WO2017133973A1

WO2017133973A1 - Improved washing performance using an alpha-amylase from bacillus cereus

Info

Publication number: WO2017133973A1
Application number: PCT/EP2017/051743
Authority: WO
Inventors: Daniela HERBST; Nina Mussmann; Timothy O'connell; Inken PRÜSER; Anna Krüger; Neele Meyer; Garabed Antranikian; Anke Peters
Original assignee: Henkel Ag & Co. Kgaa
Priority date: 2016-02-03
Filing date: 2017-01-27
Publication date: 2017-08-10
Also published as: DE102016201643A1

Abstract

The invention relates to alpha-amylases which comprise an amino acid sequence that, over its entire length, shares at least 70% sequence identity with the amino acid sequence of SEQ ID NO. 1, and to the production and use thereof. Said type of alpha-amylases have a good cleaning performance.

Description

"Improved washing performance by an alpha-amylase from Bacillus cereus"

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

Uses of these alpha-amylases and agents containing them, in particular detergents and cleaners.

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

(Endohydrolysis). 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. The 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 - -

10.8). Its optimum temperature is 45 ° C, the enzyme also having activity at lower temperatures, for example 15-20 ° C.

Despite the many known alpha-amylases, there continues to be a need for alpha-amylase variants which have altered biochemical properties and thereby have improved performance in industrial applications, especially in detergents or cleaners.

Surprisingly, it has now been found that an alpha-amylase from Bacillus cereus or a sufficiently similar alpha-amylase (based on the sequence identity), 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%

Sequence identity to the amino acid sequence given in SEQ ID NO: 1 on their

Total length has.

An 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.

As further subjects of the invention are with the alpha-amylases of 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

Purification process, and uses defined alpha-amylases according to the invention.

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. Thus, with particularly preferred embodiments of the invention, performance-enhanced alpha-amylase variants are provided. Such advantageous

Embodiments of alpha-amylases according to the invention thus enable improved

Washing results on starch-containing stains in a wide temperature range.

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.

In various embodiments of the invention, the alpha-amylase comprises a

Amino acid sequence corresponding to the amino acid sequence given in SEQ ID NO: 1 over its total length to 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%, 99.0%, 99.2%, 99.4%, 99.5%, 99.6% or 99.8% is identical.

The identity of nucleic acid or amino acid sequences is determined by a sequence comparison. This 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. For example, the Clustal series (see, for example, Chenna et al., 2003, Multiple Sequence Alignment with the Clinical Series of Programs, Nucleic Acid Research 31, 3497-3500), 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. Also possible are alignment comparisons (alignments) 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.

Such a comparison also allows a statement about the similarity of the compared sequences to each other. It is usually given in percent identity, that is, the proportion of identical nucleotides or amino acid residues at the same or in an alignment corresponding positions. The broader concept of homology involves conserved amino acid substitutions in the consideration of amino acid sequences, that is, amino acids with similar chemical activity, since these usually have similar chemical properties within the protein

Exercise activities. Therefore, the similarity of the sequences compared may also be stated as percent homology or percent similarity. 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.

In a further embodiment of the invention, 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

Has reference washing performance. 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

Cleaning degree of the washed textiles. For example, the washing process for 60 minutes at a temperature of 30 ° C and the water have a water hardness between 15.5 and 16.5 ° (German hardness). The concentration of alpha-amylase in the for this - -

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%

Preservatives, 2% ethanol, 0.2% dye transfer inhibitor, remainder demineralized water. Preferably, 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.

In the context of the invention, the determination of the cleaning performance at 30 ° C under

Use of a liquid detergent as indicated above, wherein the washing process is preferably carried out for 60 minutes.

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. Usually, 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.

In general, 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

enzymatic activity of alpha-amylase. - -

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

Residual activity (see M. Bender et al., J. Am. Chem. Soc., 88, 24 (1966), pp. 5890-5913).

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

The subject of the invention therefore forms 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.

Compared to the alpha-amylase described in SEQ ID NO: 1, 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.). Furthermore, inventive

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. For this purpose, in particular the surface charges and / or the isoelectric point of the molecules and thereby their interactions with the substrate can be changed. Thus, for example, the net charge of the enzymes can be changed in order to influence the substrate binding, in particular for use in detergents and cleaners. Alternatively or additionally, 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. The term "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. Conservative amino acid substitutions within the scope of the invention include, for example: G = A = S, l = V = L = M, D = E, N = Q, K = R, Y = F, S = T, G = A = I = V = L = M = Y = F = W = P = S = T.

Alternatively or additionally, the alpha-amylase is characterized in that it is obtainable from an alpha-amylase according to the invention as starting molecule by fragmentation, deletion, insertion or substitution mutagenesis and comprises an amino acid sequence over a length of at least 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 51 1 or 512 contiguous amino acids matches the parent molecule.

Thus it is possible, for example, to delete individual amino acids at the termini or in the loops of the enzyme, without this losing or reducing the catalytic activity. Furthermore, such fragmentation, deletion, insertion or substitution mutagenesis can also reduce, for example, the allergenicity of the enzymes concerned and thus improve their overall applicability. Advantageously, even after mutagenesis, 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

Starting enzyme. Other substitutions can also show beneficial effects. Both single and multiple contiguous amino acids can be substituted for other amino acids.

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. - -

Further confirmation of the correct assignment of the amino acids to be altered, i.

in particular their functional correspondence, can provide comparative experiments, according to which the two positions assigned to each other on the basis of an alignment in both compared alpha-amylases are changed in the same way and it is observed whether the enzymatic activity is changed in the same way in both. If, for example, an amino acid exchange in a specific position of the Bacillus cereus alpha-amylase according to SEQ ID NO: 1 is accompanied by a change in an enzymatic parameter, for example by an increase in the M value, then a corresponding change in the enzymatic parameter, for example also an increase in the M value, observed in an alpha-amylase variant according to the invention, whose amino acid exchange was achieved by the same introduced amino acid, it is to be seen here a confirmation of the correct assignment.

In particular, according to the invention, fragments of the amylase having the amino acid sequence according to SEQ ID NO: 1, which are shortened at the N-terminus such that the signal peptide, i.

in particular, the N-terminal 27 amino acids are no longer included. Also of these truncated fragments variants can be used in various embodiments of the invention, which to the starting from the amino acid sequence given in SEQ ID NO: 1 truncated by 27 N-terminal amino acids, over the total length to at least 70%, 71%, 72nd %, 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.

All these facts are also applicable to the process of the invention for the production of an alpha-amylase. Accordingly, 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 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510 or 513 contiguous amino acids matches the parent molecule.

All embodiments also apply to the inventive method. - -

In further embodiments of the invention, 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. For an increase in stability during storage and / or during use, for example during the washing process, causes the enzymatic activity to last longer and thus improve the cleaning performance. In principle, all stabilization options described in the prior art and / or appropriate considerations come into consideration. Preference is given to those stabilizations which are achieved via mutations of the enzyme itself, since such stabilizations do not require any further working steps following the recovery of the enzyme. Examples of sequence changes suitable for this purpose are mentioned above. Other suitable sequence changes are known from the prior art.

Options for stabilization include:

Protection against the influence of denaturing agents, such as surfactants, on mutations causing an alteration of the amino acid sequence on or at the surface of the protein;

Replacement of amino acids located near the N-terminus against those likely to contact non-covalent interactions with the rest of the molecule, thus contributing to the maintenance of the globular structure.

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.

For the purposes of the present application, derivatives are understood as meaning those proteins whose pure amino acid chain has been chemically modified. Such

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. Similarly, derivatization is the covalent bond to a

to understand macromolecular carrier, or even a non-covalent inclusion in suitable macromolecular cage structures. 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.

Derivatives of a protein according to the invention can also be used in the broadest sense

Preparations of these proteins are understood. Depending on the extraction, processing or preparation, 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.

Therefore, all preparations of a protein according to the invention are also according to the invention. This is also independent of whether or not it actually exhibits this enzymatic activity in a particular preparation. Because it may be desired that it has no or only low activity during storage, and unfolds its enzymatic function only at the time of use. This can be controlled, for example, via appropriate accompanying substances.

In particular, the joint preparation of alpha-amylases with specific inhibitors is possible in this regard.

With regard to all the above-described 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. In preferred embodiments, the

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. - -

These 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 acid sequences to determine beyond doubt, since despite the degeneracy of the genetic code individual codons defined amino acids are assigned. Therefore, the person skilled in the art can start by coding for an amino acid sequence for this amino acid sequence

Determine 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

Amino acids understood by the respective organism. There may be bottlenecks in protein biosynthesis when the codons lying on the nucleic acid in the

Organism of a comparatively small number of loaded tRNA molecules

face. Although coding for the same amino acid, this results in a codon being translated less efficiently in the organism than a synonymous codon encoding the same amino acid. Due to the presence of a higher number of tRNA molecules for the synonymous codon, it can be more efficiently translated in the organism.

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. 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.

For the purposes of the present invention, 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. In the context of the present invention, 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. In particular, expression is influenced by the promoter (s) that regulate transcription. In principle, 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. In the present case, at least one promoter for the expression of a nucleic acid according to the invention is made available and used for its expression. Furthermore, 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-β-D-thiogalactopyranoside (IPTG), which is used as an activator of the bacterial lactose operon (lac operon). In contrast to expression vectors, the nucleic acid contained is not expressed in cloning vectors.

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. Preferably, a nucleic acid according to the invention or a vector according to the invention is transformed into a microorganism, which then has a

represents host cell according to the invention. Alternatively, individual components, i.

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

Components are then supplemented accordingly. Methods of transforming cells are well established in the art and well known to those skilled in the art. In principle, all cells, that is to say prokaryotic or eukaryotic cells, are suitable as host cells. Preference is given to those host cells that can be handled genetically advantageous, which, for example, the

Transformation with the nucleic acid or the vector and its stable establishment, for example, unicellular fungi or bacteria. Furthermore, 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. Furthermore, the alpha-amylases can be modified by the cells producing them after their preparation, for example by attachment of sugar molecules,

Formylations, aminations, etc. Such post-translational modifications may functionally affect alpha-amylase.

Further preferred embodiments 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.

For example, by controlled addition of chemical compounds that serve as activators, by changing the culture conditions or when reaching a specific cell density, these can be excited for expression. This enables an economical production of the proteins according to the invention. An example of such a compound is IPTG as described above.

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

Experience. For a specific production, 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.

In Gram-negative bacteria, such as Escherichia coli, a large number of proteins are secreted into the periplasmic space, ie into the compartment between the two membranes enclosing the cells. This can be advantageous for special applications. Furthermore, 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. In contrast, 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. In addition, 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.

In a further embodiment 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

Stenotrophomonas maltophilia.

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. In contrast to prokaryotic cells, eukaryotic cells are capable of producing the protein formed

post-translationally modify. Examples thereof are fungi such as Actinomycetes or yeasts such as Saccharomyces or Kluyveromyces. This may be particularly advantageous, for example, if the proteins are to undergo specific modifications in the context of their synthesis that enable such systems. 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. Such oligosaccharide modifications may be desirable, for example, to lower the allergenicity of an expressed protein. Also, coexpression with the enzymes naturally produced by such cells, such as cellulases, may be advantageous. Furthermore, for example, 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. In the first case, 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

a) cultivating a host cell according to the invention, and

b) isolating the alpha-amylase from the culture medium or from the host cell.

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 based on a corresponding process for the production of an alpha-amylase according to the invention provide embodiments thereof

Invention subject matter.

Fermentation processes, which are characterized in that the fermentation is carried out via a feed strategy, come in particular into consideration. Here are the

Supplied with media components consumed by ongoing cultivation.

As a result, considerable increases can be achieved both in the cell density and in the cell mass or dry mass and / or in particular in the activity of the alpha-amylase of interest. Furthermore, 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. Alternatively, without secretion, isolation of the alpha-amylase from the host cell, i. a purification of the same from the

Cell mass, carried out, for example, by precipitation with ammonium sulfate or ethanol, or by chromatographic purification. - -

All of the above issues may be combined into methods to produce alpha-amylases of the invention.

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

Detergent in the context of the invention also 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. Among the latter, i.a. calculated the fabric softener.

The washing or cleaning agents according to the invention, which may be in the form of homogeneous solutions or suspensions in the form of powdered solids, 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.

In particular, 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. In particular, by the combination of an alpha- - -

Amylase with a surfactant and / or a builder (builder) and / or a

Peroxygen compound and / or a bleach activator, such a synergism can be achieved.

Advantageous ingredients of agents according to the invention are disclosed in International

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.

In further embodiments of the invention, the agent is characterized in that it

(a) 1-85 wt%, preferably 5-65 wt%, of surfactants; and or

(b) 0-45 wt.%, preferably 0.1-15 wt.% builder (builders); and or

(c) 0.0005-15 wt%, preferably 0.001-5 wt%, of protease; and or

(d) 0.0005-15% by weight, preferably 0.001-5% by weight, of lipase; and or

(e) 0.00005-15% by weight, preferably 0.0001-5% by weight, of mannanase; and or

(f) 0.00005-15 wt.%, preferably 0.0001-5 wt.%, of cellulase / pectate lyase; and or

(g) 0.00005-15 g / wash load, preferably 0.0001-5 g / wash load, xanthan lyase; and or

(h) 0.00005-15 g / wash load, preferably 0.00005-15 g / wash load, endoglucanase capable of digesting xanthan gum.

An agent according to the invention advantageously contains the alpha-amylase in an amount of from 2 μg to 20 mg, preferably from 5 μg to 17.5 mg, more preferably from 20 μg to 15 mg and very particularly preferably from 50 μg to 10 mg per g of the composition. In addition, that can

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. Further, 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. Furthermore, 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.

In further embodiments of the invention, 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

(e) is divided into several components.

These embodiments of the present 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. Alternatively, 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.

Furthermore, the 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, β-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. Increasing preference 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. Particularly preferably, 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. Very particularly preferably, 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.

In various embodiments, 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.

These include both manual and mechanical processes, with mechanical processes being preferred. 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. The same applies to processes for cleaning all other materials than textiles, especially hard surfaces. 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

method according to the invention applies.

Since 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

Invention subject matter.

Alternative embodiments of this subject matter of the invention are also processes for the treatment of textile raw materials or for textile care, in which at least one

Process step an alpha-amylase according to the invention is active. Below are methods for - -

Textile raw materials, fibers or textiles with natural ingredients preferred, and especially for those with wool or silk.

In a further aspect, 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.

All aspects, objects, and embodiments described for alpha-amylase and agents containing it are also applicable to the described methods and uses. Therefore, reference is made at this point expressly to the disclosure in the appropriate place with the statement that this disclosure also applies to the above uses and methods according to the invention.

- -

Examples

Example 1: Identification of the amylase

From 20 bacillus strains, a metagenome database was prepared, which was examined for amylase activity. In this case, 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 produced by E. coli amylase was on different

starchy textiles examined for their washing performance. The found sequence

differs significantly from the sequences of amylases, which were previously used in detergents and cleaners.

Example 2: Activity assay

For the determination of the 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. for 3 minutes compared with a photometric control value (blank value). Calibration was performed on an enzyme standard of known activity (e.g., Maxamyl® / Purastar® 2900 Genencor 2900 TAU / g). The evaluation was carried out by determining the absorption difference dE (405 nm) per min against the enzyme concentration of the standard.

Example 3: Washing test and results

A wash test was performed on the purified supernatant from E. coli containing the wild-type alpha-amylase from Bacillus cereus.

Conditions: 30 ° C, 16 ° dH water, 1 h;

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.

stains:

1. C-S-26 Cornstarch 053

2. C-S-27 potato starch 048

3. C-S-28 rice starch 135

4. C-S-29 tapioca starch 024

Prepare excised tissue (diameter = 10 mm) in a microtiter plate, preheat the wash liquor to 30 ° C, final concentration 4.7 g / L;

Apply the lye and enzyme to the stain, incubate for 1 h at 30 ° C and 600 rpm; - - Then rinse the soiling several times with clear water, leave to dry and determine the brightness with a colorimeter.

The lighter the tissue, the better the cleaning performance. Measured here is the L value = brightness, the higher the brighter.

It is washed with a common liquid detergent without enzymes (see Table 2).

Sample 1: only detergent as benchmark (comparison reference)

Sample 2: detergent plus Bacillus cereus alpha-amylase (according to the invention)

The results are shown in Table 1.

Table 1: Results of the washing tests

It becomes clear that amylase performs well on all four soils. A significant improvement in performance is said to be as low as 1 unit, with an improvement of up to 3.9 units. As a negative control, the boiled, purified supernatant from the production organism E. coli was washed (99 ° C for 30 min), showing no washing performance.

Table 2: Used detergent matrix

The matrix used contained no optical brighteners, perfumes, dyes or enzymes.

Claims

claims

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.

2. alpha-amylase, characterized in that

(a) it is obtainable from an alpha-amylase according to claim 1 as starting molecule by single or multiple conservative amino acid substitution; and or

(b) it is obtainable from an alpha-amylase according to claim 1 as a starting molecule by fragmentation, deletion, insertion or substitution mutagenesis and a

A length of at least 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510 or 513

contiguous amino acids matches the parent molecule.

3. A process for producing an alpha-amylase according to claim 1 or 2 comprising

Providing a parent alpha-amylase having at least 70% sequence identity to the amino acid sequence given in SEQ ID NO: 1 over its entire length.

The method of claim 3, further comprising one or both of the following

Steps:

(a) introducing a single or multiple conservative amino acid substitution;

(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 360, 370, 380, 390, 400, 410, 420, 430, 440 in length , 450, 460, 470, 480, 490, 500, 510 or 513 contiguous amino acids with the

Matched starting molecule.

5. Nucleic acid encoding an alpha-amylase according to any one of claims 1 or 2 or encoding a obtained according to a method of claims 3 or 4 alpha-amylase.

6. Vector comprising a nucleic acid according to claim 5, in particular a cloning vector or an expression vector.

A non-human host cell which comprises a nucleic acid according to claim 5 or a vector according to claim 6, or which comprises an alpha-amylase according to any one of claims 1 or 2, or an alpha-amylase obtained according to a method of claims 3 or 4 including, in particular, one that secretes the alpha-amylase into the medium surrounding the host cell.

8. A method for producing an alpha-amylase comprising

a) culturing a host cell according to claim 7

b) isolating the alpha-amylase from the culture medium or from the host cell.

9. agent, in particular a detergent or cleaning agent, characterized in that it contains at least one alpha-amylase according to any one of claims 1 or 2 or obtained by a process of claims 3 or 4 alpha-amylase, in particular is the alpha-amylase in an amount up to 1 wt .-% based on active protein.

10. A process for the cleaning of textiles or hard surfaces, characterized in that in at least one process step, an agent according to claim 9 is applied, or that in at least one process step, an alpha-amylase according to any one of claims 1 or 2 or one of a method of Claims 3 or 4 obtained alpha-amylase is catalytically active.

1 1. Use of an alpha-amylase according to any one of claims 1 or 2 or a obtainable by a process of claims 3 or 4 alpha-amylase in a washing or

Detergent for removal of starch-containing soiling.