WO2017133974A1 - Performance de lavage améliorée grâce à une alpha-amylase de bacillus cereus - Google Patents

Performance de lavage améliorée grâce à une alpha-amylase de bacillus cereus Download PDF

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WO2017133974A1
WO2017133974A1 PCT/EP2017/051747 EP2017051747W WO2017133974A1 WO 2017133974 A1 WO2017133974 A1 WO 2017133974A1 EP 2017051747 W EP2017051747 W EP 2017051747W WO 2017133974 A1 WO2017133974 A1 WO 2017133974A1
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Prior art keywords
alpha
amylase
amino acid
acid sequence
amylases
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English (en)
Inventor
Daniela HERBST
Nina Mussmann
Timothy O'connell
Inken PRÜSER
Anna Krüger
Neele Meyer
Garabed Antranikian
Anke Peters
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Henkel AG and Co KGaA
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Henkel AG and Co KGaA
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    • 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.)

Definitions

  • the invention is in the field of enzyme technology.
  • the invention relates in particular to alpha-amylases and their preparation whose amino acid sequence, which can be used in particular with regard to use in detergents and cleaning agents, all sufficiently similar alpha-amylases with a correspondingly similar sequence according to SEQ ID NO: 1 and for them coding nucleic acids.
  • the invention further relates to methods and
  • Alpha-amylases are among the most technically important enzymes. Their use for detergents and cleaners is established industrially and they can be contained in modern, powerful detergents and cleaners.
  • An alpha-amylase is an enzyme that catalyzes the hydrolysis of the internal a (1-4) glycoside bonds of amylose but not the cleavage of terminal or a (1-6) glycoside bonds.
  • Alpha-amylases are therefore a group of esterases (E.C. 3.2.1.1.).
  • Alpha-amylases catalyze the cleavage of starch, glycogen, and other oligo- and polysaccharides that have a (1-4) -glycoside linkage. In this respect, alpha-amylases counteract starch residues in the laundry and catalyze their hydrolysis
  • Alpha-amylases with broad substrate spectra are used in particular where inhomogeneous raw materials or substrate mixtures have to be reacted, that is, for example, in detergents and cleaning agents, since contaminants can consist of differently structured starch molecules and oligosaccharides.
  • alpha-amylases used in the washing or cleaning agents known from the prior art are usually of microbial origin and are generally derived from bacteria or fungi, for example the genera Bacillus, Pseudomonas, Acinetobacter, Micrococcus, Humicola, Trichoderma or Trichosporon, in particular Bacillus , Alpha-amylases are usually produced by biotechnological methods known per se by suitable microorganisms, for example by transgenic expression hosts of the genera Bacillus or by filamentous fungi.
  • a particularly extensively characterized alpha-amylase is one from the alkalophilic Bacillus sp. Strain TS-23, which hydrolyzes at least five types of starch (Lin et al., Biotechnol Appl Biochem, 28: 61-68, 1998).
  • the alpha-amylase from Bacillus sp. Strain TS-23 has a pH optimum of 9, although it is stable over a broad range of pH (ie, pH 4.7 to - - -
  • alpha-amylase variants which have altered biochemical properties and thereby provide improved performance in industrial applications, particularly in detergents or cleaners.
  • an alpha-amylase from Bacillus cereus or a sufficiently similar alpha-amylase particularly suitable for use in detergents or cleaners, since they are a wide range of starch substrates under standard Washing conditions hydrolyzed.
  • the invention therefore in a first aspect comprises an alpha-amylase comprising an amino acid sequence which has at least 70% sequence identity with the amino acid sequence given in SEQ ID NO: 1 over its entire length.
  • Another object of the invention is a process for producing an alpha-amylase comprising providing a parent alpha-amylase which is at least 70%
  • alpha-amylase in the sense of the present patent application therefore comprises both the alpha-amylase as such and an alpha-amylase prepared by a method according to the invention. All statements on alpha-amylase therefore relate both to the alpha-amylase as a substance and to the corresponding processes, in particular the production process of the alpha-amyalse.
  • alpha-amylases according to the invention or the production process for alpha-amylases according to the invention for these alpha-amylases encoding nucleic acids, alpha-amylases according to the invention or
  • Nucleic acids containing non-human host cells and alpha-amylases according to the invention comprising agents, in particular detergents and cleaners, washing and
  • the present invention is based on the surprising finding of the inventors that an alpha-amylase according to the invention from Bacillus cereus, which comprises at least 70% identical amino acid sequence to the amino acid sequence given in SEQ ID NO: 1, the hydrolysis of a broad spectrum of starch substrates under standard washing conditions - - causes.
  • This is particularly surprising insofar as hitherto for none of the alpha-amylases from Bacillus cereus the use in detergents or cleaning agents has been described.
  • the alpha-amylases according to the invention have a high stability in detergents or cleaners, for example with respect to surfactants and / or bleaches and / or with respect to temperature influences, and / or against acidic or alkaline conditions and / or with respect to pH changes and / or against denaturing or oxidizing agents and / or against proteolytic degradation and / or against a change in the redox ratios.
  • performance-enhanced alpha-amylase variants are provided. Such advantageous
  • An alpha-amylase according to the invention has an enzymatic activity, that is to say it is capable of hydrolysing starch and oligosaccharides, in particular in a washing or cleaning agent.
  • An alpha-amylase according to the invention is therefore an enzyme which catalyzes the hydrolysis of a (1-4) -glycoside bonds in glycoside substrates and thereby is able to cleave starch or oligosaccharides. Furthermore, it is at a
  • alpha-amylase according to the invention is preferably a mature alpha-amylase, i. to the catalytically active molecule without signal and / or propeptide (s). Unless otherwise stated, the sequences given refer to each mature (processed) enzymes.
  • the alpha-amylase comprises a
  • sequence comparison is based on the BLAST algorithm established and commonly used in the prior art (see, for example, Altschul, SF, Gish, W., Miller, W., Myers, EW & Lipman, DJ. (1990) "Basic local alignment search Biol. 215: 403-410; and Altschul, Stephan F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Hheng Zhang, Webb Miller, and David J.
  • Lipman (1997): "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs"; Nucleic Acids Res., 25, pp.3389-3402) and is in principle accomplished by similar sequences of nucleotides or amino acids in the nucleic acid or amino acid sequences of each other be assigned. A tabular assignment of the respective positions is referred to as alignment.
  • Another algorithm available in the prior art is the FASTA algorithm. Sequence comparisons (alignments), - - Especially multiple sequence comparisons are created with computer programs.
  • T-Coffee see, for example, Notredame et al (2000): T-Coffee: A novel method for multiple sequence alignments, J. Mol. Biol. 302, 205-217 or programs based on these programs or algorithms.
  • alignment comparisons with the computer program Vector NTI® Suite 10.3 (Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, California, USA) with the default parameters whose AlignX module for sequence comparisons is based on ClustalW.
  • Identity and / or homology information can be made about whole polypeptides or genes or only over individual regions. Homologous or identical regions of different nucleic acid or amino acid sequences are therefore defined by matches in the sequences. Such areas often have identical functions. They can be small and comprise only a few nucleotides or amino acids. Often, such small regions exert essential functions for the overall activity of the protein. It may therefore be useful to relate sequence matches only to individual, possibly small areas. Unless otherwise indicated, identity or homology information in the present application, however, refers to the total length of the particular nucleic acid or amino acid sequence indicated.
  • the alpha-amylase is characterized in that its purification performance is not significantly reduced over that of an alpha-amylase comprising an amino acid sequence corresponding to the amino acid sequences set forth in SEQ ID NO: 1, i. at least 70%, 75%, 80%, 85%, 90%, 95% of
  • the cleaning performance can be determined in a washing system containing a detergent in a dosage between 4.5 and 7.0 grams per liter of wash liquor and the alpha-amylase, wherein the alpha-amylases to be compared
  • Concentration (based on active protein) are used and the cleaning performance against a cotton soiling is determined by measuring the
  • Detersive detergent is from 0.001-1 wt.%, Preferably from 0.001-0, 1 wt.%, And more preferably from 0.01 to 0.06 wt.%, Based on active, purified protein.
  • a preferred liquid detergent for such a washing system is composed as follows (all figures in weight percent): 7% alkylbenzenesulfonic acid, 9% anionic surfactants, 4% Na salts of C12-C18 fatty acids, 7% nonionic surfactants, 0, 7% phosphonates, 3.2%
  • Citric acid 3.0% NaOH, 0.04% defoamer, 5.7% 1, 2-propanediol, 0, 1%
  • the dosage of the liquid detergent is between 4.5 and 6.0 grams per liter of wash liquor, for example, 4.7, 4.9 or 5.9 grams per liter of wash liquor. Preference is given to washing in a pH range between pH 8 and pH 10.5, preferably between pH 8 and pH 9.
  • the degree of whiteness i. the brightening of the stains, as a measure of the cleaning performance is determined by optical measurement methods, preferably photometrically.
  • a suitable device for this purpose is for example the spectrometer Minolta CM508d.
  • the devices used for the measurement are previously calibrated with a white standard, preferably a supplied white standard.
  • the activity-equivalent use of the respective alpha-amylase ensures that, even if the ratio of active substance to total protein (the values of the specific activity) diverge, the respective enzymatic properties, for example the cleaning performance of certain soils, are compared.
  • a low specific activity can be compensated by adding a larger amount of protein.
  • the alpha-amylase activity is determined in a customary manner, preferably by an optical measuring method, preferably a photometric method.
  • the appropriate test involves the alpha-amylase-dependent cleavage of the substrate para-nitrophenyl maltoheptaoside. This is cleaved by the alpha-amylase into para-nitrophenyl oligosaccharide.
  • the para-nitrophenyl oligosaccharide is in turn catalyzed by the enzymes glucoamylase and alpha-glucosidase to glucose and para-nitrophenol.
  • the presence of para-nitrophenol can be measured using a photometer, e.g. of the Tecan Sunrise device and the XFLUOR software, at 405 nm and thus allows a conclusion on the
  • the protein concentration can be determined by known methods, for example, the BCA method (bicinchoninic acid, 2,2'-biquinolyl-4,4'-dicarboxylic acid) or the biuret method (AG Gornall, CS Bardawill and MM David, J. Biol. Chem., 177 (1948), pp. 751-766).
  • the determination of the active protein concentration can in this regard via a titration of the active sites using a suitable irreversible inhibitor and determination of the
  • Proteins can be grouped into groups of immunologically related proteins by reaction with an antiserum or antibody.
  • the members of such a group are characterized by having the same antigenic determinant recognized by an antibody. They are therefore structurally so similar to each other that they are recognized by an antiserum or specific antibodies. Another one
  • alpha-amylases which are characterized in that they have at least one and increasingly preferably two, three or four matching antigenic determinants with an alpha-amylase according to the invention. Due to their immunological similarity, such alpha-amylases are structurally so similar to the alpha-amylases according to the invention that a similar function is to be assumed as well.
  • alpha-amylases according to the invention may have further amino acid changes, in particular amino acid substitutions, insertions or deletions.
  • Such alpha-amylases are, for example, by targeted genetic alteration, i. by mutagenesis, further developed and optimized for specific applications or specific properties (for example, in terms of catalytic activity, stability, etc.).
  • Nucleic acids are introduced into recombination approaches and thus used to generate completely novel alpha-amylases or other polypeptides.
  • the goal is to introduce into the known molecules targeted mutations such as substitutions, insertions or deletions, for example, to improve the cleaning performance of enzymes of the invention.
  • targeted mutations such as substitutions, insertions or deletions, for example, to improve the cleaning performance of enzymes of the invention.
  • the surface charges and / or the isoelectric point of the molecules and thereby their interactions with the substrate can be changed.
  • the net charge of the enzymes can be changed in order to influence the substrate binding, in particular for use in detergents and cleaners.
  • the stability of the alpha-amylase can be further increased by one or more corresponding mutations, thereby improving its cleaning performance.
  • Advantageous properties of individual mutations, eg individual substitutions may be complementary.
  • An alpha-amylase which has already been optimized with regard to certain properties, for example with regard to its activity and / or its tolerance with respect to the substrate spectrum, can therefore be further developed within the scope of the invention.
  • Another object of the invention is therefore an alpha-amylase, which is characterized in that it is obtainable from an alpha-amylase as described above as the starting molecule by one or more conservative amino acid substitution.
  • conservative amino acid substitution means the substitution of one amino acid residue for another amino acid residue, which substitution does not result in a change in polarity or charge at the position of the exchanged amino acid, e.g. Example, the replacement of a nonpolar amino acid residue against another nonpolar amino acid residue.
  • the alpha-amylase is characterized in that it is obtainable from an alpha-amylase according to the invention as the starting molecule by fragmentation, deletion, insertion or substitution mutagenesis and comprises an amino acid sequence which is at least 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 645, 650, 651, 652, 653, 654, 655, 656, 657, or 658
  • the enzymes retain their catalytic activity, i. their catalytic activity is at least equal to that of the starting enzyme, i. In a preferred embodiment, the catalytic activity is at least 80, preferably at least 90% of the activity of the
  • the further amino acid positions are hereby defined by an alignment of the amino acid sequence of an alpha-amylase according to the invention with the amino acid sequence of the alpha-amylase from Bacillus cereus, as indicated in SEQ ID NO: 1. Furthermore, the assignment of the positions depends on the mature (mature) protein. This assignment is also to be used in particular if the amino acid sequence of an alpha-amylase according to the invention comprises a higher number of amino acid residues than the Bacillus cereus alpha-amylase according to SEQ ID NO: 1. Starting from said positions in the amino acid sequence of the Bacillus alpha-amylase cereus are the change positions in an alpha-amylase according to the invention those which are just assigned to these positions in an alignment. - -
  • N-terminal 10-30 amino acids are no longer included.
  • these truncated fragments variants can be used in various embodiments of the invention, which at least 70% of the total length of the amino acid sequence given in SEQ ID NO: 1 truncated by 10-30 N-terminal amino acids, 71% , 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88 %, 89%, 90%, 90.5%, 91%, 91, 5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95, 5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 98.8%, 99.0%, 99.2%, 99.4%, 99.5% %, 99.6% or 99.8% are identical.
  • a method according to the invention further comprises one or more of the following method steps: a) introduction of a single or multiple conservative amino acid substitution into an initial alpha-amylase according to SEQ ID NO: 1; b) altering the amino acid sequence by fragmentation, deletion, insertion or substitution mutagenesis such that the alpha-amylase comprises an amino acid sequence of at least 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 645, 650, 651, 652, 653, 654, 655, 656, 657 or 658
  • the alpha-amylase or the alpha-amylase produced by a process according to the invention is still at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91, 5%, 92 %, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 98.8%, 99%, 99.2%, 99.4%, 99.5%, 99.6% or 99.8% identical to the amino acid sequence given in SEQ ID NO: 1 via their Overall length.
  • Another object of the invention is a previously described alpha-amylase, which is additionally stabilized, in particular by one or more mutations, for example substitutions, or by coupling to a polymer.
  • a polymer for example, ethylene glycol, ethylene glycol, ethylene glycol, ethylene glycol, ethylene glycol, ethylene glycol, ethylene glycol, ethylene glycol, ethylene glycol, ethylene glycol, ethylene glycol, ethylene glycosulftas, g., g., g., g., g., tys, a poly(ethylene glycol) glyceride, asethyl, glyceride, ethylene glycol, ethylene glycol, ethylene glycol, ethylene glycol, ethylene glycol, ethylene glycol, ethylene glycol, ethylene glycol, ethylene glycol, ethylene glycol, ethylene glycol, ethylene glycol, glyceride, ethylene glycol, ethylene glycol, glyceride
  • Preferred embodiments are those in which the enzyme is stabilized in several ways, as several stabilizing mutations act additive or synergistic.
  • Another object of the invention is an alpha-amylase as described above, which is characterized in that it has at least one chemical modification.
  • An alpha-amylase having such a change is termed a derivative, i. the alpha-amylase is derivatized.
  • derivatives are understood as meaning those proteins whose pure amino acid chain has been chemically modified.
  • Derivatizations can be made, for example, in vivo by the host cell expressing the protein. In this regard, couplings of low molecular weight compounds such as lipids or oligosaccharides are particularly noteworthy. However, derivatizations can also be carried out in vitro, for example by the chemical transformation of a side chain of an amino acid or by covalent binding of another compound to the protein. For example, the - -
  • Coupling of amines to carboxyl groups of an enzyme to change the isoelectric point possible Such another compound may also be another protein that is bound to a protein of the invention via bifunctional chemical compounds, for example.
  • derivatization is the covalent bond to a
  • Derivatizations may, for example, affect the substrate specificity or binding strength to the substrate or cause a temporary blockage of the enzymatic activity when the coupled substance is an inhibitor. This can be useful, for example, for the period of storage. Such modifications may further affect stability or enzymatic activity. They can also serve to reduce the allergenicity and / or immunogenicity of the protein and thus, for example, increase its skin compatibility. For example, couplings with macromolecular compounds, for example, polyethylene glycol, can improve the protein in terms of stability and / or skin tolerance.
  • a protein may be associated with various other substances, for example from the culture of the producing microorganisms.
  • a protein may also have been deliberately added to other substances, for example to increase its storage stability.
  • alpha-amylases or alpha-amylase variants and / or derivatives particular preference is given in the context of the present invention to those whose catalytic activity and / or their substrate tolerance correspond to those of the alpha-amylase according to SEQ ID NO: 1, wherein the catalytic activity and the
  • Substrate tolerance can be determined as described above.
  • a further subject of the invention is a nucleic acid which codes for an alpha-amylase according to the invention, as well as a vector containing such a nucleic acid, in particular a cloning vector or an expression vector.
  • a nucleic acid which codes for an alpha-amylase according to the invention, as well as a vector containing such a nucleic acid, in particular a cloning vector or an expression vector.
  • Nucleic acid is a nucleic acid according to SEQ ID NO: 2. Accordingly, a particularly preferred vector according to the invention is a vector comprising a nucleic acid according to SEQ ID NO: 2. - -
  • DNA or RNA molecules may be DNA or RNA molecules. They can be present as a single strand, as a single strand that is complementary to this single strand, or as a double strand. Especially in the case of DNA molecules, the sequences of both complementary strands must be taken into account in all three possible reading frames. Furthermore, it should be noted that different codons, so base triplets, can code for the same amino acids, so that a particular amino acid sequence can be encoded by several different nucleic acids. Due to this degeneracy of the genetic code, all nucleic acid sequences are included in this subject of the invention which can encode any of the alpha-amylases described above. The expert is able to do this
  • nucleic acids easily. Furthermore, with nucleic acids according to the invention, one or more codons may be replaced by synonymous codons. This aspect relates in particular to the heterologous expression of the enzymes according to the invention. Thus, every organism, for example a host cell of a production strain, has a certain codon usage. Under codon use is the translation of the genetic code in
  • a person skilled in the art can use well-known methods such as chemical synthesis or the polymerase chain reaction (PCR) in combination with molecular biological and / or proteinchemical standard methods, using known DNA and / or amino acid sequences, the corresponding nucleic acids to complete genes manufacture.
  • PCR polymerase chain reaction
  • Such methods are for example from Sambrook, J., Fritsch, E.F. and Maniatis, T. 2001. Molecular cloning: a laboratory manual, 3rd Edition Cold Spring Laboratory Press.
  • vectors are understood as consisting of nucleic acids which contain a nucleic acid according to the invention as a characteristic nucleic acid region. They can establish these in a species or cell line over several generations or cell divisions as a stable genetic element.
  • Vectors, especially when used in bacteria, are special plasmids, ie circular genetic elements.
  • a nucleic acid according to the invention is cloned into a vector.
  • the vectors include, for example, those whose origin is bacterial - -
  • Plasmids, viruses or bacteriophages are, or predominantly synthetic vectors or plasmids with elements of various origins. With the other genetic elements present in each case, vectors are able to establish themselves as stable units in the relevant host cells over several generations. They may be extra chromosomally present as separate units or integrated into a chromosome or chromosomal DNA.
  • Expression vectors comprise nucleic acid sequences which enable them to replicate in the host cells containing them, preferably microorganisms, particularly preferably bacteria, and to express a contained nucleic acid there.
  • expression is influenced by the promoter (s) that regulate transcription.
  • the expression may be effected by the natural promoter originally located in front of the nucleic acid to be expressed, but also by a promoter of the host cell provided on the expression vector or also by a modified or completely different promoter of another organism or another host cell.
  • at least one promoter for the expression of a nucleic acid according to the invention is made available and used for its expression.
  • expression vectors can be regulatable, for example by changing the culturing conditions or when a specific cell density of the host cells contained therein is reached or by addition of specific substances, in particular activators of gene expression.
  • An example of such a substance is the galactose derivative isopropyl- ⁇ -D-thiogalactopyranoside (IPTG), which is used as an activator of the bacterial lactose operon (lac operon).
  • IPTG galactose derivative isopropyl- ⁇ -D-thiogalactopyranoside
  • lac operon lac operon
  • a further subject of the invention is a non-human host cell which contains a nucleic acid according to the invention or a vector according to the invention, or which contains an alpha-amylase according to the invention, in particular one which secretes the alpha-amylase into the medium surrounding the host cell.
  • a nucleic acid according to the invention or a vector according to the invention is transformed into a microorganism, which then has a
  • Nucleic acid parts or fragments of a nucleic acid according to the invention are introduced into a host cell such that the resulting host cell contains a nucleic acid according to the invention or a vector according to the invention. This procedure is particularly suitable when the host cell already contains one or more constituents of a nucleic acid according to the invention or a vector according to the invention and the others
  • preferred host cells are characterized - - Good microbiological and biotechnological handling. This concerns, for example, easy culturing, high growth rates, low demands on fermentation media and good production and secretion rates for foreign proteins.
  • Preferred host cells according to the invention secrete the (transgenially) expressed protein into the medium surrounding the host cells.
  • the alpha-amylases can be modified by the cells producing them after their preparation, for example by attachment of sugar molecules,
  • inventions are those host cells which are regulatable in their activity due to genetic regulatory elements which are provided, for example, on the vector, but may also be present in these cells from the outset.
  • Preferred host cells are prokaryotic or bacterial cells. Bacteria are characterized by short generation times and low demands on cultivation conditions. As a result, inexpensive cultivation methods or production methods can be established. In addition, the expert has a rich in bacteria in the fermentation technology
  • gram-negative or gram-positive bacteria may be suitable for a wide variety of reasons to be determined experimentally in individual cases, such as nutrient sources, product formation rate, time requirement, etc.
  • Gram-negative bacteria such as Escherichia coli
  • Gram-negative bacteria can also be designed such that they eject the expressed proteins not only into the periplasmic space but into the medium surrounding the bacterium.
  • Gram-positive bacteria such as Bacilli or Actinomycetes or other representatives of Actinomycetales have no outer membrane, so that secreted proteins readily into the medium surrounding the bacteria, usually the
  • Nutrient medium can be dispensed, from which the expressed proteins can be purified. They can be isolated directly from the medium or further processed.
  • Gram-positive bacteria are related or identical to most of the organisms of origin for technically important enzymes and usually form even comparable enzymes, so they have a similar codon use and their protein synthesizer is naturally aligned accordingly. - -
  • Host cells according to the invention may be altered in their requirements of the culture conditions, have different or additional selection markers or express other or additional proteins. In particular, it may also be those host cells which express several proteins or enzymes transgene.
  • the present invention is applicable in principle to all microorganisms, in particular to all fermentable microorganisms, particularly preferably those of the genus Bacillus, and results in the production of proteins according to the invention by the use of such microorganisms. Such microorganisms then represent host cells in the sense of the invention.
  • the host cell is characterized in that it is a bacterium, preferably one selected from the genera of Escherichia, Klebsiella, Bacillus, Staphylococcus, Corynebacterium, Arthrobacter, Streptomyces, Stenotrophomonas and Pseudomonas, more preferably one selected from the group consisting of Escherichia coli, Klebsiella planticola, Bacillus licheniformis, Bacillus lentus, Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus alcalophilus, Bacillus globigii, Bacillus gibsonii, Bacillus clausii, Bacillus halodurans, Bacillus pumilus, Staphylococcus carnosus, Corynebacterium glutamicum , Arthrobacter oxidans, Streptomyces lividans, Streptomyces coelicolor and
  • the host cell may also be a eukaryotic cell, which is characterized in that it has a cell nucleus.
  • a further subject of the invention therefore represents a host cell, which is characterized in that it has a cell nucleus.
  • eukaryotic cells are capable of producing the protein formed
  • fungi such as Actinomycetes or yeasts such as Saccharomyces or Kluyveromyces.
  • yeasts such as Saccharomyces or Kluyveromyces.
  • Modifications that eukaryotic systems perform, especially in connection with protein synthesis include, for example, the binding of low molecular weight compounds such as membrane anchors or oligosaccharides.
  • oligosaccharide modifications may be desirable, for example, to lower the allergenicity of an expressed protein.
  • coexpression with the enzymes naturally produced by such cells, such as cellulases may be advantageous.
  • thermophilic fungal expression systems may be particularly suitable for the expression of temperature-resistant proteins or variants.
  • the host cells according to the invention are conventionally cultivated and fermented, for example in discontinuous or continuous systems.
  • a - Inoculated suitable nutrient medium with the host cells and harvested the product after an experimentally determined period of time from the medium.
  • Continuous fermentations are characterized by achieving a flow equilibrium, in which over a relatively long period of time cells partly die out but also regrow and at the same time the protein formed can be removed from the medium.
  • Host cells according to the invention are preferably used to produce alpha-amylases according to the invention.
  • Another object of the invention is therefore a method for producing an alpha-amylase comprising
  • This subject invention preferably comprises fermentation processes. Fermentation processes are known per se from the prior art and represent the actual large-scale production step, usually followed by a suitable purification method of the product produced, for example the alpha-amylase according to the invention. All
  • Fermentation processes which are characterized in that the fermentation is carried out via a feed strategy, come in particular into consideration.
  • the fermentation is carried out via a feed strategy
  • the fermentation can also be designed so that undesired metabolic products are filtered out or neutralized by the addition of buffer or suitable counterions.
  • the produced alpha-amylase can be harvested from the fermentation medium.
  • Such a fermentation process is resistant to isolation of the alpha-amylase from the host cell, i. however, requires the provision of suitable host cells or one or more suitable secretion markers or mechanisms and / or transport systems for the host cells to secrete the alpha-amylase into the fermentation medium.
  • suitable host cells or one or more suitable secretion markers or mechanisms and / or transport systems for the host cells to secrete the alpha-amylase into the fermentation medium.
  • isolation of the alpha-amylase from the host cell i. a purification of the same from the
  • Another object of the invention is an agent which is characterized in that it contains an alpha-amylase according to the invention as described above.
  • the agent is preferably a washing or cleaning agent.
  • This subject matter of the invention includes all conceivable types of detergents or cleaners, both concentrates and undiluted agents, for use on a commercial scale, in the washing machine or in hand washing or cleaning. These include detergents for textiles, carpets, or natural fibers for which the
  • Label detergent is used. These include, for example, dishwashing detergents for dishwashers or manual dishwashing detergents or cleaners for hard surfaces such as metal, glass, porcelain, ceramics, tiles, stone, painted surfaces, plastics, wood or leather, for which the term detergent is used, ie in addition to manual and machine Dishwashing agents, for example, scouring agents, glass cleaners, toilet scenters, etc.
  • the washing and cleaning agents in the invention also include washing aids which are added to the actual detergent in the manual or machine textile laundry to achieve a further effect. Furthermore count to washing and
  • Textilvor- and post-treatment agent ie those means with which the garment is brought into contact before the actual laundry, for example, to dissolve stubborn dirt, and also such agents, the laundry downstream in one of the actual textile laundry further give desirable properties such as comfortable grip, crease resistance or low static charge.
  • the fabric softener i.a. calculated the fabric softener.
  • the washing or cleaning agents according to the invention may contain, in addition to an alpha-amylase according to the invention, all known ingredients customary in such agents, preferably at least one further ingredient in the composition is available.
  • the agents according to the invention may in particular contain surfactants, builders, peroxygen compounds or bleach activators. In addition, they may contain water-miscible organic solvents, further enzymes, sequestering agents, electrolytes, pH regulators and / or further auxiliaries such as optical brighteners, grayness inhibitors, foam regulators, as well as dyes and fragrances, and combinations thereof.
  • a combination of an alpha-amylase according to the invention with one or more further ingredients of the composition is advantageous, since in preferred embodiments according to the invention such an agent has an improved cleaning performance by virtue of resulting synergisms.
  • Peroxygen compound and / or a bleach activator such a synergism can be achieved.
  • Patent Application WO2009 / 121725 beginning on page 5, penultimate paragraph, and ending on page 13 after the second paragraph. This disclosure is incorporated herein by reference and the disclosure is incorporated herein by reference.
  • the agent is characterized in that it
  • An agent according to the invention advantageously contains the alpha-amylase in an amount of from 2 ⁇ g to 20 mg, preferably from 5 ⁇ g to 17.5 mg, more preferably from 20 ⁇ g to 15 mg and very particularly preferably from 50 ⁇ g to 10 mg per g of the composition.
  • the alpha-amylase in an amount of from 2 ⁇ g to 20 mg, preferably from 5 ⁇ g to 17.5 mg, more preferably from 20 ⁇ g to 15 mg and very particularly preferably from 50 ⁇ g to 10 mg per g of the composition.
  • composition according to the invention the alpha-amylase advantageously in an amount of 0.00005-15 wt .-% based on the active enzyme and the total weight of the composition, preferably from 0.0001-5 wt .-% and particularly preferably from 0.001-1 wt .-% contain.
  • the alpha-amylase contained in the agent, and / or other ingredients of the composition may be coated with a substance impermeable to the enzyme at room temperature or in the absence of water, which becomes permeable to the enzyme under conditions of use of the agent.
  • Such an embodiment of the invention is thus characterized in that the alpha-amylase is coated with a substance which is impermeable to the alpha-amylase at room temperature or in the absence of water.
  • the washing or cleaning agent itself may be packaged in a container, preferably an air-permeable container, from which it is released shortly before use or during the washing process.
  • the agent is characterized in that it
  • (A) is in solid form, in particular as a free-flowing powder having a bulk density of 300 g / l to 1200 g / l, in particular 500 g / l to 900 g / l, or
  • (b) is in pasty or liquid form, and / or - -
  • (c) is in the form of a gel or pouch, and / or
  • (d) is present as a one-component system, or
  • compositions according to the invention include all solid, powdered, liquid, gelatinous or paste-like administration forms of compositions according to the invention, which if appropriate can also consist of several phases and can be present in compressed or uncompressed form.
  • the agent can be present as a free-flowing powder, in particular with a bulk density of 300 g / l to 1200 g / l, in particular 500 g / l to 900 g / l or 600 g / l to 850 g / l.
  • the solid dosage forms of the composition also include extrudates, granules, tablets or pouches.
  • the agent can also be liquid, gelatinous or pasty, for example in the form of a non-aqueous liquid detergent or a non-aqueous paste or in the form of an aqueous liquid detergent or a water-containing paste.
  • agent may be present as a one-component system. Such funds consist of one phase. Alternatively, an agent can also consist of several phases. Such an agent is therefore divided into several components.
  • Detergents or cleaning agents according to the invention may contain exclusively an alpha-amylase. Alternatively, they may also contain other hydrolytic enzymes or other enzymes in a concentration effective for the effectiveness of the agent. Another
  • Embodiments of the invention thus represent agents which further comprise one or more further enzymes.
  • Other enzymes which can preferably be used as further enzymes are all enzymes which can develop a catalytic activity in the agent according to the invention, in particular one
  • Protease lipase, cellulase, hemicellulase, mannanase, tannase, xylanase, xanthanase, xyloglucanase, ⁇ -glucosidase, pectinase, carrageenase, perhydrolase, oxidase, oxidoreductase or other - distinguishable from the alpha-amylases of the invention - alpha-amylases, and mixtures thereof.
  • Further enzymes are advantageously contained in the agent in each case in an amount of 1 ⁇ 10 -8 to 5 percent by weight, based on active protein.
  • each additional enzyme is in an amount of 1 x 10 -3 ⁇ 7 wt .-%, of 0.00001-1 wt .-%, of 0.00005 to 0.5 wt .-%, from 0.0001 to 0, 1 wt .-% and particularly preferably from 0.0001 to 0.05 wt .-% in agents according to the invention, based on active protein.
  • the enzymes show synergistic cleaning performance against certain stains or stains, ie the enzymes contained in the middle composition mutually support each other in their cleaning performance.
  • such a synergism is present between the alpha-amylase according to the invention and another enzyme of an agent according to the invention, including in particular between said alpha-amylase and a lipase and / or a protease and / or a mannanase and / or a cellulase and / or a pectinase.
  • Synergistic effects can occur not only between different enzymes, but also between one or more enzymes and other ingredients of the composition according to the invention.
  • Another object of the invention is a process for the cleaning of textiles or hard surfaces, which is characterized in that in at least one process step, an inventive agent is applied, or that in at least one process step, an alpha-amylase according to the invention is catalytically active, in particular such that the alpha-amylase is used in an amount of from 4C to 4g, preferably from 5C to 3g, more preferably from 10C to 2g and most preferably from 20C to 1g.
  • the method described above is characterized in that the alpha-amylase at a temperature of 0-100 ° C, preferably 0-60 ° C, more preferably 20- 45 ° C and most preferably used at 40 ° C. becomes.
  • Processes for the purification of textiles are generally distinguished by the fact that various cleaning-active substances are applied to the fabric in several process steps
  • the material to be cleaned is applied and washed off after the action time, or the material to be cleaned is treated in any other way with a detergent or a solution or dilution of this agent.
  • a detergent or a solution or dilution of this agent is applied in any other way with a detergent or a solution or dilution of this agent.
  • All conceivable washing or cleaning processes can be enriched in at least one of the process steps by the use of a washing or cleaning agent or an alpha-amylase according to the invention and then represent embodiments of the present invention.
  • All facts, objects and embodiments which are essential for alpha- Amylases and agents containing them are also applicable to this subject of the invention. Therefore, reference is made at this point expressly to the disclosure in the appropriate place with the statement that this disclosure also for the above
  • alpha-amylases according to the invention naturally already have a hydrolytic activity and also unfold them in media which otherwise have no cleaning power, as for example in bare buffer, a single and / or the sole step of such a method may be that, if desired, the only detergent-active component an alpha-amylase according to the invention is brought into contact with the soiling, preferably in a buffer solution or in water. This represents another embodiment of this
  • the present invention relates to the use of an alpha-amylase according to the invention or an alpha-amylase obtainable by a process according to the invention in a washing or cleaning agent for removing starch-containing soils.
  • a metagenome database was prepared, which was examined for amylase activity.
  • a wild-type enzyme annotated as alpha-amylase from Bacillus cereus, was discovered.
  • the corresponding gene could be isolated, transformed into E. coli and subsequently expressed therein.
  • the E. coli produced amylase was tested for washing performance on various starchy fabrics.
  • the sequence found differs significantly from the sequences of amylases previously used in detergents and cleaners.
  • amylolytic activity of amylases of the invention a modified para-nitrophenyl maltoheptaoside was used whose terminal glucose unit was blocked by a benzylidene group. From this molecule is released by the amylase para-nitrophenyl oligosaccharide, which in turn is converted by the enzymes glucoamylase and alpha-glucosidase to glucose and para-nitrophenol. Thus, the amount of released para-nitrophenol is proportional to the activity of the amylase.
  • the measurement is carried out, for example, using the Quick-Start® test kit from Abbott (Abbott Park, Illinois, USA). The increase in absorbance (405 nm) in the test mixture was determined at 37 ° C.
  • a wash test was performed on the purified supernatant from E. coli containing the wild-type alpha-amylase from Bacillus cereus.
  • Enzyme concentration 0.186 TAU / ml (determination of amylase activity with benzylidene-blocked para-nitrophenol-maltoheptaoside); this corresponds to the amount of amylase usually used in detergents.
  • Sample 2 detergent plus Bacillus cereus alpha-amylase (according to the invention)
  • amylase performs well on all four soils. From a significant performance improvement one speaks already from 1 unit, here were achieved up to 3.7 units improvement. As a negative control, the boiled, purified
  • the matrix used contained no optical brighteners, perfumes, dyes or enzymes.

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Abstract

L'invention concerne des alpha-amylases comprenant une séquence d'acides aminés qui présente sur sa longueur totale au moins 70 % d'identité de séquence avec la séquence d'acides aminés indiquée dans SEQ ID NO:1, ainsi que leur production et leur utilisation. De telles alpha-amylases offrent une bonne performance de nettoyage.
PCT/EP2017/051747 2016-02-03 2017-01-27 Performance de lavage améliorée grâce à une alpha-amylase de bacillus cereus Ceased WO2017133974A1 (fr)

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DE102016201637.7A DE102016201637A1 (de) 2016-02-03 2016-02-03 Verbesserte Waschleistung durch eine alpha-Amylase aus Bacillus cereus
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021163011A2 (fr) 2020-02-10 2021-08-19 Novozymes A/S Variants d'alpha-amylase et polynucléotides codant pour ceux-ci
WO2021163015A1 (fr) 2020-02-10 2021-08-19 Novozymes A/S Procédé de production d'éthanol à partir d'amidon brut à l'aide de variants d'alpha-amylase

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WO2009108941A2 (fr) * 2008-02-29 2009-09-03 University Of Central Florida Research Foundation, Inc. Production et utilisation de matériaux dégradant les plantes
US20130149770A1 (en) * 2008-06-06 2013-06-13 Danisco Us Inc. Variant Alpha-Amylases from Bacillus Subtilis and Methods of Uses, Thereof

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DE102008017103A1 (de) 2008-04-02 2009-10-08 Henkel Ag & Co. Kgaa Wasch- und Reinigungsmittel enthaltend Proteasen aus Xanthomonas

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WO2009108941A2 (fr) * 2008-02-29 2009-09-03 University Of Central Florida Research Foundation, Inc. Production et utilisation de matériaux dégradant les plantes
US20130149770A1 (en) * 2008-06-06 2013-06-13 Danisco Us Inc. Variant Alpha-Amylases from Bacillus Subtilis and Methods of Uses, Thereof

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DATABASE EMBL [online] 2 June 2010 (2010-06-02), "Bacillus cereus strain XHJ-2-6 amylase gene, complete cds.", XP002768704, retrieved from EBI accession no. EM_STD:GU979530 Database accession no. GU979530 *
DATABASE Geneseq [online] 21 January 2010 (2010-01-21), "Plant biomass degrading enzyme #557.", retrieved from EBI accession no. GSP:AXR39553 Database accession no. AXR39553 *
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021163011A2 (fr) 2020-02-10 2021-08-19 Novozymes A/S Variants d'alpha-amylase et polynucléotides codant pour ceux-ci
WO2021163015A1 (fr) 2020-02-10 2021-08-19 Novozymes A/S Procédé de production d'éthanol à partir d'amidon brut à l'aide de variants d'alpha-amylase

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