EP1054957A1 - Amylase bacillaire alcaline - Google Patents

Amylase bacillaire alcaline

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Publication number
EP1054957A1
EP1054957A1 EP99903592A EP99903592A EP1054957A1 EP 1054957 A1 EP1054957 A1 EP 1054957A1 EP 99903592 A EP99903592 A EP 99903592A EP 99903592 A EP99903592 A EP 99903592A EP 1054957 A1 EP1054957 A1 EP 1054957A1
Authority
EP
European Patent Office
Prior art keywords
amylase
alpha
dna sequence
activity
bacillus
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99903592A
Other languages
German (de)
English (en)
Inventor
Helle Outtrup
Bjarne Roenfeldt Nielsen
Lisbeth Hedegaard
Jens Toenne Andersen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novozymes AS
Original Assignee
Novo Nordisk AS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novo Nordisk AS filed Critical Novo Nordisk AS
Publication of EP1054957A1 publication Critical patent/EP1054957A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2414—Alpha-amylase (3.2.1.1.)
    • C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase

Definitions

  • the present invention relates to amylases having improved washing performance m an alkaline detergent solution at low temperature .
  • a-amylase enzymes have been used for a variety of different purposes, the most important of which are starch liquefaction, textile desizing, starch modification m the paper and pulp industry, and for brewing and baking.
  • a further use of a-amylases, which is becoming increasingly important is the removal of starchy stains during washing with a detergent at alkaline pH.
  • a-amylase products examples include Termamyl ® , BAN ® and Fungamyl ® , all available from Novo Nordisk A/S, Denmark. These and similar products from other commercial sources have an acidic to a neutral pH optimum, typically m the range of from pH 5 to pH 7.5, and they do not display optimal activity m detergent solutions at alkaline pH.
  • WO 95/26397 discloses an a-amylase from a Bacillus strain which has optimum activity at pH 8.
  • WO 96/23873 describes variants of Bacillus amylases with improved performance under washing conditions.
  • alpha-amylase which is: a) a polypeptide produced by Bacillus sp . NCIMB 40916, or b) a polypeptide having an ammo acid sequence as shown m positions 32-532 of SEQ ID NO: 2, or c) a polypeptide encoded by the alpha-amylase encoding part of the DNA sequence cloned into a plasmid present m Escheri chi a col l DSM 12662, or d) an analogue of the polypeptide defined m (a) or (b) which :
  • the invention provides an a-amylase having one or more of the following characteristics:
  • thermostability such that it retains more than 90 % of its activity after 20 minutes incubation at 25°C m a solution of 3 g/1 of a test detergent containing 20% STPP, 25% Na2S04,
  • the invention also provides an isolated DNA sequence which encodes an alpha-amylase, wherem the alpha-amylase is that described above, or where the DNA sequence comprises: a) the DNA sequence shown m positions 94-1596 of SEQ ID NO : 1 , or b) an analogue of the DNA sequence defined m a) which l) is at least 60 % homologous with said DNA sequence, or n) hybridizes with said DNA sequence at least 55°C.
  • Other aspects of the invention provide a recombmant expression vector comprising the DNA sequence, and a cell transformed with the DNA sequence or the recombmant expression vector.
  • the invention also provides a method for producing an alpha-amylase by cultivating the cell and a detergent composition comprising said alpha-amylase.
  • Fig. 1 shows a pH activity profile of the amylase from NCIMB 40916 compared to two prior-art amylases (SP722 and Termamyl) .
  • Fig. 2 shows a temperature activity profile of the amylase from NCIMB 40916.
  • Fig. 3 shows the stability of the amylase from NCIMB 40916 after incubation at various temperatures.
  • Fig. 4 shows the cloning vector pSJ1678 described Example 1.
  • Figs. 5 and 6 show results of washing tests described in Example 4.
  • the alpha-amylase of the invention may be derived from a strain of Bacillus .
  • a preferred strain is Bacillus sp . NCIMB 40916. This strain was deposited on 28 January 1998 by the inventors under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure at the National Collections of Industrial and Marine Bacteria Limited (NCIMB), 23 St. Machar Drive, Aberdeen AB2 1RY, Scotland, United Kingdom.
  • a Escheri - chia coli strain termed JA388 containing the alpha amylase gene cloned m plasmid pJA388 has also been deposited on 3 February 1999 under the terms of the Budapest Treaty with the Deutshe Sammmlung von Microorganismen und Zellkulturen GmbH (DSMZ) , Mascheroder Weg lb, D-38124 Braunschweig DE, and given the accession number DSM 12662.
  • DSMZ Deutshe Sammmlung von Microorganismen und Zellkulturen GmbH
  • the alpha-amylase of the invention can be produced by cultivating a suitable amylase-producmg strain of Bacillus or the transformed host cell of the invention m a suitable nutrient medium, and recovering the alpha-amylase from the cul - ture medium.
  • the medium used to cultivate the cells may be any conventional medium suitable for growing the host cell m question and obtaining expression of the a-amylase of the invention. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. as described m catalogues of the American Type Culture Collection) .
  • the a-amylase secreted from the host cells may conveniently be recovered from the culture medium by well-known procedures, including separating the cells from the medium by centrifugation or filtration, and precipitating protemaceous components of the medium by means of a salt such as ammonium sulphate, followed by the use of chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.
  • a preferred alpha-amylase is derived from Bacillus sp . NCIMB 40916. It can be produced as described m the examples. The ammo acid sequence of the amylase and of the DNA encoding it are shown m SEQ ID NO: 1 and 2. The following characteristics were found for an amylase of the mvention (purified alpha-amylase from NCIMB 40916) :
  • a pH-activity curve is shown m Fig. 1, taking the activity at pH 7.3 as 100%. It was determined using the Phadebas assay usmg 50 mM Britten-Robmson buffer ad usted to various pH-values.
  • the pH profiles of two prior- art Bacillus amylases (Termamyl derived from B . licheniformis and SP722 produced according to WO 95/26397) were measured under the same conditions are also shown m Figure 1.
  • Figure 1 shows that the amylase of the invention has about 10 times higher activity than the two prior-art amylases at pH 9.5.
  • Fig. 1 also shows that the optimum activity is about pH 9.5.
  • a temperature-activity curve was measured usmg the Phadebas assay at various temperatures with 50 mM Britten- Robmson buffer adjusted to pH 9.5. The results are shown m Fig. 2. It is seen from Fig. 2 that the amylase of the invention has optimum activity at about 55°C.
  • Stability of the amylase was measured at pH 10.5 (50 mM CAPS) at 22, 37, 45, 55 and 65 °C after 30 minutes incubation.
  • the enzyme was diluted to 40 NU/ml m buffer and 25 microlitre sample was incubated at the respective temperatures. After 30 minutes, the samples was chilled on ice and residual activity was determined.
  • a stability profile of the amylase of the mvention and a prior-art amylase (SP722) is shown m Figure 3.
  • the ammo acid sequence homology may be determined as the degree of identity between the two sequences indicating a derivation of the first sequence from the second.
  • the homology may suitably be determined by means of computer programs known m the art.
  • FASTA provided m GCG version 8 (Needleman, S.B. and Wunsch, CD., (1970), Journal of Molecular Biology, 48, 443-453) may be used with the following settings: Scoring matrix: GenRunData :blosum50. cmp, Variable pa factor used Gap creation penalty: 12, Gap extension penalty: 2.
  • m GCG version 8 Needleman, S.B. and Wunsch, CD., (1970), Journal of Molecular Biology, 48, 443-453
  • Scoring matrix GenRunData :blosum50. cmp
  • Variable pa factor used Gap creation penalty: 12
  • Gap extension penalty 2.
  • Gap from GCG version 9 may be used with a translated version 8 peptide scoring matrix, a gap creation penalty of 30, a gap extension penalty of 1 using ntol ' s matrix
  • the ammo acid sequence exhibits a degree of identity preferably of at least 60%, preferably at least 70%, more preferably at least 80%, especially at least 90% or at least 95%, with the ammo acid sequence shown m positions 32-532 of SEQ ID NO: 2.
  • the DNA sequence homology may be determined as the degree of identity between the two sequences indicating a derivation of the first sequence from the second.
  • the homology may suitably be determined by means of computer programs known m the art such as GAP provided m the GCG program package (described above) .
  • GAP uses the method of Needleman/Wunsch/Sellers to make alignments.
  • the DNA construct of the present invention comprises a DNA sequence exhibiting a degree of identity preferably of at least 60%, preferably at least 70%, more preferably at least 80%, especially at least 90% or at least 95%, with the nucleic acid sequence shown m positions 94-1596 of SEQ ID NO: 1.
  • the hybridization is used to indicate that a given DNA sequence is analogous to a nucleotide probe corresponding to SEQ ID NO : 1.
  • the hybridization conditions are described m detail below.
  • Suitable conditions for determining hybridization between a nucleotide probe and a homologous DNA or RNA sequence involves presoakmg of the filter containing the DNA fragments or RNA to hybridize m 5 x SSC (standard saline citrate) for 10 mm, and prehybridization of the filter m a solution of 5 x SSC (Sambrook et al . 1989), 5 x Denhardt ' s solution (Sambrook et al . 1989), 0.5 % SDS and 100 ⁇ g/ml of denatured sonicated salmon sperm DNA (Sambrook et al . 1989), followed by hybridization m the same solution containing a random-primed (Femberg, A. P.
  • Molecules to which the oligonucleotide probe hybridizes under these conditions are detected using a x-ray film.
  • the expression vector of the invention 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 the a-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.
  • 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, m ichromosome or an artificial chromosome.
  • the vector may be one which, when introduced mto a host cell, is integrated mto the host cell genome and replicated together with the chromosome (s) into which it has been integrated.
  • the DNA sequence should be operably connected to a suitable promoter sequence.
  • the promoter may be any DNA sequence which shows transcriptional activity m the host cell of choice and may be derived from genes encoding pro- terns either homologous or heterologous to the host cell .
  • suitable promoters for directing the transcription of the DNA sequence encoding an a-amylase of the invention, especially a bacterial host are the promoter of the lac operon of E .
  • coli the Streptomyces coeli color agarase gene dagA promoters, the promoters of the Bacillus licheniformi s a- amylase gene ⁇ amyL) , the promoters of the Bacillus stearothermophilus maltogenic amylase gene ⁇ amyM) , the promoters of the Bacillus Amyloliquefaciens a-a ylase ( amyQ) , the promoters of the Bacillus subtili s xylA and xylB genes etc.
  • useful promoters are those derived from the gene encoding A .
  • oryzae TAKA amylase Rhizomucor wiehei aspartic protemase, A . niger neutral a-amylase, A . niger acid stable a-amylase, A . niger glu- coamylase, Rhizomucor miehei lipase, A . oryzae alkaline protease, A . oryzae triose phosphate isomerase or A . nidulans acetamidase .
  • the expression vector of the invention may also comprise a suitable transcription terminator and, m eukaryotes, poly- adenylation sequences operably connected to the DNA sequence encoding the a-amylase of the invention. Termination and poly- adenylation sequences may suitably be derived from the same sources as the promoter.
  • the vector may further comprise a DNA sequence enabling the vector to replicate m the host cell m question.
  • a DNA sequence enabling the vector to replicate m the host cell m question.
  • sequences are the origins of replication of plasmids pUC19, pACYC177, pUBHO, pE194, pAMBl and pIJ702.
  • the vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect m the host cell, such as the dal genes from B . subtili s or B . li chemfor- mis , or one which confers antibiotic resistance such as ampi- cillm, kanamycm, chloramphenicol or tetracyclm resistance.
  • a selectable marker e.g. a gene the product of which complements a defect m the host cell, such as the dal genes from B . subtili s or B . li chemfor- mis , or one which confers antibiotic resistance such as ampi- cillm, kanamycm, chloramphenicol or tetracyclm resistance.
  • the vector may comprise Aspergillus selection markers such as amdS, argB, niaD and sC, a marker giving rise to hygromyc resistance, or the selection may be accomplished by co-
  • the cell of the invention is advantageously used as a host cell m the recombinant production of the a-amylase of the invention.
  • the cell may be transformed with the DNA construct of the invention encoding the amylase, conveniently by integrating the DNA construct (m one or more copies) m the host chromosome. This integration is generally considered to be an advantage as the DNA sequence is more likely to be stably maintained 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 m 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. 10
  • Examples of bacterial host cells which, on cultivation, are capable of producing the enzyme of the invention are gram-positive bacteria such as strains of Bacillus, such as strains of B . subtilis , B . li chenifor i s , B . lentus, B . clausn , B . brevi s, B . s tearothermophilus, B . alkalophilus , B . amyloliquefaciens, B . coagulans , B . circulans , B . lautus , B . mega terium or B . thuringi ensi s, or strains of Streptomyces, such as S . lividans or S .
  • Bacillus such as strains of B . subtilis , B . li chenifor i s , B . lentus, B . clausn , B . brevi s, B . s tearother
  • the transformation of the bacteria may be effected by protoplast transformation, electroporation, conjugation, or by using competent cells m a manner known per se (cf. Sambrook et al . , supra) .
  • the yeast organism may favourably be selected from a species of Saccharomyces or Schizosaccharomyces , e.g. Saccharomyces cerevi siae .
  • the filamentous fungus may advantageously 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 m a manner known per se . A suitable procedure for transformation of Aspergillus host cells is described m EP 238 023.
  • the a- amylases of the invention are well suited for use m a variety of industrial processes, m particular the enzyme finds potential applications as a component m detergents, e.g. laundry and hard surface cleaning detergent compositions, but it may also be useful the production of sweeteners and ethanol from starch.
  • m detergents e.g. laundry and hard surface cleaning detergent compositions
  • sweeteners and ethanol from starch.
  • it may be used m conventional starch- converting processes, such as liquefaction and saccharification processes described US Patent No. 3,912,590 and EP patent publications Nos . 252,730 and 63,909.
  • the alkaline a-amylase of the invention may also be used in the production of lignocellulosic materials, such as pulp, paper and cardboard, from starch reinforced waste paper and cardboard, especially where repulp g occurs at pH above 7 and 11
  • amylases can facilitate the disintegration of the waste material through degradation of the reinforcing starch.
  • the a- amylase of the mvention is especially useful m a process for producing a paper akmg pulp from starch-coated printed paper. The process may be performed as described m WO 95/14807, comprising the following steps: a) disintegrating the paper to produce a pulp, b) treating with a starch-degrading enzyme before, during or after step a) , and c) separating ink particles from the pulp after steps a) and b) .
  • the a-amylases of the invention may also be very useful m modifying starch where enzymatically modified starch is used papermakmg together with alkaline fillers such as calcium carbonate, kaolin and clays. With the alkaline a-amylases of the invention it becomes possible to modify the starch the presence of the filler thus allowing for a simpler integrated process .
  • the a-amylase of the invention may also be very useful m textile desizmg.
  • a- amylases are traditionally used as auxiliaries m the desizmg process to facilitate the removal of starch-contammg size which has served as a protective coatmg on weft yarns during weaving. Complete removal of the size coatmg after weaving is important to ensure optimum results m the subsequent processes, m which the fabric is scoured, bleached and dyed. Enzymatic starch break-down is preferred because it does not involve any harmful effect on the fiber material . In order to reduce processing cost and increase mill throughput, the desizmg processing is sometimes combined with the scouring and bleaching steps.
  • non-enzymatic auxiliaries such as alkali or oxidation agents are typically used to break down the starch, because traditional a-amylases are not very compatible with high pH levels and bleaching agents.
  • the non- enzymatic breakdown of the starch size does lead to some fiber damage because of the rather aggressive chemicals used. Accordingly, it would be desirable to use the a-amylases of the 12
  • the a-amylases may be used alone or m combination with a cellulase when desizmg cellulose-containing fabric or textile .
  • the a-amylases of the invention may also be very useful m a beer-making process; the a-amylases will typically be added during the mashing process.
  • the a-amylase may typically be a component of a detergent composition, e.g., a laundry detergent composition.
  • a detergent composition e.g., a laundry detergent composition.
  • the detergent composition may be included m the detergent composition m the form of a non-dustmg granulate, a stabilized liquid, or a protected enzyme.
  • Non-dustmg granulates may be produced, e.g., as disclosed m US 4,106,991 and 4,661,452 (both to Novo Industri A/S) and may optionally be coated by methods known m the art.
  • waxy coatmg 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 m which the alcohol contains from 12 to 20 carbon atoms and m which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and t ⁇ glycerides of fatty acids.
  • PEG poly (ethylene oxide) products
  • PEG polyethyleneglycol
  • 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.
  • a polyol such as propylene glycol
  • sugar or sugar alcohol a sugar or sugar alcohol
  • lactic acid or boric acid a polyol
  • Other enzyme stabilizers are well known m the art.
  • Protected enzymes may be prepared according to the method disclosed m EP 238,216.
  • the properties of the alpha-amylase of the invention make it particularly suitable for use m alkaline detergents, e.g. at pH 9.5-10.5, and for washing at low temperatures, e.g. 20- 40°C.
  • the detergent composition of the invention may be any convenient form, e.g. as powder, granules, paste or liquid.
  • liquid detergent may be aqueous, typically containing up to 70% water and 0-30% organic solvent, or non-aqueous.
  • the detergent composition comprises one or more surfactants, 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-olefmsulfonate (AOS) , alkyl sulfate (fatty alcohol sulfate) (AS), alcohol ethoxysulfate (AEOS or AES) , secondary alkanesulfonates (SAS) , alpha-sulfo fatty acid methyl esters, alkyl- or alkenylsuccmic acid, or soap.
  • anionic surfactant such as linear alkylbenzene- sulfonate (LAS) , alpha-olefmsulfonate (AOS) , alkyl sulfate (fatty alcohol sulfate) (AS), alcohol ethoxysul
  • nonionic surfactant such as alcohol ethoxylate (AEO or AE) , alcohol propoxylate, carboxylated alcohol ethoxylates, nonylphenol ethoxylate, alkylpolygly- coside, alkyldimethylamme oxide, ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide, or polyhydroxy alkyl fatty acid amide (e.g. as described m WO 92/06154) .
  • AEO or AE alcohol ethoxylate
  • alcohol propoxylate carboxylated alcohol ethoxylates
  • nonylphenol ethoxylate nonylphenol ethoxylate
  • alkylpolygly- coside alkyldimethylamme oxide
  • ethoxylated fatty acid monoethanolamide e.g. as described m WO 92/06154
  • polyhydroxy alkyl fatty acid amide e.g. as described m WO 92/06154
  • the detergent composition may additionally comprise one or more other enzymes, such as pullulanase, esterase, lipase, cutmase, protease, cellulase, peroxidase, or oxidase, e.g., laccase.
  • enzymes such as pullulanase, esterase, lipase, cutmase, protease, cellulase, peroxidase, or oxidase, e.g., laccase.
  • the detergent contains 1-65% of a detergent builder or complexmg agent such as zeolite, diphosphate, tri- phosphate, phosphonate, citrate, mtrilotriacetic acid (NTA) , ethylenediammetetraacetic acid (EDTA) , diethylenet ⁇ - ammepentaacetic acid (DTMPA) , alkyl- or alkenylsuccmic acid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst) .
  • a detergent builder or complexmg agent such as zeolite, diphosphate, tri- phosphate, phosphonate, citrate, mtrilotriacetic acid (NTA) , ethylenediammetetraacetic acid (EDTA) , diethylenet ⁇ - ammepentaacetic acid (DTMPA) , alkyl- or alkenylsuccmic acid, soluble silicates or layered silicates (e.g.
  • the detergent builders may be subdivided into phosphorus- containing and non-phosphorous-containing types.
  • phosphorus-containing inorganic alkaline detergent builders include the water-soluble salts, especially alkali metal pyrophosphates, orthophosphates , polyphosphates and phospho- nates .
  • 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 alummo silicates of which zeolites is the best known representative. 14
  • suitable organic builders include alkali metal, ammonium or substituted ammonium salts of succmates, malonates, fatty acid malonates, fatty acid sulphonates, carboxymethoxy succmates, polyacetates , carboxylates , polycar- boxylates, ammopolycarboxylates 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 (vmylpyrrolidone) (PVP) , polyethyleneglycol (PEG), poly (vinyl alcohol) (PVA) , poly- carboxylates such as polyacrylates, polymaleates , maleic/acrylic acid copolymers and lauryl methacrylate/acrylic acid copolymers.
  • CMC carboxymethylcellulose
  • PVP poly (vmylpyrrolidone)
  • PEG polyethyleneglycol
  • PVA poly (vinyl alcohol)
  • poly- carboxylates such as polyacrylates, polymaleates , maleic/acrylic acid copolymers and lauryl methacrylate/acrylic acid copolymers.
  • the detergent composition may contain bleaching agents of the chlorme/bromme-type or the oxygen-type.
  • the bleaching agents may be coated or encapsulated.
  • Examples of inorganic chlorme/bromme-type bleaches are lithium, sodium or calcium hypochlo ⁇ te or hypobromite as well as chlorinated t ⁇ sodium phosphate.
  • Examples of organic chlorme/bromme-type bleaches are heterocyclic N-bromo and N- chloro lmides such as trichloroisocyanu ⁇ c , t ⁇ bromoiso- cyanuric, dibromoisocyanuric and dichloroisocyanuric acids, and salts thereof with water solubiliz g cations such as potassium and sodium. Hydantom compounds are also suitable.
  • the bleaching system may also comprise peroxyacids of, e.g., the amide, lmide, or sulfone type.
  • the oxygen-type bleach may be an inorganic persalt, preferably with a bleach activator, or a peroxy acid compound.
  • inorganic persalts are alkali metal perborates, both tetrahydrates and monohydrates, alkali metal percarbonates , persilicates and perphosphates .
  • the activator may be tetraacetylethylenediamme (TAED) or nonanoyloxybenzene- sulfonate (NOBS) .
  • the enzymes of the detergent composition of the 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 15
  • the composition may be formulated as described in, e.g., WO 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 including clays, deflocculant material, foam boosters, foam depressors, suds suppressors, anti -corrosion agents, soil -suspending agents, anti-soil-redeposition agents, dyes, dehydrating agents, bactericides , optical brighteners, or perfume.
  • fabric conditioners including clays, deflocculant material, foam boosters, foam depressors, suds suppressors, anti -corrosion agents, soil -suspending agents, anti-soil-redeposition agents, dyes, dehydrating agents, bactericides , optical brighteners, or perfume.
  • the pH (measured in aqueous solution at use concentration) will usually be neutral or alkaline, e.g. in the range of 7-11. More specifically, the alpha-amylase of the invention may be incorporated in any of the detergent formulations described in WO 96/23873.
  • the a-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 a-amylase may be added in an amount corresponding to 0.00001-1 mg (calculated as pure enzyme protein) of a- amylase per liter of wash liquor.
  • Escherichia coli SJ2 is described in Diderichsen, B., Wedsted, U. , Hedegaard, L., Jensen, B.R., Sj ⁇ holm, C. (1990) Journal of Bacteriology, Vol.172, No. 8, p. 4315-4321.
  • the gene bank vector was pSJ1678 which is further disclosed in W094/19454 which is hereby incorporated by reference. 16
  • the gene bank vector pSJ1678 is further disclosed m WO 94/19454 which is hereby incorporated by reference.
  • A/P (Asia/Pacific) Model Detergent has the following composition: 20% STPP (sodium tripolyphosphate) , 25% Na 2 S0 4 ,
  • DNA was isolated from Bacillus sp . NCIMB 40916 by the method of Pitcher, D. G., Saunders,N. A., and Owen, R. J.
  • This gene library was screened for alpha amylase activity and the strain showing alpha amylase activity was termed JA388.
  • This Escheri chia coli strain JA388 which was deposited and given the accession number DSM 12662, and which harbours the plasmid termed pJA388 encoding the alpha amylase was used for DNA sequencing.
  • the Alpha amylase gene cloned in plasmid pJA388 was characterized by DNA sequencing by primer walking, using the
  • Taq deoxytermmal cycle sequencing kit (Perkm Elmer, USA) , fluorescent labeled terminators and appropriate oligonucleotides as primers.
  • the signal peptide and the mature alpha amylase are presented m SEQ ID NO: 2.
  • the deduced N-termmal sequence was verified by sequencing 34 ammo acids at the N-termmal of the protein.
  • E . coli JA388 harboring plasmid pJA388 was cultivated over night m LB-broth containing chloramphenicol 10 ⁇ g/ml , 37 °C, 250 rpm. Cells were harvested from 2.7 1 culture broth by centrifugation at 6000 rpm for 15 minutes. The mtracellular located amylase was released from the cells by using the following osmotic shock procedure:
  • the enzyme solution was applied on a Pharmacia Q Sepharose column FF previously equilibrated m EKV-buffer, pH 7, and the column was washed with EKV-buffer. Bound proteins were eluted with a linear NaCl gradient from 0-500 mM over 10 column volumes. Amylase containing fractions were pooled and dialyzed aga st EKV-buffer over night. The solution was then applied on a Q Sepharose column FF previously equilibrated m EKV-buffer, pH 8, the column was washed with EKV-buffer, and the amylase was eluted with a linear NaCl gradient from 0-500 mM over 10 column volumes. Amylase containing fractions were pooled. A Phenyl Superose column previously equilibrated m EKV- buffer, pH 8 containing 1 M ammonium sulfate was loaded with 18
  • the enzyme solution added ammonium sulfate to a concentration of 1 M. Unbound material was washed out with the ammonium sulfate buffer and the column was eluted with a linear NaCl gradient from 1-0 M ammonium sulfate over 20 column volumes. Amylase containing fractions were pooled.
  • the purified amylase was analyzed by SDS-PAGE and only one band was obtained after staining with Coomasie Blue.
  • Example 4 Washing test Washing performance was evaluated by washing soiled test swatches for 15 minutes at 25°C m a detergent solution with the alpha-amylase of the invention.
  • the detergent used was the A/P Model Detergent described above at 3 g/1 having pH 10.5, or a commercial detergent from Malaysia (FAB Total from Colgate) at 3 g/1 having a pH of approx. 9.7.
  • the purified amylase of Example 3 was added to the detergent solution at the concentration indicated below.
  • the test swatches were soiled with orange rice starch (CS-28 swatches available from CFT, Center for Test Material, Holland) .
  • ⁇ R remission of the swatch washed with the alpha-amylase minus the remission of a swatch washed at the same conditions without the alpha-amylase.
  • Alpha-amylase ⁇ R ⁇ R concentration Model detergent Malaysian detergent (mg enzyme protein

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Abstract

La présente invention concerne des amylases présentant un pouvoir lavant accru dans une solution détergente alcaline à basse température. L'invention concerne plus particulièrement des amylases de l'espèce Bacillus se caractérisant par une efficacité accrue dans des solutions alcalines, plus particulièrement dans des solutions détergentes alcalines dont le pH se situe autour de 9-11.
EP99903592A 1998-02-18 1999-02-15 Amylase bacillaire alcaline Withdrawn EP1054957A1 (fr)

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WO1999042567A1 (fr) 1999-08-26

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