from Bacillus strain NCIB 12512) at

2545°C (•) , at 55°C (*) and at 65°C (x) , the test being performed as described in Example 4.

DETAILED DESCRIPTION OF THE INVENTION

The -.-Amylases of the Invention

One embodiment of the present invention provides an 3oc.-amylase having a specific activity at least 25% higher or at least 35% higher or at least 45% higher or at least 55% higher or at least 65% higher or at least 75% or at least 25-75% higher than the specific activity of Termamyl ^® at a temperature in the range of 25°C to 55°C or at a temperature in the range

of 25°C to 35°C or at a temperature in the range of 35°C to 45°C or at a temperature in the range of 45°C to 55°C and at a pH value in the range of pH 8 to pH 10 or at a pH value in the range of pH 8 to 8.5 or at a pH value in the range of pH 8.5 to 59.0 or at a pH value in the range of pH 9.0 to 9.5 or at a pH value in the range of pH 9.5 to 10.0, measured by the α.-amylase activity assay as described herein.

It has surprisingly been found that preferred novel α-amylases of the invention may be characterized by having a 0 specific activity at least 25% higher than the specific activity of Termamyl ^® at any temperature in the range of 25°C to 55°C and at any pH value in the range of from pH 8 to pH 10, measured by the ce-amylase activity assay as described herein.

Compared with known o.-amylases it is very remarkable 5 how well the α-amylases of the invention perform at pH 10; accordingly in a preferred embodiment the α-amylase is charac¬ terized by having a specific activity at least 25% higher than the specific activity of Termamyl ^® at any temperature in the range of 25°C to 55°C and at pH 10, using the o.-amylase 0 activity assay as described herein.

In another aspect the invention relates to an a- amylase comprising the amino acid sequence shown in SEQ ID No.

1 or an o;-amylase being at least 80% homologous with the amino acid sequence (SEQ ID No.l), preferably being at least 85% 5 homologous with SEQ ID No. 1, more preferably being at least 90% homologous with SEQ ID No.l.

A polypeptide is considered to be X% homologous to the parent α-amylase if a comparison of the respective amino acid sequences, performed via known algorithms, such as the one 0described by Lipman and Pearson in Science 227, 1985, p. 1435, reveals an identity of X% .

In a further aspect the invention relates to an a- amylase comprising the amino acid sequence shown in SEQ ID No.

2 or an α-amylase being at least 80% homologous with the amino 5 acid sequence (SEQ ID No.2), preferably being at least 85% homologous with SEQ ID No. 2, more preferably being at least 90% homologous with SEQ ID No.2.

In another embodiment the invention relates to an - amylase comprising an N-terminal amino acid sequence identical to that shown in SEQ ID No. 3 or an -amylase being at least 80% homologous with SEQ ID No.3 in the N-terminal, preferably 5being at least 90% homologous with SEQ ID No.3 in the N- terminal .

Preferred α-amylases of the invention are obtainable from an alkaliphilic Bacillus species, particularly from one of the Bacillus strains NCIB 12289, NCIB 12512, NCIB 12513 and DSM

109375. In the context of the present invention, the term "obtainable from" is intended not only to indicate an α-amylase produced by a Bacillus strain but also an a-amylase encoded by a DNA sequence isolated from such a Bacillus strain and produced in a host organism transformed with said DNA sequence.

15 The strain NCIB 12289 is described in detail in EP

0 277 216. The strain NCIB 12289 has been deposited according to the Budapest Treaty on the International Recognition of the Deposits of Microorganisms for the Purpose of Patent Pro¬ cedures, on 8 July 1986 at The National Collection of Indus- 0 trial Bacteria (NCIB) under accession no. NCIB 12289.

The strain NCIB 12512 is described in detail in EP 0 277 216. The strain NCIB 12512 has been deposited according to the Budapest Treaty on the International Recognition of the Deposits of Microorganisms for the Purpose of Patent Pro- 5cedures, on 5 August 1987 at The National Collection of Industrial Bacteria (NCIB) under accession no. NCIB 12512.

The strain NCIB 12513 is described in detail in EP 0 277 216. The strain NCIB 12513 has been deposited according to the Budapest Treaty on the International Recognition of the 0Deposits of Microorganisms for the Purpose of Patent Pro¬ cedures, on 5 August 1987 at The National Collection of Industrial Bacteria (NCIB) under accession no. NCIB 12513.

The strain DSM 9375 has been deposited according to the Budapest Treaty on the International Recognition of the 5Deposits of Microorganisms for the Purpose of Patent Procedures, on 16 August 1994 at Deutsche Sammlung von Mikroor- ganismen und Zellkulturen GmbH (DSM) under Accession No. DSM

Cloning a DNA sequence encoding an ce-amylase

The DNA sequence encoding an ce-amylase of the invention may be isolated from any cell or microorganism producing the ce-amylase in question, using various methods well known in the art. First, a genomic DNA and/or cDNA library should be constructed using chromosomal DNA or messenger RNA from the organism that produces the ce-amylase to be studied. Then, if the amino acid sequence of the ce-amylase is known, homologous, labelled oligonucleotide probes may be synthesized and used to identify ce-amylase-encoding clones from a genomic library prepared from the organism in question. Alternatively, a labelled oligonucleotide probe containing sequences homolo¬ gous to a known ce-amylase gene could be used as a probe to identify ce-amylase-encoding clones, using hybridization and washing conditions of lower stringency. According to the present invention preferred probes may be constructed on the basis of SEQ ID No. 1 or on the basis of SEQ ID No. 2 or on the basis of SEQ ID No. 4 or on the basis of SEQ ID No 5. Yet another method for identifying ce-amylase-encoding clones would involve inserting fragments of genomic DNA into an expression vector, such as a plasmid, transforming ce-amylase- negative bacteria with the resulting genomic DNA library, and then plating the transformed bacteria onto agar containing a substrate for ce-amylase, thereby allowing clones expressing the ce-amylase to be identified.

Alternatively, the DNA sequence encoding the enzyme may be prepared synthetically by established standard methods, e.g. the phosphoamidite method described by S.L. Beaucage and M.H. Caruthers in Tetrahedron Letters 22, 1981, pp. 1859-1869 or the method described by Matthes et al . in The EMBO J. 3., 1984, pp. 801-805. In the phosphoamidite method, oligonucleoti- des are synthesized, e.g. in an automatic DNA synthesizer, purified, annealed, ligated and cloned in appropriate vectors. Finally, the DNA sequence may be of mixed genomic and synthetic origin, mixed synthetic and cDNA origin or mixed

genomic and cDNA origin, prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate, the frag¬ ments corresponding to various parts of the entire DNA sequence) , in accordance with standard techniques. The DNA sequence may also be prepared by polymerase chain reaction (PCR) using specific primers, for instance as described in US 4,683,202 or R.K. Saiki et al . in Science 239, 1988, pp. 487- 491.

Expression of ce-amylase According to the invention, an ce-amylase-encoding DNA sequence produced by methods described above, or by any alternative methods known in the art, can be expressed, in enzyme form, using an expression vector which typically includes control sequences encoding a promoter, operator, ribosome binding site, translation initiation signal, and, optionally, a repressor gene or various activator genes.

The recombinant expression vector carrying the DNA sequence encoding an ce-amylase of the invention may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, a bacteriophage or an extrachromosomal element, minichromosome or an artificial chromosome. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome (s) into which it has been integrated. In the vector, the DNA sequence should be operably connected to a suitable promoter sequence. The promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding pro¬ teins either homologous or heterologous to the host cell . Examples of suitable promoters for directing the transcription of the DNA sequence encoding an ce-amylase of the invention,

especially in a bacterial host, are the promoter of the lac operon of E.coli, the Streptomyces coelicolor agarase gene dagA promoters, the promoters of the Bacillus licheniformis ce- amylase gene (amyL) , the promoters of the Bacillus stearother- smophilus maltogenic amylase gene (amvM) , the promoters of the Bacillus Amyloliquefaciens ce-amylase (amvO) , the promoters of the Bacillus subtilis xylA and xylB genes etc. For transcrip¬ tion in a fungal host, examples of useful promoters are those derived from the gene encoding A. orvzae TAKA amylase, Rhizo- lomucor miehei aspartic proteinase, A. niger neutral ce-amylase, A. niger acid stable ce-amylase, A. niger glucoamylase, Rhizo- mucor miehei lipase, A. orvzae alkaline protease, A. orvzae triose phosphate isomerase or A. nidulans acetamidase.

The expression vector of the invention may also

15 comprise a suitable transcription terminator and, in eukaryotes, polyadenylation sequences operably connected to the DNA sequence encoding the ce-amylase of the invention. Termina¬ tion and polyadenylation sequences may suitably be derived from the same sources as the promoter. 0 The vector may further comprise a DNA sequence enabling the vector to replicate in the host cell in question. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUBHO, pE194, pAMBl and pIJ702.

The vector may also comprise a selectable marker, 5 e.g., a gene the product of which complements a defect in the host cell, such as the dal genes from B. subtilis or B. li¬ cheniformis. or one which confers antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracyclin resis¬ tance. Furthermore, the vector may comprise Aspergillus 0 selection markers such as amdS, argB, niaD and sC, a marker giving rise to hygromycin resistance, or the selection may be accomplished by co-transformation, e.g., as described in WO 91/17243.

While intracellular expression may be advantageous 5 in some respects, e.g., when using certain bacteria as host cells, it is generally preferred that the expression is ex¬ tracellular.

Procedures suitable for constructing vectors of the invention encoding an ce-amylase and containing the promoter, terminator and other elements, respectively, are well known to persons skilled in the art (cf., for instance, Sambrook et al . in Molecular Cloning: A Laboratory Manual, 2nd Ed. , Cold Spring Harbor, 1989) .

The cell of the invention, either comprising a DNA construct or an expression vector of the invention as defined above, is advantageously used as a host cell in the recombinant production of an ce-amylase of the invention. The cell may be transformed with the DNA construct of the invention encoding the ce-amylase conveniently by integrating the DNA construct (in one or more copies) in the host chromosome. This integration is generally considered to be an advantage as the DNA sequence is more likely to be stably maintained in the cell. Integration of the DNA constructs into the host chromosome may be performed according to conventional methods, e.g., by homologous or heterologous recombination. Alternatively, the cell may be transformed with an expression vector as described above in connection with the different types of host cells.

The cell of the invention may be a cell of a higher organism such as a mammal or an insect, but is preferably a microbial cell, e.g., a bacterial or a fungal (including yeast) cell. Examples of suitable bacteria are grampositive bacteria such as Bacillus subtilis. Bacillus licheniformis. Bacillus lentus. Bacillus brevis. Bacillus stearothermophilus, Bacillus alkalophilus. Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans. Bacillus lautus, Bacillus megaterium. Bacillus thuringiensis, or Streptomyces lividans or

Streptomyces murinus, or gramnegative bacteria such as E.coli .

The transformation of the bacteria may, for instance, be effected by protoplast transformation or by using competent cells in a manner known per se. The yeast organism may favourably be selected from a species of Saccharomyces or Schizosaccharomyces, e.g., Saccharomvces cerevisiae. The filamentous fungus may advan-

tageously belong to a species of Aspergillus, e.g. , Aspergillus oryzae or Aspergillus niger. Fungal cells may be transformed by a process involving protoplast formation and transformation of the protoplasts followed by regeneration of the cell wall in a manner known per se. A suitable procedure for transformation of Aspergillus host cells is described in EP 238 023.

In a yet further aspect, the present invention relates to a method of producing an ce-amylase of the invention, which method comprises cultivating a host cell as described above under conditions conducive to the production of the ce- amylase and recovering the ce-amylase from the cells and/or cul¬ ture medium.

.The medium used to cultivate the cells may be any conventional medium suitable for growing the host cell in question and obtaining expression of the ce-amylase of the invention. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g., as described in catalogues of the American Type Culture Collection) . The ce-amylase secreted from the host cells may con¬ veniently be recovered from the culture medium by well-known procedures, including separating the cells from the medium by centrifugation or filtration, and precipitating proteinaceous components of the medium by means of a salt such as ammonium sulphate, followed by the use of chromatographie- procedures such as ion exchange chromatography, affinity chromatography, or the like.

Assay for ce-Amylase Activity ce-Amylase activity was determined by a method employing Phadebas ^® tablets as substrate. Phadebas tablets (Phadebas ^® Amylase Test, supplied by Pharmacia Diagnostic) contain a cross-linked insoluble blue-coloured starch polymer which has been mixed with bovine serum albumin and a buffer substance and tabletted. For every single measurement one tablet is suspended in a tube containing 5 ml 50 mM Britton-Robinson buffer (50 mM

acetic acid, 50 mM phosphoric acid, 50 mM boric acid, 0.1 mM CaCl ₂ , pH adjusted to the value of interest with NaOH) . The test is performed in a water bath at the temperature of interest. The ce-amylase to be tested is diluted in x ml of 50 mM Britton-Robinson buffer. 1 ml of this ce-amylase solution is added to the 5 ml 50 mM Britton-Robinson buffer. The starch is hydrolysed by the ce-amylase giving soluble blue fragments. The absorbance of the resulting blue solution, measured spectropho- tometrically at 620 nm, is a function of the ce-amylase ac- tivity.

It is important that the measured 620 nm absorbance after 10 or 15 minutes of incubation (testing time) is in the range of 0.2 to 2.0 absorbance units at 620 nm. In this absorbance range there is linearity between activity and absorbance (Lambert-Beer law) . The dilution of the enzyme must therefore be adjusted to fit this criterion.

Under a specified set of conditions (temp., pH, reaction time, buffer conditions) 1 mg of a given ce-amylase will hydrolyse a certain amount of substrate and a blue colour will be produced. The colour intensity is measured at 620 nm. The measured absorbance is directly proportional to the specific activity (activity/mg of pure ce-amylase protein) of the ce-amylase in question under the given set of conditions. Thus, by testing different ce-amylases of interest (including Termamyl ^® , the ce-amylase used for reference) under identical conditions, the specific activity of each of the ce-amylases at a given temperature and at a given pH can be compared directly, and the ratio of the specific activity of each of the ce- amylases of interest relative to the specific activity of Termamyl ^® can be determined.

Industrial Applications

Owing to their activity at alkaline pH values, the ce-amylases of the invention are well suited for use in a variety of industrial processes, in particular the enzyme finds potential applications as a component in washing, dishwashing and hard surface cleaning detergent compositions, but it may

also be useful in the production of sweeteners and ethanol from starch. Conditions for conventional starch-converting processes and liquefaction and/or saccharification processes are de¬ scribed in, for instance, US Patent No. 3,912,590 and EP patent publications Nos. 252,730 and 63,909.

Being alkaline the ce-amylases of the invention also possess valuable properties in the production of lignocel- lulosic materials, such as pulp, paper and cardboard, from starch reinforced waste paper and cardboard, especially where repulping occurs at pH above 7 and where amylases can facili¬ tate the disintegration of the waste material through degrada¬ tion of the reinforcing starch. The ce-amylases of the invention are especially useful in the deinking/recycling processes of making paper out of old starch-coated or starch-containing printed paper. It is usually desirable to remove the printing ink in order to produce new paper of high brightness; examples of how the ce-amylases of the invention may be used in this way are described in PCT/DK 94/00437.

The ce-amylases of the invention may also be very useful in modifying starch where enzymatically modified starch is used in papermaking together with alkaline fillers such as calcium carbonate, kaolin and clays. With the alkaline ce- amylases of the invention it becomes possible to modify the starch in the presence of the filler thus allowing for a simpler integrated process.

The ce-amylases of the invention may also be very useful in textile desizing. In the textile processing industry, ce-amylases are traditionally used as auxiliaries in the desizing process to facilitate the removal of starch-containing size which has served as a protective coating on weft yarns during weaving.

Complete removal of the size coating after weaving is important to ensure optimum results in the subsequent processes, in which the fabric is scoured, bleached and dyed. Enzymatic starch break-down is preferred because it does not involve any harmful effect on the fibre material .

In order to reduce processing cost and increase mill

throughput, the desizing processing is sometimes combined with the scouring and bleaching steps. In such cases, non-enzymatic auxiliaries such as alkali or oxidation agents are typically used to break down the starch, because traditional ce-amylases are not very compatible with high pH levels and bleaching agents. The non-enzymatic breakdown of the starch size does lead to some fibre damage because of the rather aggressive chemicals used.

Accordingly, it would be desirable to use the ce- amylases of the invention as they have an improved performance in alkaline solutions. The ce-amylases may be used alone or in combination with a cellulase when desizing cellulose-containing fabric or textile.

The ce-amylases of the invention may also be very useful in a beer-making process; the ce-amylases will typically be added during the mashing process.

Detergent Compositions

According to the invention, the ce-amylases may typically be a component of a detergent composition, e.g., a laundry detergent composition or a dishwashing detergent composition. As such, it may be included in the detergent composition in the form of a non-dusting granulate, a stabili¬ zed liquid, or a protected enzyme. Non-dusting granulates may be produced, e.g., as disclosed in US 4,106,991 and 4,661,452 (both to Novo Industri A/S) and may optionally be coated by methods known in the art . Examples of waxy coating materials are poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molecular weights of 1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluid bed techniques are given in patent GB 1483591. Liquid enzyme preparations may, for instance, be stabilized by adding a polyol such as propylene

glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods. Other enzyme stabilizers are well known in the art. Protected enzymes may be prepared according to the method disclosed in EP 238,216. The detergent composition of the invention may be in any convenient form, e.g. as powder, granules, paste or liquid. A liquid detergent may be aqueous, typically containing up to 70% water and 0-30% organic solvent, or nonaqueous.

The detergent composition comprises one or more surf- actants, each of which may be anionic, nonionic, cationic, or amphoteric (zwitterionic) . The detergent will usually contain 0-50% of anionic surfactant such as linear alkylbenzene- sulfonate (LAS) , alpha-olefinsulfonate (AOS) , alkyl sulfate (fatty alcohol sulfate) (AS) , alcohol ethoxysulfate (AEOS or AES) , secondary alkanesulfonates (SAS) , alpha-sulfo fatty acid methyl esters, alkyl- or alkenylsuccinic acid, or soap. It may also contain 0-40% of nonionic surfactant such as alcohol ethoxylate (AEO or AE) , alcohol propoxylate, carboxylated alcohol ethoxylates, nonylphenol ethoxylate, alkylpolygly- coside, alkyldimethylamine oxide, ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide, or polyhydroxy alkyl fatty acid amide (e.g. as described in WO 92/06154) .

The detergent composition may additionally comprise one or more other enzymes, such as pullulanase, esterase, lipase, cutinase, protease, cellulase, peroxidase, or oxidase, e.g., laccase.

Normally the detergent contains 1-65% of a detergent builder, but some dishwashing detergents may contain even up to 90% of a detergent builder, or complexing agent such as zeolite, diphosphate, triphosphate, phosphonate, citrate, nitrilotriacetic acid (NTA) , ethylenediaminetetraacetic acid

(EDTA) , diethylenetriaminepentaacetic acid (DTMPA) , alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates

(e.g. SKS-6 from Hoechst) . The detergent builders may be subdivided into phosphorus-containing and non-phosphorous-containing types. Examples of phosphorus-containing inorganic alkaline detergent

builders include the water-soluble salts, especially alkali metal pyrophosphates, orthophosphates, polyphosphates and phosphonates . Examples of non-phosphorus-containing inorganic builders include water-soluble alkali metal carbonates, borates and silicates as well as layered disilicates and the various types of water-insoluble crystalline or amorphous alumino silicates of which zeolites is the best known representative.

Examples of suitable organic builders include alkali metal, ammonium or substituted ammonium salts of succinates, malonates, fatty acid malonates, fatty acid sulphonates, carboxymethoxy succinates, polyacetates, carboxylates, polycar- boxylates, aminopolycarboxylates and polyacetyl carboxylates.

The detergent may also be unbuilt, i.e. essentially free of detergent builder. The detergent may comprise one or more polymers.

Examples are carboxymethylcellulose (CMC) , poly(vinyl- pyrrolidone) (PVP) , polyethyleneglycol (PEG) , poly(vinyl alcohol) (PVA) , polycarboxylates such as polyacrylates, polymaleates, maleic/acrylic acid copolymers and lauryl methacrylate/acrylic acid copolymers.

The detergent composition may contain bleaching agents of the chlorine/bromine-type or the oxygen-type. The bleaching agents may be coated or incapsulated. Examples of inorganic chlorine/bromine-type bleaches are lithium, sodium or calcium hypochlorite or hypobromite as well as chlorinated trisodium phosphate. The bleaching system may also comprise a H-0 ₂ source such as perborate or percarbonate which may be combined with a peracid-forming bleach activator such as tetraacetylethylenediamine (TAED) or nonanoyloxybenzene- sulfonate (NOBS) .

Examples of organic chlorine/bromine-type bleaches are heterocyclic N-bromo and N-chloro imides such as trichloro- isocyanuric, tribromoisocyanuric, dibromoisocyanuric and dichloroisocyanuric acids, and salts thereof with water solubilizing cations such as potassium and sodium. Hydantoin compounds are also suitable. The bleaching system may also comprise peroxyacids of, e.g., the amide, imide, or sulfone

type .

In dishwashing detergents the oxygen bleaches are preferred, for example in the form of an inorganic persalt, preferably with a bleach precursor or as a peroxy acid com- 5 pound. Typical examples of suitable peroxy bleach compounds are alkali metal perborates, both tetrahydrates and monohydrates, alkali metal percarbonates, persilicates and perphosphates. Preferred activator materials are TAED or NOBS.

The enzymes of the detergent composition of the

10 invention may be stabilized using conventional stabilizing agents, e.g. a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative such as, e.g., an aromatic borate ester, and the composition may be formulated as described in, e.g., WO

15 92/19709 and WO 92/19708. The enzymes of the invention may also be stabilized by adding reversible enzyme inhibitors, e.g., of the protein type as described in EP 0 544 777 Bl.

The detergent may also contain other conventional detergent ingredients such as, e.g., fabric conditioners in-

20 eluding clays, deflocculant material, foam boosters/foam depressors (in dishwashing detergents foam depressors) , suds suppressors, anti-corrosion agents, soil-suspending agents, anti-soil-redeposition agents, dyes, dehydrating agents, bactericides, optical brighteners, or perfume.

25 The pH (measured in aqueous solution at use con¬ centration) will usually be neutral or alkaline, e.g. in the range of 7-11.

Particular forms of laundry detergent compositions within the scope of the invention include:

301) A detergent composition formulated as a granulate having a bulk density of at least 600 g/1 comprising

35

2) A detergent composition formulated as a granulate having a bulk density of at least 600 g/1 comprising

53) A detergent composition formulated as a granulate having a bulk density of at least 600 g/1 comprising

0

5

0

4) A detergent composition formulated as a granulate having a bulk density of at least 600 g/1 comprising

6) An aqueous structured liquid detergent composition compris¬ ing

7) A detergent composition formulated as a granulate having a bulk density of at least 600 g/1 comprising

8) A detergent composition formulated as a granulate comprising

259) A detergent composition formulated as a granulate comprising

30

1010) An aqueous liquid detergent composition comprising

11) An aqueous liquid detergent composition comprising

5

12) A detergent composition formulated as a granulate having a bulk density of at least 600 g/1 comprising

13) Detergent formulations as described in 1) - 12) wherein all or part of the linear alkylbenzenesulfonate is replaced by (C ₁₂ - C ₁₈ ) alkyl sulfate.

14) A detergent composition formulated as a granulate having a bulk density of at least 600 g/1 comprising

15) A detergent composition formulated as a granulate having a bulk density of at least 600 g/1 comprising

16) Detergent formulations as described in 1) - 15) which 10 contain a stabilized or encapsulated peracid, either as an additional component or as a substitute for already specified bleach systems.

17) Detergent compositions as described in 1) , 3) , 7) , 9) and 12) wherein perborate is replaced by percarbonate.

15 18) Detergent compositions as described in 1) , 3) , 7) , 9) , 12) , 14) and 15) which additionally contain a manganese catalyst. The manganese catalyst may, e.g., be one of the compounds described in "Efficient manganese catalysts for low-temperature bleaching", Nature 369, 1994, pp. 637-639.

2019) Detergent composition formulated as a nonaqueous detergent liquid comprising a liquid nonionic surfactant such as, e.g., linear alkoxylated primary alcohol, a builder system (e.g. phosphate) , enzyme and alkali. The detergent may also comprise anionic surfactant and/or a bleach system.

25 Particular forms of dishwashing detergent composi¬ tions within the scope of the invention include:

1) POWDER AUTOMATIC DISHWASHING COMPOSITION

2) POWDER AUTOMATIC DISHWASHING COMPOSITION

3) POWDER AUTOMATIC DISHWASHING COMPOSITION

4) POWDER AUTOMATIC DISHWASHING COMPOSITION

5) POWDER AU OMATIC DISHWASHING COMPOSITION

56) POWDER AND LIQUID DISHWASHING COMPOSITION WITH CLEANING SURFACTANT SYSTEM

0

5

0

5

5

7) NON-AQUEOUS LIQUID AUTOMATIC DISHWASHING COMPOSITION

8) NON-AQUEOUS LIQUID DISHWASHING COMPOSITION

9) THIXOTROPIC LIQUID AUTOMATIC DISHWASHING COMPOSITION

H2__ C__JA fatty acid 0.5%

0) LIQUID AUTOMATIC DISHWASHING COMPOSITION

11) LIQUID AUTOMATIC DISHWASHING COMPOSITION CONTAINING PROTECTED BLEACH PARTICLES

11) Automatic dishwashing compositions as described in 1) , 2) , 3) , 4) , 6) and 10) , wherein perborate is replaced by per¬ carbonate.

12) Automatic dishwashing compositions as described in 1) - 6) which additionally contain a manganese catalyst. The manganese catalyst may, e.g., be one of the compounds described in "Efficient manganese catalysts for low-temperature bleaching", Nature 369. 1994, pp. 637-639.

The ce-amylases of the invention may be incorporated in concentrations conventionally employed in detergents. It is at present contemplated that, in the detergent composition of the invention, the ce-amylase may be added in an amount corre- sponding to 0.00001-1 mg (calculated as pure enzyme protein) of ce-amylase per liter of wash/dishwash liquor.

The present invention is further illustrated in the following examples which are not intended to be in any way limiting to the scope of the invention as claimed.

EXAMPLE 1

ce-amylase Preparations from Bacillus strains NCIB 12289, NCIB 12513, DSM 9375 and NCIB 12512. Fermentation: Each of the above mentioned Bacillus strains was incubated at 26°C on a rotary shaking table (300 r.p.m.) in 500 ml baffled Erlenmeyer flasks containing 100 ml of BP-X medium + 0.1 M Carbonate buffer pH 9.0. BP-X medium: Potato starch 100 g Ground barley 50 g Soybean flour 20 g Sodium caseinate 10 g Na ₂ HP0 ₄ X 12 H ₂ 0 9 g Termamyl ^® 60L* 0.1 g Pluronic ^® 0.1 g

*) available from Novo Nordisk A/S.

The starch in the medium was liquified by slowly heating the medium from 60°C to 85°C for 30 minutes. After this the temperature of the medium was quickly raised to 95°C for 10 minutes and then cooled. Lastly the medium was sterilized by heating at 121°C for 40 minutes.

Purification of ce-amylase from NCIB 12289, DSM 9375 and NCIB 12512. After 5 days of incubation the culture broth was filtrated and concentrated using a Filtron™ ultrafiltration module with 3KD membranes and washed with deionized water until the conductivity was 1 mS/cm. The pH was adjusted to pH 5.9 with 10% (v/v) acetic acid. A S-sepharose FF column was equilibrated in EKV-buffer, pH 5.9. If not otherwise stated, the purification buffer was 100 mM boric acid, 10 mM succinic acid, 2 mM CaCl ₂ , (EKV-buffer) adjusted to the indicated pH with NaOH.

The enzyme solution was applied to the column, the column was washed with EKV-buffer, pH 5.9, and the amylase was

eluted with a linear NaCl gradient (0-> 500 mM NaCl) . Amylase containing fractions were pooled and the pH adjusted to pH 7 with 3% (w/v) NaOH.

A chelate agarose column was loaded with Cu++ and equilibrated in the following manner: 50 mM CuS0 ₄ , pH 5 was pumped ^" on to the column until the whole column was blue, then excess of Cu++-ions were removed by washing the column with 500 mM imidazol, pH 7, and finally the column was equilibrated with EKV-buffer, pH 7. The amylase pool from the S-sepharose column was applied to the Cu++-loaded Chelate agarose column, the column was washed with EKV-buffer, pH 7, and the enzyme was eluted with a linear gradient of imidazol (0-> 500 mM imidazol) . Amylase containing fractions were pooled and a solution of saturated ammonium sulphate was added to give a final concentration of 1M (NH ₄ ) ₂ S0 ₄ in the pool.

A phenyl sepharose column was equilibrated in EKV- buffer + 1M (NH ₄ ) ₂ S0 ₄ , pH 7. The amylase pool from the Cu++- column was applied to the hydrophobic interaction column. Binding experiments had shown that the amylase is a rather hydrophobic enzyme, and hence binds tightly to the phenyl column. Protein which did not bind as tightly to the column was washed off the column with EKV-buffer, pH 7. The amylase was step-eluted from the column with EKV-buffer + 25% (v/v) isopropanol. The amylase containing pool was adjusted to pH 9.5 with 3% (w/v) NaOH and diluted 5 times with deionized water.

A Q-sepharose HP column was equilibrated in 20 mM Tris-HCl, pH 9.5. The amylase pool from the phenyl sepharose column was applied to the column and the column was washed with 20 mM Tris-HCl, pH 9.5. The amylase was eluted with a linear gradient of NaCl (0 -> 250 mM NaCl) .

The amylase peak was adjusted to pH 7 with 10% (v/v) acetic acid.

A Cu++-loaded chelating sepharose FF column (loaded with Cu++ as described for the chelate agarose column) was equilibrated with EKV-buffer, pH 7. The amylase peak from the Q-sepharose column was applied to the column, and the column was washed thoroughly with EKV-buffer, pH 7. The amylase was

eluted with a steep linear gradient of imidazol (0 -> 500 mM imidazol) .

The purified amylase was purity checked by SDS-PAGE electrophoresis. The coomassie stained gel had only one band.

Purification of ce-amylase from NCIB 12513

After 5 days of incubation the culture broth was filtrated and concentrated using a Filtron™ ultrafiltration module with 3KD membranes. The concentrated solution was filtrated and saturated to 20% w/w with ammoniumsulfate. The solution was then batch absorbed using a AFFI-T™ matrix from Kem-En-Tec A/S. The amylase was eluted using 25% isopropanol in 20 mM Tris pH 7.5 after wash of the matrix with deionized water. The eluted enzyme was subjected to dialysis (20 mM Tris pH 8.5) and a stepwise batch adsorption on Q-sepharose FF for colour removal was made.

A chelate agarose column was loaded with Cu++ and equilibrated in the following manner: 50 mM CuS0 ₄ , pH 5 was pumped on to the column until the whole column was blue, then excess of Cu++-ions was removed by washing the column with 500 mM imidazol, pH 7, and finally the column was equilibrated with 50 mM borate buffer, pH 7.

In spite of the low pi (5.8) the amylase was not bound to the Q-sepharose FF at pH 8.5.

The run through from the Q-sepharose FF column was applied on the Cu-chelating agarose and eluted using 250 mM imidazol, 20 mM Tris pH 7.0 and the eluted column was dialysed against 50 mM borate buffer pH 7.0. The pH was adjusted to pH 9.5 and the dialysed solution was bound on a Q-sepharose HP and eluted over 10 columns using a linear gradient from 0-250 mM NaCl . Amylase containing fractions were pooled and a solution of saturated ammonium sulphate was added to give a final concentration of 20% w/w, and the fractions were applied on a phenyl sepharose column. The column was washed using deionized water and eluted using 25% isopropanol in 50 mM borate buffer pH 7.0.

The purified amylase was purity checked by SDS-PAGE

electrophoresis. The coomassie stained gel had only one band.

EXAMPLE 2

Physical-Chemical Properties of the ce-Amylases

The ce-amylase obtained from Bacillus strain NCIB 5 12289, fermented and purified as described in Example 1, was found to possess the following properties:

A pi of about 8.8-9.0 as determined by isoelectric focusing on LKB Ampholine ^® PAG plates (3.5-9.5) - meaning that said plates are useful in the pi range of 3.5 to 9.5. 0 A molecular weight of approximately 55 kD as deter¬ mined by SDS-PAGE.

A pH profile as shown in Fig. 1, which was determined at 37°C in the pH range of from 4 to 10.5. The assay for ce- amylase activity described previously was used, using Britton- 5 Robinson buffer adjusted to predetermined pH values. It appears from Fig. 1 that the enzyme possesses ce-amylase activity at all pH values of from 4 to 10.5, having optimum at pH 7.5-8.5, and at least 60% of the maximum activity at pH 9.5.

Amino acid sequence of the ce-amylase was determined using standard methods for obtaining and sequencing peptides, for reference see Findlay & Geisow (Eds.) , Protein Seguencing - a Practical Approach, 1989, IRL Press.

The N-terminal amino acid sequence was found to be : His-His-Asn-Gly-Thr-Asn-Gly-Thr-Met-Met-Gln-Tyr-Phe-Glu-Trp- Tyr-Leu-Pro-Asn-Asp (SEQ ID No. 3) .

The ce-amylases obtained from Bacillus strains NCIB 12512 and DSM 9375, fermented and purified as described in Example 1, were found to possess the same pi (8.8-9.0) , the same molecular weight (55 kD) , and the same N-terminal sequence (SEQ ID No. 3) as the ce-amylase obtained from NCIB 12289; so it can be concluded that the ce-amylases obtained from NCIB 12289, NCIB 12512 and DSM 9375 have the following common features:

(a) A pi of about 8.6-9.3 determined by isoelectric focusing on LKB Ampholine ^® PAG plates;

b) A molecular weight of approximately 55 kD as determined by SDS-PAGE; c) An N-terminal amino acid with the amino acid sequence as shown in ID No. 3.

^" The full amino acid sequence of the Bacillus strain NCIB 12512 ce-amylase is disclosed in SEQ ID No. 1 of the present invention. The full DNA sequence of the Bacillus strain NCIB 12512 ce-amylase is disclosed in SEQ ID No. 4 of the present invention. The ce-amylase obtained from Bacillus strain NCIB

12513, fermented and purified as described in Example 1, was found to possess a pi of about 5.8 and a molecular weight of approximately 55 kD.

The full amino acid sequence of the Bacillus strain NCIB 12513 ce-amylase is disclosed in SEQ ID No. 2 of the present invention. The full DNA sequence of the Bacillus strain NCIB 12513 ce-amylase is disclosed in SEQ ID No. 5 of the present invention.

EXAMPLE 3

pH and Temperatures Profiles of the ce-Amylases according to the Invention Compared to Termamyl ^® .

A pH profile of an ce-amylase obtained from Bacillus strain NCIB 12512 (I) , of an ce-amylase obtained from Bacillus strain NCIB 12513 (II) and of Termamyl ^® (III) were determined at 55°C in the pH interval of from 4 to 10.5. The ce-amylases of the invention were fermented and purified as described in Example 1 and Termamyl ^® was obtained from Novo Nordisk A/S. The assay for ce-amylase activity described previously was used, using 50 mM Britton-Robinson buffer adjusted to predetermined pH values and a reaction time of 15 minutes. The results are presented in Fig. 2. It appears from Fig. 2 that the ce-amylases of the invention possess ce-amylase activity at all pH values of from pH 4 to pH 10.5, having optimum at pH 7.5-8.5.

A temperature profile of an ce-amylase obtained from

Bacillus strain NCIB 12512 (I) , of an ce-amylase obtained from Bacillus strain NCIB 12513 (II) and of Termamyl ^® (III) were determined at pH 10.0 in the temperature interval of from 25°C to 95°C. The ce-amylases of the invention were fermented and purified as described in Example 1 and Termamyl ^® was obtained from Novo Nordisk A/S. The assay for ce-amylase activity described previously was used, using 50 mM Britton-Robinson buffer adjusted to pH 10.0 and a reaction time of 10 minutes. The results are presented in Fig. 3. It appears from Fig. 3 that the ce-amylases of the invention possess ce-amylase activity at all temperature values of from 25°C to 85°C, having optimum at 45°C-55°C, and that the specific activity of the ce-amylase of the invention is 25% higher than the specific activity of Termamyl ^® at any temperature in the temperature interval of from 25°C to 55°C.

EXAMPLE 4

Dishwashing Performance of novel ce-amylases ce-amylases of the invention obtained from Bacillus strain NCIB 12289 and from Bacillus strain 12512 as described in Example 1, were tested using the following test for deter¬ gent amylases for automatic dishwashing:

Plates were dipped in hot corn starch and glasses were soiled by pouring corn starch from one glass to another. The plates and glasses were left to dry overnight and then washed in a dishwasher under the following conditions:

Amylase dosage: 0-0.50 mg of enzyme protein per litre of washing liquor Detergent : Commercial European

Detergent dosage: 4.0 g per litre of washing liquor Dishwashing: 45°C, 55°C or 65°C program, Cylinda pH: 10.1 during dishwashing.

Evaluation/Rating System:

Removal of starch film (RSF) from the plates and

glasses was evaluated after colouring the items with iodine (iodine turns starch blue) . The following rating scale was used:

*) unwashed

After each item had been evaluated according to the above mentioned rating system, the total value of the scores obtained was divided by the total number of items. The result¬ ing RSF-value was then plotted against the mg ce-amylase protein used per litre of washing liquor.

Results :

Bacillus strain NCIB 12289 ce-amylase: This ce-amylase was tested at 55°C and the results are shown in Fig. 4. It can be seen from Fig. 4 that an RSF value of between 3 and 4 is obtained at an enzyme dosage of 0.1 mg of ce-amylase protein per litre of washing liquor.

Bacillus strain NCIB 12512 ce-amylase: This ce-amylase was tested at 45°C (• ) , at 55°C (*) and at 65°C (x) , and the results are shown in Fig. 5. It can be seen from Fig. 5 that an RSF value of between 3 and 4.5 is obtained at an enzyme dosage of 0.1 mg of ce-amylase protein per litre of washing liquor (the RSF-value increasing with increasing temperature) .

EXAMPLE 5

Mini Dishwashing Performance of Novel ce-Amylases

The following mini dishwashing assay was used: A suspension of starchy material was boiled and cooled to 20°C. The cooled starch suspension was applied on small, individually identified glass plates (approx. 2 x 2 cm) and dried at a temperature in the range of 60-140°C in a drying cabinet. The individual plates were then weighed. For assay purposes, a solution of standard European-type automatic dishwashing detergent (5 g/1) having a temperature of 55°C was prepared. The detergent was allowed a dissolution time of 1 minute, after which the amylase in question was added to the detergent solution (contained in a beaker equipped with magnetic stir¬ ring) so as to give an enzyme concentration of 0.5 mg/1. At the same time, the weighed glass plates, held in small supporting clamps, were immersed in a substantially vertical position in the amylase/detergent solution, which was then stirred for 15 minutes at 55°C. The glass plates were then removed from the amylase/detergent solution, rinsed with distilled water, dried at 60°C in a drying cabinet and re-weighed. The performance of the amylase in question [expressed as an index relative to Termamyl (index 100)] was then determined from the difference in weight of the glass plates before and after treatment, as follows :

Index = ^we i ^< ? ^nt loss for plate treated with ce-amylage . ₁₀₀ weight loss for plate treated with Termamyl

Results

The above described mini dishwashing test was performed at pH 10.0 with Termamyl ^® , the novel ce-amylase from NCIB 12513 and the novel ce-amylase from NCIB 12512 (the novel ce-amylases obtained as described in Example 1) . The tests gave the following results :

Termamyl ^® Index: 100 ce-amylase (NCIB 12512) Index: 163

ce-amylase (NCIB 12513) Index: 175

Surprisingly, the performance in the mini dishwashing test is proportional with the specific activity at pH 10.0, 55°C as can be seen from Fig. 3: Termamyl ^® Spec, activity: 2200 U/mg ce-amylase (NCIB 12512) Spec, activity: 4400 U/mg ce-amylase (NCIB 12513) Spec, activity: 5200 U/mg.

EXAMPLE 6

Laundry washing

Detergent: Commercial US heavy duty granulate detergent (HDG)

Detergent dosage 2 g/1

ce-amylase dosage: 0.2 mg enzyme protein/1

Soil: Potato starch colored with Cibacron Blue

3GA on cotton

Water hardness: 9°dH Time: 15 minutes Temperature: 40°C

Evaluation: Reflectance at 660 nm. The delta reflectance was calculated from the reflectance obtained for a swatch having been washed with the relevant enzyme and the reflectance obtained for a swatch washed without enzyme. More specifically, the delta reflectance is the reflectance obtained with enzyme minus the reflectance obtained without enzyme.

Results

The above described laundry washing test was per¬ formed with Termamyl ^® , the novel ce-amylase from NCIB 12513 and the novel ce-amylase from NCIB 12512 (the novel ce-amylases

obtained as described in Example 1) . The tests gave the following results:

Termamyl ^® Index: 100 ce-amylase (NCIB 12512) Index: 145 ce-amylase (NCIB 12513) Index: 133

From the results presented above it is evident that the ce-amylases of the invention exert a considerably improved starch removal capacity relative to Termamyl, in other words that the ce-amylases of the invention have an improved laundry washing performance compared to that of Termamyl.

EXAMPLE 7

Catalytic Efficiency of the Bacillus Strain NCIB 12512 ce- Amylase and the Bacillus Strain NCIB 12513 ce-Amylase Compared with Termamyl ^® . The kinetics of hydrolysis catalyzed by the ce- amylases of the invention and by Termamyl ^® at various substrate concentrations were determined using the Somogyi-Nelson method (described below) with amylose (Merck 4561) and amylopectin (Sigma A7780) as substrates. The hydrolysis velocities were measured under different substrate concentrations (1%, 0.5%, 0.3%, 0.25% and 0.2%) .

The number of reducing sugars were measured using the Somogyi-Nelson method, and determined as glucose eqv. made/mg of amylase x h giving the hydrolysis velocity. The data were plotted according to the Michaelis-Menten and Lineweaver-Burk equations. From these equations V_ _ιaχ /K _rn can easily be calculated by using the following approximation:

LSI

V = V. x -SJ +Ϊ .

isi 2 -H_nax

When [S] < < K_, : V = V _maχ x K_, = K_, ^" x [S]

At a given substrate concentration, that substrate concentration being less than K,., the expression V_ _ιaχ /K _m is equivalent to the catalytic efficiency of a given ce- amylase. In Table 1 below V-^/K,. is calculated for three different ce-amylases.

Table 1.

Catalytic efficiency [V _maχ /K _π| ] determined at 55°C, pH 7.3 in 50 mM Britton-Robinson buffer

The catalytic efficiency of ce-amylase (NCIB 12513) and ce-amylase (NCIB 12512) have shown to be surprisingly high towards both Amylopectin and Amylose compared to Termamyl . Especially the high catalytic efficienty towards amylose is considered to be of significant importance for the improved specific activities and dishwash/laundry performance compared to Termamyl .

Linear amylose molecules can align themselves next to each other and form interchain hydrogenbonds through the hydroxyl groups. This network of amylose molecules has crystal¬ line characteristics and are difficult to solubilize and hydrolyze by any known amylase.

Somogyi Method for the Determination of Reducing Sugars

The method is based on the principle that the sugar reduces cupric ions to cuprous oxide which reacts with arsenate molybdate reagent to produce a blue colour which is measured spectrophotometrically. The solution which is to be examined must contain between 50 and 600 mg of glucose per litre.

1 ml of sugar solution is mixed with 1 ml of copper

reagent and placed in a boiling water bath for 20 minutes. The resulting mixture is cooled and admixed with 1 ml of Nelson's colour reagent and 10 ml of deionized water. The absorbancy at 520 nm is measured. 5 In the region 0-2 the absorbance is proportional to the amount of sugar, which may thus be calculated as follows:

mg glucose/1 = 100 (sample - blank)

(standard - blank)

% glucose = (sample - blank)

10 100 (standard - blank)

REAGENTS

1. Somogyi' s copper reagent 35.1 g of Na ₂ HP0 ₄ .2H ₂ 0, and

40.0 g of potassium sodium tartrate (KNaC ₄ H ₄ 0 ₂ .4H ₂ 0) 15 are dissolved in

700 ml of deionized water.

100 ml of 1 N sodium hydroxide and

80 ml of 10% cupric sulphate (CuS0 ₄ .5H ₂ 0) are added,

180 g of anhydrous sodium sulphate are dissolved in the mix- 20 ture, and the volume is brought to 1 litre with deionized water.

2. Nelson's colour reagent

50 g of ammonium molybdate are dissolved in 900 ml of deionized water. Then 2542 ml of concentrated sulphuric acid (Merck) are added, fol¬ lowed by

6 g of disodium hydrogen arsenate heptahydrate dissolved in 50 ml of deionized water, and the volume is brought to 1 litre with deionized water.

30 The solution must stand for 24-48 hours at 37°C before use. It must be stored in the dark in a brown glass bottle with a glass stopper.

3 . Standard

100 mg of glucose (May & Baker, anhydrous) are dissolved in 1 litre of deionized water.

Reference: J. Biol. Chem. 153 , 375 (1944)

SEQϋE-SICE LISTING

(1) GENERAL ΗSDFORMATICN:

(i) APPLICANT:

(A) NAME: NOVO NCRDISK A/S (B) STREET: Novo Alle

(C) CITY: Bagsvaerd

(E) CDϋNIRY: Denmark

(F) POSTAL CEDE (ZIP) : DK-2880

(G) τπr,FPHTftlR- +45 44 44 88 88 (H) TELEFAX: 445 44 49 05 55

(I) TELEX: 37173

(ii) TITLE OF ΓNVENITCN: AI_KAL3NE BACT LUS AMYLASE (iii) NUMBER OF S__C_0__NCES : 5 (iv) CπVFOTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) CEMPUTER: IBM PC cαipatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFIWARE: Patentln Release #1.0, Version #1.25 (EPO)

(2) _NF_RMA_[TCN F R SEQ 3D NO: 1:

(i) SEQOENCE C__ARACTE_RISTICS:

(A) LEN5IH: 485 arnino acids

(B) 1YPE: amino acid

(C) STF-ANDECNESS: single (D) TOPODDGY: linear

(ii) Dl-ECULE TΪFE: peptide

(xi) SIIQϋENCE DESCRIPπCN: SEQ ID NO: 1:

His His Asn Gly Thr Asn Gly Thr Met Met Gin Tyr Phe Glu Trp Tyr 1 5 10 15 Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Asp Aεp Ala Ala

20 25 30

Asn Leu Lys Ser Lys Gly He Thr Ala Val Trp He Pro Pro Ala Trp 35 40 45

Lys Gly Thr Ser Gin Asn Aεp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60

Asp Leu Gly Glu Phe Asn Gin Lys Gly Thr Val Arg Thr Lys Tyr Gly 65 70 75 80

Thr Arg Asn Gin Leu Gin Ala Ala Val Thr Ser Leu Lys Asn Asn Gly 85 90 95

He Gin Val Tyr Gly Aεp Val Val Mat Asn His Lys Gly Gly Ala Aεp 100 105 110

Gly Thr Glu He Val Asn Ala Val Glu Val Asn Arg Ser Asn Arg Asn 115 120 125

Gin Glu Thr Ser Gly Glu Tyr Ala He Glu Ala Tφ Thr Lys Phe Asp 130 135 140

Phe Pro Gly Arg Gly Asn Asn His Ser Ser Phe Lys Tφ Arg Tφ Tyr 145 150 155 160

His Phe Asp Gly Thr Asp Tφ Aεp Gin Ser Arg Gin Leu Gin Asn Lys 165 170 175

He Tyr Lys Phe Arg Gly Thr Gly Lys Ala Tφ Aεp Tφ Glu Val Asp 180 185 190 Thr Glu Asn Gly Asn Tyr Aεp Tyr Lsu Met Tyr Ala Asp Val Asp Met 195 200 205

Aεp His Pro Glu Val He His Glu Leu Arg Asn Tφ Gly Val Tφ Tyr 210 215 220

Thr Asn Thr Lsu Asn Leu Asp Gly Phe Arg He Asp Ala Val Lys His 225 230 235 240

He Lys Tyr Ser Phe Thr Arg Aεp Tφ Leu Thr His Val Arg Asn Thr 245 250

Thr Gly Lys Pro Met Phe Ala Val Ala Glu Phe Tφ Lys Asn Aεp Leu 260 265 270

Gly Ala He Glu Asn Tyr Leu Asn Lys Thr Ser Tφ Asn His Ser Val 275 280 285

Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly 290 295 300

Gly Tyr Tyr Asp Met Arg Asn He Leu Asn Gly Ser Val Val Gin Lys 305 310 315 320

His Pro Thr His Ala Val Thr Phe Veil Asp Asn His Asp Ser Gin Pro 325 330 335

Gly Glu Ala Leu Glu Ser Phe Val Gin Gin Tφ Phe Lys Pro Leu Ala 340 345 350

Tyr Ala Leu Val Leu Thr Arg Glu G n Gly Tyr Pro Ser Val Phe Tyr 355 360 365

Gly Aεp Tyr Tyr Gly He Pro Thr His Gly Val Pro Ala Met Lys Ser

370 375 380

Lys He Asp Pro Leu Leu Gin Ala Arg Gin Thr Phe Ala Tyr Gly Thr 385 390 395 400

Gin His Aεp Tyr Phe Asp His His Aεp He He Gly Tφ Thr Arg Glu 405 410 415

Gly Asn Ser Ser His Pro Asn Ser Gly Leu Ala Thr He IVfet Ser Aεp 420 425 430

Gly Pro Gly Gly Asn Lys Tφ Mat Tyr Val Gly Lys Asn Lys Ala Gly 435 440 445 Gin Val Tφ Arg Asp He Thr Gly Asn Arg Thr Gly Thr Val Thr He 450 455 460

Asn Ala Aεp Gly Tφ Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser 465 470 475 480

Val Tφ Val Lys Gin 485

(2) II-?CRMATTCN FOR. SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 485 amino acids

(B) TYPE: amino acid (C) SIRANDECNESS: single

(D) TOP0ICG- ^" : linear

(ii) jyO-IECULE TYPE: peptide

(xi) SEQUENCE DESCRIPITCN: SEQ ID NO: 2:

His His Asn Gly Thr Asn Gly Thr Met Mat Gin Tyr Phe Glu Tφ His 1 5 10 15

Leu Pro Asn Asp Gly Asn His Tφ Asn Arg Leu Arg Aεp Aεp Ala Sear 20 25 30

Asn Leu Arg Asn Arg Gly He Thr Ala He Tφ He Pro Pro Ala Tφ 35 40 45 Lys Gly Thr Ser Gin Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60

Asp Leu Gly Glu Phe Asn Gin Lys Gly Thr Val Arg Thr Lys Tyr Gly 65 70 75 80

Thr Arg Ser Gin Leu Glu Sear Ala He His Ala Leu Lys Asn Asn Gly 85 90 95

Val Gin Val Tyr Gly Aεp Val Val Met Asn His Lys Gly Gly Ala Asp 100 105 110

Ala Thr Glu Asn Val Leu Ala Val Glu Val Asn Pro Asn Asn Arg Asn 115 120 125

Gin Glu He Ser Gly Aεp Tyr Thr He Glu Ala Tφ Thr Lys Phe Aεp 130 135 140

Phe Pro Gly Arg Gly Asn Thr Tyr Ser Aεp Phe Lys Trp Arg Tφ Tyr 145 150 155 160

His Phe Asp Gly Val Asp Tφ Asp Gin Ser Arg Gin Phe Gin Asn Arg 165 170 175

He Tyr Lys Phe Arg Gly Asp Gly Lys Ala Tφ Asp Tφ Glu Val Aεp 180 185 190

Ser Glu Asn Gly Asn Tyr Aεp Tyr Leu Met Tyr Ala Asp Val Asp Nfet 195 200 205 Asp His Pro Glu Val Val Asn Glu Leu Arg Arg Tφ Gly Glu Tφ Tyr 210 215 220

Thr Asn Thr Leu Asn Leu Asp Gly Phe Arg He Aεp Ala Val Lys His 225 230 235 240

He Lys Tyr Ser Phe Thr Arg Asp Tφ Leu Thr His Val Arg Asn Ala 245 250

Thr Gly Lys Glu Met Phe Ala Val Ala Glu Phe Tφ Lys Asn Asp Leu 260 265 270

Gly Ala Leu Glu Asn Tyr Leu Asn Lys Thr Asn Tφ Asn His Ser Val 275 280 285

Phe Aεp Val Pro Leu His Tyr Asn Lsu Tyr Asn Ala Ser Asn Ser Gly 290 295 300

Gly Asn Tyr Asp Met Ala Lys Lsu Lsu Asn Gly Thr Val Val Gin Lys 305 310 315 320

His Pro Mat His Ala Val Thr Phe Val Aεp Asn His Asp Ser Gin Pro 325 330 335

Gly Glu Ser Lsu Glu Ser Phe Val Gin Glu Tφ Phe Lys Pro Leu Ala 340 345 350

Tyr Ala Leu He Leu Thr Arg Glu Gin Gly Tyr Pro Ser Val Phe Tyr 355 360 365

Gly Asp Tyr Tyr Gly He Pro Thr His Ser Val Pro Ala Met Lys Ala 370 375 380

Lys He Asp Pro He Leu Glu Ala Arg Gin Asn Phe Ala Tyr Gly Thr

385 390 395 400

Gin His Aεp Tyr Phe Asp His His Asn He He Gly Tφ Thr Arg Glu 405 410 415

Gly Asn Thr Thr His Pro Asn Ser Gly Lsu Ala Thr He Mst Ser Asp 420 425 430

Gly Pro Gly Gly Glu Lys Tφ Met Tyr Val Gly Gin Asn Lys Ala Gly 435 440 445

Gn Val Tφ His Asp He Thr Gly Asn Lys Pro Gly Thr Val Thr He 450 455 460 Asn Ala Aεp Gly Tφ Ala Asn Phe Ser Val Asn Gly Gly Ser Val Ser 465 470 475 480

He Tφ Val Lys Arg 485

(2) I ^MATTCN FOR. SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS:

(A) LENGIH: 20 amino acids

(C) S]_AND-_ENESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ 3D NO: 3 :

His His Asn Gly Thr Asn Gly Thr Met Met Gin Tyr Phe Glu Tφ Tyr 1 5 10 15

Leu Pro Asn Asp 20

(2) -NPCRMATTCN F R SEQ 3D NO: 4 :

(i) SEQUENCE C__ R2^CTTE_RISπCS :

(A) LENGTH: 1455 base pairs

(B) TYPE: nucleic acid (C) _3__F?ANDEC_NESS: single

(D) TOP01CGY: linear

(ii) MDI-ECUIJE TYPE: ENA (genomic)

(xi) SEQUENCE DESCRIFTTCN: SEQ ID NO: 4 : CS-]X_A__AA-_G CW_CAAATGG TACT-ATGATG CAA__ATITCG AATOGi TT GCX_AAATGAC 60

GGΪ__V_I _IT GGftACAGGIT GAQGS_IGAC GC_ _CTAACT TAAAGAGTAA AGGGATAACA 120

GCTCTATGGA TOXACCIGC A_-GGAAGC3GG ACTIOOC-AGA AIGATGTAGG TTA_I _X_AGCC 180

TATGATTTAT AIC^OCTTGG AGAGriTAAC CAGAAGGQGA CGGITCGTAC AAAAIAIGGA 240

ACACGCAAOC AGCTACAG3C TT_π_rjD3ACC TCITTAAAAA ATAAC03CAT TCAGGTATAT 300 QGIX-AIGTCG TCAIGAATCA __AAAOC_IGGA CCAGAIGGIA CGGAAAITGT AAATGCOG__A 360

GAAGT3AATC QG_^GCAADCG AAAGCAGGAA ACCTCAGGAG AGIMGCAAT AGAAGCGIGG 420

ACAAAGΓΠG Aτττ_ riGG AAGAQGAAAT AAO_AT_T_CA GCΓΓTAAGIG GCGC GGTAT 480

CZ-ITTTGATG α_ACAGATTG CX-ATCAGICA 0-XXAGC TC AAAACAAAAT A3_ __AAA_[TC 540

AGGX3GAAC_ _ ^} G(_AAGGX_tTG G g GGGAA GIU.ATACAG _^GAA_D3GC_AA σa_K3ftCD_r 600 CTI-_IO-_A-[G O ^OGIQG T_-_π__-ICAC QC-AGAAGIAA TAC__] GAACT TAGAAACIGG 660

QG_ _IOIOGΓ ATAOSAAIAC ACTGAAOC T GA3 _GATTTA GAAT-AGATGC AGIGAAACAT 720

A3-AAAATATΑ GCTITACGAG A-_-^_πGGCTT ACACATCIGC CI5V__ACX_AC AGGTAAAOCA 780

ATGITTGCAG TGCOGAGIT TTGGAAAAAT (_?_XTIGGIG CAATIGAAAA CTA1T1GAAT 840

AAAACAAGIT CGAATCACTC QG33ITTGAT GI.π_TC3X-C ACTATAATTT C^IACAATGCA. 900 _X-TAAΪAGCG C-lLUriM'JA T__-TAT-_AGA AATATITTAA ATOJITLTGI' GC1GCAAAAA 960

C5-ICCAACAC AIC _GTTAC TiTT -lTGAT AACX-ATGATT CTCAGOCCQG QC_ _C_ATTG 1020

GAA_K_CT_TG TTCAACAAIG GTI-J^A OC CTIGCATATG C_-3 K-GITCT GA__AAGQGAA 1080

CAAG-JITATC C TOCCTATT TIA_ _QC3GAT TACTACQGTA T -OCAAGOGA TGGIC£TGCI- 1140

GX_TAIGAAAT CTAAAATAGA GCC1X_TTCIG CAG3CACGIC AAACTTTTGC CTAD ΞGTACG 1200 CAGCAIGAIT A 'iTTUATCA TCATC^TATT ATO3GITOGA CAAGAGAGGG AAAT-^-CTCC 1260

CATCX-AAATT CAC-3CX TIGC CAOCKITATG TO--_-IGGIC (_AGGIOGTAA CAAATGGAIG 1320

T T ^ΠΞGG AAAATAAAGC CKSGACAAGIT TO_AGAGATA TIACO3C_AAA TAQ__¥_AGGC 1380

ADCX-TCACAA TIAAIGCAGA CGGATG9GGT AATTTCTCIG TTAAHXΞGAGG CIOI_- TΑC. 1440

CHTIQGSIGA AGCAA 1455 (2) 3__ -RMATTCN ECR SEQ 3D NO: 5 : (I) SEQUENCE CHAE-^CTERISITCS :

(A) LENGIH: 1455 base pairs

(B) TYPE: nucleic acid

(C) _3-_RANDEDS__SS: single (D) TOPOLOGY: linear

(ii) MDIIECULE TYPE: ENA (genomic)

(xi) SEQUENCE DESCRIPITCN: SEQ 3D NO: 5 :

CATCATAAIG GG&CAAAΪGG C-ACS-IGAIG CAATALTIT AATOGCACTT GOT-AATGAT 60

GQG__ T_ACT QG_V_IA_-ATT AAGAC-ATGAT GCTAGIAATC __AA__AAA_____. AGGTAIAACC 120 C AITΓGGA ττα_GCCTGC CTGGAAAQGG ACΓΠCGCAAA AI AI-TOGG CSCATOGAGOC ι ^β o

TAIC-ΩCTTT GC-AATΓTAAT CAAAAGOGGA CIΞG_Tα_TAC TAAGIAIGOG 240

ACACΠIAGIC AATTGC-AGIC TC<-_- _K_CAT GCΓITΓAAAGA. ATAATΌGCGΓ TCAAGΓITAT 300

GGGG.-IGTAG _X__-T3AACX_A TAAAQGAGGA GCTC3__GCTA CAGAAAACGT TCTIGC GIC 360

GAGGIGAATC CAAAIAAGCG GAATCAAGftA A___-_XπQGQG ACTACACAAT TGAGGCTIOG 420 ACTAAGITTG A__TTTCO-_G GAGGGGIAAT AC__3_AC CAG ACTTTAAATG GCT_ _TG3TAT 480

CA_πT_?Cr_g_IG CΠGI__---I Q-MCAATCA CI-ACAATICC AAAATCGTAΓ CTACAAATTC 540

C-3 D-_A_-G GIAAGGCATG QGA__TGQC_AA CEIAGATIU3G AAAAIG3AAA T3-ATGATTAT 600

TTAA-KΞTATG C_ _ATGIAGA TA_I_GATCAT CX33GAQG__AG TAAAIGAGCT TAGAAC-AIGG 660

GG-^GAAIQGT ATACAAAIAC AT-AAATCTT (.ΪATOGATTTA O-ATO-iATGC GGIC^AGCAT 720 AITAAAIATA GCTTTACAO-. TGA-CTGGTTG ACπ_A__GIAA (3AAACGCAAC C43GAAAAGAA 780

ATO3T-I-CTG TIGCTGAATT TTGGAAAAAT GA-.TTAGSIG GCTR-GAGAA CTATTTAAAT 840

AAAACAAACT C -AATC_ATTC TGICITIGAT GnXTXXCITC AITAIAATCT TTATAACGCG 900

TCAAAIAGIG (.AGCJCAACIA TOACMOGCA AAALTILTIA TC ^CGG TGITCAAAAG 960

CATO-AATGC AIGCXI3__AAC TLTLULUGFT AATCACGAIT CT_AACX_TGG QGAATCATTA 1020 GAATCATTTG TACAAGAAIG G-TTAAGOCA C IGCTTATG CGLT'LA LTTI' AACAAGAGAA 1080

CAAG3CTATC OC__X^CICTT CTA_KΞGIGAC TA-_TA_K-GAA TTOCAACACA TAGIGTCDCA 1140

C _AA__GAAAG CXZAAGATIGA TCCAAICTTA GAGGOΞCGIC AAAAT373TGC AIAIGGAACA 1200

CAA___ _IGA__T A LTLTJAOCA _X_A__AATA-_A ATO3C_A_[GGA CAO-JIGAAGG AAATAOCACG 1260

CAICCCAATT C_AG__^CITGC GACTATCAIG TCGC_A3T_GGC CAGGG3GAGA G-W.IQGATG 1320 TAΑ_TAQ3GC AAAAIAAAGC AGGTGAAGIT -OGC_ -3I_ACA .AACTIGGAAA TAAACCAGGA 1380

A__AGITACGA TCAAIGCAGA TU_A_[GQGCT AATTTTTCAG TAAAIGGAGG ATCTG_TTQC 1440

A_ΓΠGOG_GA AACGA 1455

Intβrnationβl Application No: PCT/

Form CTMO/m (January 1M1)