CN101381712A - Amylase variant - Google Patents

Abstract

Translated fromChinese

本发明公开了亲本α－淀粉酶变体，其(i)具有选自SEQ ID No.1－3和SEQID No.7所示氨基酸序列的氨基酸序列；或(ii)与这些氨基酸序列中的一种或多种显示出至少80％同源性；和/或显示与抗体的免疫交叉反应性，所述抗体针对具有这些氨基酸序列之一的α－淀粉酶产生；和/或由与探针杂交的DNA序列编码，所述探针与编码具有这些氨基酸序列之一的α－淀粉酶之DNA序列相同；在该变体中亲本α－淀粉酶的至少一个氨基酸残基已被缺失和/或取代；和/或相对亲本α－淀粉酶插入至少一个氨基酸残基；该变体具有α－淀粉酶活性且相对于亲本α－淀粉酶显示至少一种下列性质：热稳定性增加；氧化稳定性增加；Ca²⁺依赖性降低；其条件是该变体的氨基酸序列不与SEQ ID No.1－3和SEQ ID No.7中所示的任一序列相同。The present invention discloses a parent alpha-amylase variant, which (i) has an amino acid sequence selected from the amino acid sequences shown in SEQ ID No. 1-3 and SEQ ID No. 7; or (ii) is identical to one of these amino acid sequences One or more exhibits at least 80% homology; and/or exhibits immunological cross-reactivity with antibodies raised against α-amylases having one of these amino acid sequences; and/or results from hybridization with a probe The DNA sequence encoding the probe is identical to the DNA sequence encoding an alpha-amylase having one of these amino acid sequences; in this variant at least one amino acid residue of the parent alpha-amylase has been deleted and/or substituted and/or at least one amino acid residue inserted relative to the parent alpha-amylase; the variant has alpha-amylase activity and exhibits at least one of the following properties relative to the parent alpha-amylase: increased thermal stability; increased oxidative stability ; reduced Ca²⁺ dependence; provided that the amino acid sequence of the variant is not identical to any of the sequences shown in SEQ ID No. 1-3 and SEQ ID No. 7.

Description

Amylase variant

The application is to be that February 5, application number in 1996 are the applying date: 96191711.3, exercise question is: the dividing an application of " amylase variant ".

Technical field that the present invention belongs to

The present invention relates to for parent enzyme, have the character (as, the calcium ion dependency of the heat of improvement and/or oxidative stability and/or reduction) of improvement and improved washing thus and/or the alpha-amylase variants of dishwashing detergent (and/or textiles removes slurry) performance.The DNA construct of said variant and carrier and the cell that carries DNA construct the present invention also relates to encode.The invention still further relates to method that produces amylase variant and washing agent additive and the de-sludging composition that comprises amylase variant.The invention still further relates to the purposes of amylase variant on textiles goes to starch.

Background of invention

α-Dian Fenmei has been used much year industrial, has many uses, wherein the most important thing is starch liquefacation, textiles remove slurry, at starch conversion aspect papermaking and the Pulp industry and be used for wine brewing and baking.The purposes of the α-Dian Fenmei that another becomes more and more important is to remove amyloid dirt during washing or dishwashing detergent.

Attempt to make up the alpha-amylase variants have the character of the improvement of specific end use (removing slurry) in recent years as starch liquefacation and textiles.

For example, the U.S. 5,093,257 disclose the chimeric α-Dian Fenmei that comprises bacstearothermophilus α-Dian Fenmei N-terminal portions and bacillus licheniformis alpha-amylase C-terminal portions.According to record, chimeric α-Dian Fenmei has unique character, as comparing different thermostabilitys with its parent's α-Dian Fenmei.Yet all specifically described chimeric α-Dian Fenmei are compared the enzymatic activity that shows reduction with its parent's α-Dian Fenmei.

It is the hybrid amylase of Q-R-L that EP252 666 has described general formula, wherein Q is the N-terminal polypeptide residue of 55 to 60 amino-acid residues, it is a homologous with 57-terminal amino acid residues at least 75% of the bacillus amyloliquefaciens α-Dian Fenmei that has illustrated, R is the polypeptide that has illustrated, L is the C-terminal polypeptide that comprises 390 to 400 amino-acid residues, and it is a homologous with 395 C-terminal amino acid residues at least 75% of the bacillus licheniformis alpha-amylase that has illustrated.

Suzuki etc. (1989) disclose chimeric α-Dian Fenmei, and wherein the designated area of bacillus licheniformis alpha-amylase is substituted by the respective regions of bacillus amyloliquefaciens α-Dian Fenmei.In order to differentiate the zone that thermostability is worked, made up chimeric α-Dian Fenmei.Find that such zone comprises the amino-acid residue 177-186 and the amino-acid residue 255-270 of bacillus amyloliquefaciens α-Dian Fenmei.As if the change of amino-acid residue does not influence the character of enzyme except that its thermostability in chimeric α-Dian Fenmei.

WO 91/00353 discloses at least one amino-acid residue alpha-amylase mutant different with its parent's α-Dian Fenmei.Disclosed alpha-amylase mutant in said patent application is because its aminoacid replacement is applied to starch degradation and/or textiles goes to starch the character that shows improvement.Some mutant show stability and improve, but do not report or point out the improvement of enzymatic activity.Only the mutant of explanation is from the preparation of parent's bacillus licheniformis alpha-amylase as an example, and it carries one of following sudden change: H133Y or H133Y+T1491.Another sudden change of having recommended is A111T.

FR 2,676, and 456 disclose the mutant of bacillus licheniformis alpha-amylase, wherein replaced by a kind of more hydrophobic amino-acid residue at a near amino-acid residue the Histidine 133 and/or a near amino-acid residue the L-Ala 209.According to record, the alpha-amylase mutant of generation has improved thermal stability, is useful in textiles, papermaking, wine brewing and starch liquefacation industry.

EP 285 123 discloses the method for carrying out the nucleotide sequence random mutagenesis.Mentioned the nucleotide sequence of coding bacstearothermophilus α-Dian Fenmei as an example of such sequence.When sudden change, obtained under low pH value, to have the active alpha-amylase variants of improvement.

None is mentioned even is hinted and can make up the alpha-amylase mutant that the character of improvement is arranged concerning washing agent industry in above-mentioned reference.

EP 525 610 relates to the mutant enzyme that has ionic surface active agent (tensio-active agent) improved stability.Mutant enzyme is replaced generation by another kind of amino-acid residue by an amino-acid residue of the surface portion of parent enzyme.Specifically described unique mutant enzyme is a proteolytic enzyme in EP 525 610.The amylase conduct can obtain the example of the enzyme of ionic surface active agent improved stability referred, but diastatic type, origin or concrete sudden change all do not indicate.

WO 94/02597 discloses and shown improved stability and active alpha-amylase mutant in the presence of oxygenant.In mutant alpha-amylases, the amino-acid residue that one or more methionine residues have been different from halfcystine and methionine(Met) replaces.According to record, alpha-amylase mutant can be used for washing agent and/or dishwashing detergent additive and textiles and removes slurry.

WO 94 18314 discloses oxidation-stabilized alpha-amylase mutant, is included in the sudden change of bacillus licheniformis alpha-amylase M197 position.

EP 368 341 described with/not with Starch debranching enzyme and other amylolytic enzyme of being used to wash with the α-Dian Fenmei combination of dishwashing detergent.

An object of the present invention is to provide alpha-amylase variants, this variant has the critical nature of improvement with respect to its parent's α-Dian Fenmei, the washing and/or the dishwashing detergent performance of particularly relevant said variant, for example, the thermostability of increase, the oxidative stability of increase, reduction to Ca²⁺The ionic dependency and/or in suitable pH value zone (for example, laundry or dishwashing detergent) improved stability or activity.Such variant α-Dian Fenmei has superiority, and wherein, they can use under than the lower dosage of its parent's α-Dian Fenmei.And alpha-amylase variants can be removed amyloid dirt, and this dirt can not (or having any problem) be removed with present known α-Dian Fenmei washing agent enzyme.

Description of the invention

Purpose as the work on basis of the present invention is improvement (if possible) α-Dian Fenmei (particularly, the α-Dian Fenmei that can obtain from Bacillus strain specifically) stability, these α-Dian Fenmei serve as that the basis is with respect at present commercially available α-Dian Fenmei selection with its performance of removing destarching in alkaline medium (the washing agent solution that uses as typical case in laundry or dishwashing detergent).In this respect, the present inventor is surprisingly found out that: the rational modification of one or more amino-acid residues of the different zones of aminoacid sequence that in fact might be by parent's α-Dian Fenmei is improved the character of the above-mentioned type (on seeing) of such parent's α-Dian Fenmei.The present invention is found to be the basis with this.

Therefore, first aspect of the present invention relates to the variant of parent's α-Dian Fenmei, and said parent's α-Dian Fenmei is a kind of:

I) have respectively at one of aminoacid sequence shown in the SEQ of this paper ID No.1, SEQ ID No.2, SEQ ID No.3 and the SEQID No.7; Or

Ii) with at one or more aminoacid sequences shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and the SEQ ID No.7 show at least 80% homology; And/or the immune cross-reactivity of the antibody that demonstrates and produce by the anti-α-Dian Fenmei that has respectively at one of aminoacid sequence shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and the SEQ ID No.7; And/or by having respectively dna sequence encoding at the identical probe hybridization of the dna sequence dna of the α-Dian Fenmei of one of aminoacid sequence shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and the SEQ ID No.7 with coding.

The condition that alpha-amylase variants of the present invention met is: this variant be aminoacid sequence not with at the identical variant of aminoacid sequence shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 or the SEQ ID No.7.

The dna sequence dna of described first three α-Dian Fenmei aminoacid sequence of encoding shows in SEQ ID No.4 (being coded in the aminoacid sequence shown in the SEQ ID No.1), SEQ ID No.5 (being coded in the aminoacid sequence shown in the SEQ ID No.2) and SEQ ID No.6 (being coded in the aminoacid sequence shown in the SEQ ID No.3).

The aminoacid sequence and corresponding DNA sequence (being respectively SEQ ID No.4 and SEQ ID No.5) (identical with the SEQ ID NOs. of present patent application) of SEQ ID No.1 and SEQ ID No.2 parent α-Dian Fenmei are also disclosed in WO 95/26397.

Variant of the present invention is such variant, and wherein: (a) at least one amino-acid residue of said parent's α-Dian Fenmei is lacked; And/or (b) at least one amino-acid residue of said parent's α-Dian Fenmei is replaced by different amino-acid residue; And/or (c) relatively parent's α-Dian Fenmei have at least an amino-acid residue to be inserted into; Said variant has alpha-amylase activity and shows at least a following character with respect to parent's α-Dian Fenmei:

The thermostability that increases promptly keeps enzymatic activity satisfactorily under than the higher temperature of the temperature of using parent enzyme to be fit to;

The oxidative stability that increases, promptly the resistance to oxygenant (as oxygen, SYNTHETIC OPTICAL WHITNER of oxidation or the like) degraded improves;

The Ca that reduces²⁺Dependency is promptly at the Ca than parent's α-Dian Fenmei lower concentration²⁺The ability that works satisfactorily when existing.The Ca that this reduction is arranged²⁺Dependent α-Dian Fenmei is very desirable when being used for the de-sludging composition, because composition typically comprises relatively large strong material (as phosphoric acid salt, EDTA or the like) in conjunction with calcium ion like this.

The example that other available desirable character that reaches according to variant of the present invention improved or modified (comparing with said parent's α-Dian Fenmei) has:

In neutrality or than higher pH (for example, in the scope of pH value at 7-10.5 (as the scope of 8.5-10.5)) stability and/or the raising of Alpha-starch degrading activity down;

(for example, the temperature in 40-70 ℃ of scope) Alpha-starch degrading activity improves under than higher temperature;

Improve or the pH value of (for example, do washing medium, dishwashing detergent medium or textiles-go to starch medium) (as follows) of reducing iso-electric point (pI) so that the medium that said alpha-amylase variants and variant use is therein mated better;

The combination to the substrate of particular type of improvement, the specificity to substrate of improvement, and/or the specificity to substrate cracking (hydrolysis) of improvement.

If pass through algorithm known (as Lipman and Pearson, science, 227 (1985), 1435 is described) certain aminoacid sequence of carrying out and the parent's α-Dian Fenmei identity that relatively discloses X% of aminoacid sequence separately, just can think this aminoacid sequence and parent's α-Dian Fenmei X% homology.Use default GAP penalty value, can suitably use GAP computer program [the genetics computer set (1991) of 7.3 editions GCG software package (1993,6), GCG software package procedure manual, the 7th edition, 575 Science Drive, Madison, Wisconsin State, the U.S. 53711].

In the present invention, as mentioned above and " improved performance " meaning of using with dishwashing detergent of washing be the removal of the amyloid dirt (dirt that promptly comprises starch) during washing or dishwashing detergent, improved respectively.Can routine washing with the dishwashing detergent experiment in determine performance, by with said parent's α-Dian Fenmei relatively estimate improvement.The example of one small-scale " small-sized dishwashing detergent test " can be used for the indication of dishwashing detergent performance has been described in the experimental section below.

The many different characteristicss that are appreciated that alpha-amylase variants get up to facilitate its improved performance alone or in combination, and these features comprise: than work, substrate specificity, K_m(so-called " Michaelis-Menton constant " in the Michaelis-Menten equation), V_Max[the maximum conversion speed (plateau value (plateau)) on Michaelis-Menten equation basis, measured of given substrate, pI, optimum pH, optimum temperuture, hot activation, (for example to oxygenant or tensio-active agent, in scale remover) stability, or the like.The technician is clear: the performance of variant can not be predicted separately on the basis of above-mentioned feature, wash and/or the dishwashing detergent performance test and must be accompanied by.

Another aspect of the present invention relate to the dna sequence dna that comprises the alpha-amylase variants of the present invention of encoding DNA construct, carry this DNA construct recombinant expression vector, from culture, reclaim alpha-amylase variants then to produce the method for alpha-amylase variants with this DNA construct or carrier cell transformed and by under being of value to the condition that alpha-amylase variants produces, cultivating such cell.

Another aspect of the present invention relates to the method for preparing parent's alpha-amylase variants, and this variant is because the character of aforesaid improvement and parent's α-Dian Fenmei comparison sheet reveal improvement washing and/or dishwashing detergent performance.This method comprises:

A) structure comprises the cell mass of the gene of the said parent's alpha-amylase variants of encoding,

B) alpha-amylase activity of this cell mass of screening under the condition of at least a washing of simulation and/or dishwashing detergent,

C) isolate cell from the cell mass that comprises the said parent's alpha-amylase variants gene of encoding, variant is wherein compared the activity with improvement with parent's α-Dian Fenmei under the condition that step b) is selected,

D) isolated cells culturing step c on the appropriate culture medium under the condition of being fit to),

E) from the culture that step d), obtains, reclaim alpha-amylase variants.

The present invention also relates to variant (this variant is according to variant of the present invention) by a kind of method preparation in back.

In the present invention, term " is simulated at least a washing and/or dishwashing detergent condition ", and the meaning is meant such simulation, for example, prevailing temperature or pH value during washing or dishwashing detergent, or in washing or dishwashing detergent processing the chemical composition in the used de-sludging composition.Term " chemical composition " is meant a kind of composition that comprises said de-sludging composition, or the combination of two or more composition.Below the one-tenth of many different de-sludging compositions is respectively in.

The cell mass of step a) indication can be by clones coding parent α-Dian Fenmei dna sequence dna and make DNA carry out the following fixed point of this paper or random mutagenesis suitably makes up.

In the present invention, term " variant " is used interchangeably with term " mutant ".Term " variant " meaning is to comprise the hybrid α-Dian Fenmei, promptly comprises the α-Dian Fenmei of the part of at least two kinds of different Alpha-starch lytic enzymes.Like this, a kind of like this hybrid can from, for example, from a variant deutero-part or many parts of above definition; Or make up from the variant deutero-part of above definition or many parts and from parent's α-Dian Fenmei deutero-part or many parts of a kind of not improvement.In this respect, the present invention also relates to a kind of method that produces such hybrid α-Dian Fenmei, α-Dian Fenmei wherein is in having the washing and/or the dishwashing detergent performance of improvement with its arbitrary the comparing of composition enzyme (promptly comparing with any enzyme of a part of forming hybrid), and this method comprises:

A) in the terminal coding region of the C-body of the alpha-amylase gene of the terminal coding region of N-that makes a kind of alpha-amylase gene of component α-Dian Fenmei or corresponding cDNA and another kind of component α-Dian Fenmei or corresponding cDNA or vitro recombination with the formation recombinant chou,

B) select to produce the recombinant chou of the hybrid α-Dian Fenmei of comparing washing with improvement and/or dishwashing detergent performance with its component α-Dian Fenmei,

C) recombinant chou of selecting culturing step b on the appropriate culture medium under the condition of being fit to),

D) from the culture that step c), obtains, reclaim the hybrid α-Dian Fenmei.

Another aspect of the present invention relates to the purposes of alpha-amylase variants of the present invention [comprising any variant or hybrid by the preparation of one of aforesaid method] as washing agent enzyme (being to be used for washing or dishwashing detergent specifically), the present invention also relates to comprise washing agent additive and the detergent compositions and the purposes of alpha-amylase variants of the present invention on textiles goes to starch of alpha-amylase variants.

Can produce by random mutagenesis according to variant of the present invention, the invention further relates to a kind of method of the parent's of preparation alpha-amylase variants, this method comprises:

(a) dna sequence dna of random mutagenesis coding parent α-Dian Fenmei,

(b) in host cell, be expressed in the dna sequence dna that obtains sudden change in the step (a),

(c) host cell of the amylolytic enzyme of sudden change is expressed in screening, and this amylolytic enzyme is compared (for example, the Ca of character as reducing that have aforesaid improved properties with parent's α-Dian Fenmei²⁺The oxidative stability of dependency, increase, the thermostability of increase and/or the activity that under higher pH, improves).

Detailed Description Of The Invention

Representation

In description of the invention and claim, used the single-letter and the trigram code of the amino-acid residue of conventional Nucleotide single-letter sign indicating number and routine.For being easy to reference, alpha-amylase variants of the present invention is described by using following representation:

Amino acid originally: position: the amino acid of replacement

Example according to this representation is that 30 substituted lactamines can be expressed as l-asparagine in the position:

Ala 30 Asn or A30N

The L-Ala disappearance of same position is expressed as:

Ala 30^*Or A30^*

The insertion of a kind of additional amino-acid residue (as Methionin) can be expressed as:

Ala 30 AlaLys or A30AK

The disappearance of one section successive amino-acid residue is that example can be expressed as (30-33) with amino-acid residue 30-33^*

Specific α-Dian Fenmei comprises " disappearance " (promptly lacking an amino-acid residue) of comparing with other α-Dian Fenmei and produces on this position to be inserted, and this can be expressed as:

^*36 Asp or^*36D

Be illustrated in and insert an aspartic acid in the position 36

Multiple mutation separates by plus sige, that is:

Ala30Asp+Glu34Ser or A30N+E34S

Representative sudden change of 30 and 34 (wherein L-Ala and L-glutamic acid are replaced by l-asparagine and Serine respectively) in the position.

When one or more are selected one amino-acid residue and can be inserted into given position, can be expressed as:

A30N, E or

A30N or A30E

And, the position that ought be suitable for modifying is specified herein, and when being proposed, can be regarded as any other amino-acid residue and all can replace the amino-acid residue that exists on that position (promptly be selected from any amino-acid residue of A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y and V-but not at the amino-acid residue of said position normal presence) without any specific modification.Like this, for example, when the modification (replacement) of 202 methionine(Met) in the position referred but when not specifying, can be understood as any other amino acid, other amino acid among promptly any A of being selected from, R, N, D, C, Q, E, G, H, I, L, K, F, P, S, T, W, Y and the V can be used for replacing methionine(Met).

Parent's α-Dian Fenmei

Mention, alpha-amylase variants of the present invention is very suitable for preparing on the basis that has respectively in parent's α-Dian Fenmei of one of aminoacid sequence shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and the SEQ ID No.7.

Having respectively can be from basophil (alkaliphilic) Bacillus strain (being respectively bacterial strain NCIB12512 and bacterial strain NCIB 12513) acquisition, their detailed description in EP 0 277 216 B1 in parent's α-Dian Fenmei of the aminoacid sequence shown in SEQ ID No.1 and the SEQ ID No.2.[referring to the experimental section (as follows) of this paper] described in the preparation of these two kinds of parent's α-Dian Fenmei, purifying and order-checking in detail in WO 95/26397.

Parent's α-Dian Fenmei with the aminoacid sequence shown in the SEQ ID No.3 can obtain and at (particularly) bacteriology magazine from bacstearothermophilus, and 166 (1986), describe among the 635-643.

Parent's α-Dian Fenmei (it is identical with the 4th sequence in Fig. 1) with the aminoacid sequence shown in the SEQ ID No.7 can from " kind #707 of bacillus ", obtain and by Tsukamoto etc. in biological chemistry and biophysical studies communication, 151 (1988), the 25-31 page or leaf is described.

Have respectively the variant of parent's α-Dian Fenmei of the aminoacid sequence shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, the SEQID No.7 except above-mentioned, comprise having the variant that shows with parent's α-Dian Fenmei of the aminoacid sequence of at least a high homology of four seed amino acid sequences of back according to other interested variant of the present invention, as at least 70% homology, preferred (aforesaid) at least 80% homology, wish at least 85% homology, preferred at least 90% homology, homology for example, 〉=95%.

As mentioned above, the further standard of differentiating suitable parent's α-Dian Fenmei is: a) α-Dian Fenmei demonstrates the immune cross-reactivity with following antibody, described antibody is at having respectively at SEQ ID No.1, SEQ ID No.2, the α-Dian Fenmei of one of aminoacid sequence shown in SEQ ID No.3 and the SEQ ID No.7 produces, and/or b) α-Dian Fenmei is by following dna sequence encoding, and described DNA and coding have respectively at SEQ ID No.1, SEQ ID No.2, the dna sequence dna and identical probe hybridization of SEQ ID No.3 and the α-Dian Fenmei of one of aminoacid sequence shown in the SEQ ID No.7.

Mention,, can use algorithm known to carry out (as Lipman and Pearson described (1985)) aminoacid sequence relatively for the mensuration of polypeptide (as enzyme) homology degree.

Can use antibody to carry out immune cross-reactivity and measure, described antibody be anti-α-Dian Fenmei with the aminoacid sequence shown in the SEQ IDNo.1 or have the α-Dian Fenmei of the aminoacid sequence shown in the SEQ ID No.2 have the α-Dian Fenmei of the aminoacid sequence shown in the SEQ ID No.3 or have the α-Dian Fenmei of the aminoacid sequence shown in the SEQ ID No.7 at least a epi-position produced or reaction with it.

But antibody mono-clonal or polyclonal can pass through methods known in the art (for example, resembling by described (1989) such as Hudson) and produce.The example of the suitable determination techniques of knowing in the art comprise the Western blotting and radially immunodiffusion(ID) measure, for example, by described (1989) such as Hudson.

With probe hybridization [above-mentioned standard b)] serves as that the basis is used to differentiate that the oligonucleotide probe of suitable parent's α-Dian Fenmei is passable, for example, suitably preparation (this α-Dian Fenmei has respectively in one of sequence shown in SEQ ID No.1, SEQ ID No.2, SEQ IDNo.3 and the SEQ ID No.7) on the basis of all or part of aminoacid sequence of such α-Dian Fenmei perhaps prepares on all or part of basis of the corresponding nucleotide sequence of this α-Dian Fenmei.

Felicity condition in order to test hybridization is included in preimpregnation among 5 * SSC, prehybridization is 1 hour in the solution of the supersound process calf thymus DNA of 20% methane amide, 5 * Denhardt ' s solution, 50mM sodium phosphate and the 50 μ g sex change of～40 ℃ pH6.8, then down and replenished in the same solution of 100 μ MATP hybridization 18 hours at～40 ℃, perhaps use other by, for example, the method described of Sambrook etc. (1989).

Sudden change is to the influence of special properties

The result who obtains from the inventor as can be seen, with respect to said parent's α-Dian Fenmei, the variation of the special properties that variant shows (for example, thermostability or oxidative stability) is relevant with the type and the position of sudden change (amino acid whose replacement, disappearance and insertion) in the variant on sizable degree.Yet, be appreciated that specific sudden change or sudden change mode cause the observations of the variation of given character never to get rid of the possibility that said sudden change also can influence other character.

Oxidative stability: for the oxidative stability that improves with respect to the alpha-amylase variants of its parent's α-Dian Fenmei, it seems needs the oxidable amino-acid residue of at least one of parent (preferably a plurality of) to be lacked especially or by the original oxidable amino-acid residue of a kind of ratio the low different amino-acid residue of oxidation sensitive is replaced.

Specifically, relevant in this oxidable amino-acid residue is halfcystine, methionine(Met), tryptophane and tyrosine.Like this, for example, under the situation of the parent's α-Dian Fenmei that comprises halfcystine, can expect cysteine residues disappearance or by the low amino-acid residue of oxidizability to be substituted in acquisition has improvement with respect to parent's α-Dian Fenmei the variant of oxidative stability be important.

Under above-mentioned situation about having respectively in parent's α-Dian Fenmei of the aminoacid sequence shown in SEQ ID No.1, SEQ ID No.2 and the SEQ ID No.7 (all these parent's α-Dian Fenmei do not comprise cysteine residues but have more methionine(Met) content), the disappearance of methionine residues or the oxidative stability that replaces for the improvement of the variant that reaches generation are especially relevant.Like this, at SEQ ID No.1, the M9 of the aminoacid sequence shown in SEQID No.2 and the SEQ ID No.7, M10, M105, M202, M208, M261, M309, M382, M430 and M440 position and/or it seems it is especially effective for improving oxidative stability in the disappearance of one or more methionine residues of the M323 position of the aminoacid sequence shown in the SEQ ID No.2 or replacement [for example, by Threonine (T) or replaced by one of above listed other amino acid] disappearance or the replacement of methionine residues of suitable (equivalent) position of another kind of α-Dian Fenmei sequence of one of other standard of above-mentioned parent's α-Dian Fenmei (or meet).

Under situation about having in parent's α-Dian Fenmei of the aminoacid sequence shown in the SEQ ID No.3, for the improvement oxidative stability be purpose lack or the relevant amino-acid residue that replaces comprise single cysteine residues (C363) and-by with at the sequence analogy shown in SEQ ID No.1 and the SEQ ID No.3-the be positioned at methionine residues of position M8, M9, M96, M200, M206, M284, M307, M311, M316 and M438.

In this respect, term " suitable position " refers to such position, in the sequence contrast with the aminoacid sequence (as in the sequence as shown in the SEQ ID No.1) of said parent's α-Dian Fenmei aminoacid sequence and said " reference " α-Dian Fenmei is that the basis makes the two common amino-acid residue/zone to fashionable, this position is the most closely corresponding to the specific position in the said reference sequence (for example, being occupied by identical amino-acid residue).

With having respectively in the relevant specific purpose sudden change of the improvement (raising) of the oxidative stability of the α-Dian Fenmei of the aminoacid sequence shown in SEQ ID No.1, SEQ ID No.2 and the SEQ ID No.7 is that one or more following methionine(Met)s replace (or its suitable replacements in the aminoacid sequence of other α-Dian Fenmei that parent's α-Dian Fenmei according to the invention requires): M202A, R, N, D, Q, E, G, H, I, L, K, F, P, S, T, W, Y, V.

The relevant methionine(Met) of in the aminoacid sequence shown in the SEQ ID No.2 other replaces: M323A, R, N, D, Q, E, G, H, I, L, K, F, P, S, T, W, Y, V.

With having in the relevant specific purpose sudden change of the improvement (raising) of the oxidative stability of the α-Dian Fenmei of the aminoacid sequence shown in the SEQ ID No.3 is that one or more following methionine(Met)s replace: M200A, R, N, D, Q, E, G, H, I, L, K, F, P, S, T, W, Y, V; M311A, R, N, D, Q, E, G, H, I, L, K, F, P, S, T, W, Y, V; And M316A, R, N, D, Q, E, G, H, I, L, K, F, P, S, T, W, Y, V.

Thermostability: with regard to the thermostability that improves alpha-amylase variants with respect to its parent's α-Dian Fenmei, at least one in the aminoacid sequence shown in the SEQ ID No.1 of disappearance, preferably two or even three following amino-acid residues (or its suitable thing) seeming caters to the need especially: F180, R181, G182, T183, G184 and K185.Correspondingly, at SEQ ID No.2, SEQ ID No.3 be respectively: F180, R181, G182, D183, G184 and K185 (SEQ ID No.2) with corresponding relevant especially (with suitable) amino-acid residue in the aminoacid sequence shown in the SEQ ID No.7; F178, R179, G180, I181, G182 and K183 (SEQ ID No.3); And F180, R181, G182, H183, G184 and K185 (SEQ ID No.7).

Such interested especially (pairwise) in pairs disappearance is as follows:

R181^*+ G182^*And T183^*+ G184^*(SEQ ID No.1);

R181^*+ G182^*And D183^*+ G184^*(SEQ ID No.2);

R179^*+ G180^*And I181^*+ G182^*(SEQ ID No.3); With

R181^*+ G182^*And H183^*+ G184 ＂ (SEQ ID No.7).

(or in the α-Dian Fenmei of the requirement of another kind parent's α-Dian Fenmei according to the invention these suitable disappearances of disappearance) in pairs.

Other important sudden change relevant with thermostability that seem is: the one or more replacements in the amino-acid residue of the P260 to I275 in the sequence shown in the SEQ ID No.1 (or its suitable replacement in another parent's α-Dian Fenmei of the present invention), and as the replacement of 269 lysine residues in the position.

With the example of the concrete sudden change that seem important relevant with respect to the thermostability of the alpha-amylase variants of said parent's α-Dian Fenmei is one or more following replacement (or its suitable replacement in another parent's α-Dian Fenmei of the present invention) at the aminoacid sequence shown in the SEQ ID No.1: K269R; P260E; R124P; M105F, I, L, V; M208F, W, Y; L217I; V206I, L, F.

For the parent's α-Dian Fenmei with the aminoacid sequence shown in the SEQ ID No.2, important further (suitable) sudden change is corresponding one or more following replacements: M1O5F, I, L, V; M208F, W, Y; L217I; V206I, L, F; K269R.

For the parent's α-Dian Fenmei with the aminoacid sequence shown in the SEQ ID No.3, important further (suitable) sudden change is corresponding one or two following replacement: M206F, W, Y; L215I.

For the parent's α-Dian Fenmei with the aminoacid sequence shown in the SEQ ID No.7, important further (suitable) sudden change is corresponding one or more following replacements: M105F, I, L, V; M208F, W, Y; L217I; K269R.

Other example of (particularly comparing the alpha-amylase variants that reaches improved thermal stability with the said parent's α-Dian Fenmei) sudden change that seems important is: the one or more following replacement in the aminoacid sequence shown in SEQ ID No.1, SEQ ID No.2 and the SEQ IDNo.7 (or its suitable replacement in another parent's α-Dian Fenmei of the present invention): A354C+V479C; L351C+M430C; N457D, E+K385R; L355D, E+M430R, K; L355D, E+I411R, K; N457D, E.

Ca²⁺Dependency: just reach the Ca that alpha-amylase variants reduces with respect to its parent's α-Dian Fenmei²⁺Dependency is [promptly with regard to obtaining a kind of variant, this variant externally calcium ion concn in the medium shows gratifying amylolytic activity when lower than parent enzyme is necessary, therefore, this variant, for example, lower than the parent for the susceptibility of the condition of subduing calcium ion (as the condition that in the medium that comprises calcium complexing agent (strengthening the material of washing agent soil removability), obtains) as some], at SEQ ID No.1, seeming especially caters to the need: Y243F to mix one or more following replacements (or the suitable replacement in another parent's α-Dian Fenmei of the present invention) in the aminoacid sequence shown in SEQ IDNo.2 and the SEQ ID No.7, K108R, K179R, K239R, K242R, K269R, D163N, D188N, D192N, D199N, D205N, D207N, D209N, E190Q, E194Q and N106D.

Under the situation of the aminoacid sequence shown in the SEQ ID No.3, desirable especially replacement correspondingly (considerably) shows as following one or more: K107R, K177R, K237R, K240R, D162N, D186N, D190N, D197N, D203N, D205N, D207N, E188Q and E192Q.

In addition, above-mentioned replaces the D residue and replaces the E residue with the Q residue with the N residue, and other is at the Ca of reduction²⁺To be said D and/or E residue replace with any other amino-acid residue in relevant replacement in the dependency.

Reaching the Ca of reduction²⁺Other replacement that seems important in the dependency is to be present in the down paired replacement of the amino-acid residue of column position: in the position 113 of the aminoacid sequence shown in SEQ ID No.1, SEQ ID No.2 and the SEQ ID No.7 and 151 andposition 351 and 430; And in the post-11.2 of the aminoacid sequence shown in the SEQ IDNo.3 and 150 and position 349 and 428 (or suitable paired replacement in another parent's α-Dian Fenmei in the present invention), i.e. the paired replacement of following amino-acid residue:

G113+N151 (relating to SEQ ID No.1); A113+T151 (relating to SEQ ID No.2 and SEQ IDNo.7); G112+T150 (relating to SEQ ID No.3); L351+M430 (relating to SEQ ID No.1, SEQID No.2 and SEQ ID No.7); L349+I428 (relating to SEQ ID No.3).

About reaching the Ca of reduction²⁺Dependency, the interested especially paired replacement of this class is as follows:

G113T+N151I (relating to SEQ ID No.1); A113T+T151I (relating to SEQ ID No.2 and SEQ ID No.7); G112T+T150I (relating to SEQ ID No.3); And

L351C+M430C (relating to SEQ ID No.1, SEQ ID No.2 and SEQ ID No.7); L349C+I428C (relating to SEQ ID No.3).

With regard to Ca²⁺Dependent suitable replacement, (these are substituted in the stabilized enzyme conformation is important in some other replacements, and can consider to pass through, for example, improve calcium ion on the calcium binding site within the Alpha-starch lytic enzyme or within conjunction with or the ability that keeps, reach this result) be: at SEQ IDNo.1, one or more following replacement in the aminoacid sequence shown in SEQ ID No.2 and the SEQ ID No.7 (or suitable replacement in another parent's α-Dian Fenmei in the present invention): G304W, F, Y, R, I, L, V, Q, N, G305A, S, N, D, Q, E, R, K; H408Q, E.

Corresponding (suitable) replacement in the aminoacid sequence shown in the SEQ ID No.3 is: G302W, F, Y, R, I, L, V, Q, N; G303A, S, N, D, Q, E, R, K.

Reaching the Ca of reduction²⁺Other sudden change that seems important in the dependency is the amino acid whose paired disappearance (i.e. two amino acid whose disappearances) that is selected from down column position: R181, G182, T183 and the G184 in the aminoacid sequence shown in the SEQ ID No.1 (or meet in the present invention in the aminoacid sequence of the another kind of α-Dian Fenmei that parent's α-Dian Fenmei requires suitable position).Paired disappearance like this is as follows:

R181^*+G182^*；T183^*+G184^*；R181^*+T183^*；G182^*+T183^*；

G182^*+G184^*；R181^*+G184^*(SEQ ID No.1)；

R181^*+G182^*；D183^*+G184^*；R181^*+D183^*；G182^*+D183^*；

G182^*+G184^*；R181^*+G184^*(SEQ ID No.2)；

R179^*+G180^*；I181^*+G182^*；R179^*+I181^*；G180^*+I181^*；

G180^*+G182^*；R179^*+G182^*(SEQ ID No.3)；

R181^*+G182^*；H183^*+G184^*；R181^*+H183^*；G182^*+H183^*；

G182^*+G184^*；R181^*+G184^*(SEQ ID No.7)；

(or these lack suitable disappearance in the another kind of α-Dian Fenmei that another kind parent's α-Dian Fenmei according to the invention requires in pairs).

Iso-electric point (pI): PRELIMINARY RESULTS shows: the scourability of α-Dian Fenmei, for example, the laundry scourability is optimum when the pI value of the approaching said α-Dian Fenmei of pH value of washings (washing medium).Produce in the time of like this, suitably that to have when better mating the alpha-amylase variants of iso-electric point (pI value) of (comparing with the iso-electric point of said parent's α-Dian Fenmei) with the pH value of medium (as washing medium) (using said enzyme therein) be desirable.

About reducing iso-electric point, the preferred sudden change in the aminoacid sequence shown in the SEQ ID No.1 comprises one or more in the following replacement: Q86E, R124P, S154D, T183D, V222E, P260E, R310A, Q346E, Q391E, N437E, K444Q and R452H.These appropriate combination that reduce the replacement of iso-electric point comprise: Q391E+K444Q; Q391E+K444Q+S154D.

Correspondingly, comprise one or more in the following replacement about the preferred sudden change that reduces iso-electric point: L85E, S153D, I181D, K220E, P258E, R308A, P344E, Q358E and S435E at the aminoacid sequence shown in the SEQ ID No.3.

With regard to improving iso-electric point, the preferred sudden change in the aminoacid sequence shown in the SEQ ID No.2 comprises one or more in the following replacement: E86Q, L; D154S; D183T, I; E222V, K; E260P; A310R; E346Q, P; E437N, S; And H452R.

Structure according to many variants of the present invention has been described in the experimental section below.

Alpha-amylase variants of the present invention, except the character with one or more above-mentioned improvement, preferably such variant, this variant have the starch hydrolysis rate higher than parent α-Dian Fenmei under low concentration of substrate.Perhaps, the variant that alpha-amylase variants of the present invention is preferably such has higher V than parent α-Dian Fenmei when this variant is tested under the same conditions_MaxAnd/or lower K_mUnder the situation of hybrid α-Dian Fenmei, " the parent's α-Dian Fenmei " that be used to compare should be one of component enzyme that has top performance.

V_MaxAnd K_m(parameter of Michaelis-Menten equation) can be measured by well-known method.

The method for preparing alpha-amylase variants

Be known in the art several being used for the method for sudden change quiding gene.After the clone that coding for alpha-diastatic dna sequence dna is discussed briefly, the specific site of discussing within coding for alpha-diastatic sequence is produced the method for sudden change.

The dna sequence dna of clones coding α-Dian Fenmei

Use the various methods of knowing in this area can from any cell that produces said α-Dian Fenmei or microorganism, separate the dna sequence dna of coding parent α-Dian Fenmei.The first, use organic chromosomal DNA or the messenger RNA(mRNA) derive from the α-Dian Fenmei that generation studies to make up genomic dna and/or cDNA library.Then, if the aminoacid sequence of α-Dian Fenmei is known, just can synthesize and use the clone of the oligonucleotide probe of homology, mark with identifier number α-Dian Fenmei the genomic library from said from the organism preparation.Perhaps, comprise the probe that can be used as the clone of identifier number α-Dian Fenmei under more undemanding hybridization and wash conditions with the labeled oligonucleotide probe of known alpha-amylase gene homologous sequence.

Another method that is used for the clone of identifier number α-Dian Fenmei comprises: genomic DNA fragment is inserted into expression vector (as plasmid) transforms the α-Dian Fenmei negative bacteria with the genome dna library that produces, then the bacterium plating that transforms to the agar that comprises the α-Dian Fenmei substrate, make thus and can differentiate express alpha-diastatic clone.

Perhaps, the dna sequence dna of codase can be by the synthetic preparation of known standard method, for example, and the method that (1984) such as phosphoamidite method of being described by S.L Beaucage and M.H.Caruthers (1981) or Matthes are described.In the phosphoamidite method, oligonucleotide for example, synthetic on an automatization dna synthesizer, purifying, annealing, connects and is cloned in the appropriate carriers.

At last, dna sequence dna can be blended genome and synthetic origin, blended synthetic and cDNA origin or blended genome and cDNA origin, can prepare according to the fragment of standard technique by connecting synthetic, genomic or cDNA origin (suitably time, corresponding to the fragment of whole DNA sequence different piece).Dna sequence dna also can use specific primer by polymerase chain reaction (PCR) preparation, for example in the U.S. 4,683,202 or the method described of R.K.Saiki etc. (1988).

Site-directed mutagenesis

In case after being separated to coding for alpha-diastatic dna sequence dna and differentiating required mutational site, just can use the synthetic oligonucleotide to import sudden change.These oligonucleotide are included in the nucleotide sequence of required mutational site flank; Insert mutant Nucleotide between synthesis phase at oligonucleotide.In specific method, in carrying the carrier of alpha-amylase gene, produce the dna single chain breach that bridging connects coding for alpha-diastatic sequence.Then, the synthesizing ribonucleotide that carries required sudden change and the homology of single stranded DNA are partly annealed.Fill remaining breach with dna polymerase i (Klenow fragment), use the T4 ligase enzyme to connect construct then.Morinaga etc. (1984) have described the object lesson of this method.The U.S. 4,760,025 discloses by carrying out small boxlike and has changed the oligonucleotide that imports the various mutations of encoding.Yet, once can import even a greater variety of sudden change any by the method for Morinaga, because can import the oligonucleotide of a lot of all lengths.

Nelson and Long (1989) have described the another kind of method that sudden change is imported coding for alpha-diastatic dna sequence dna.It comprises that segmental 3 steps of PCR that comprise required sudden change (importing as one of primer in the PCR reaction by the DNA chain that uses chemosynthesis) generate.Can carry the dna fragmentation of sudden change by from the fragment that PCR generates, separating, and then be inserted in the expression plasmid with the restriction endonuclease cracking.

Random mutagenesis

Random mutagenesis is adapted at translating at least three parts in the gene of said aminoacid sequence or carries out with location or regiospecificity random mutagenesis within whole gene.

For being the regiospecificity random mutagenesis of purpose to improve thermostability, specifically, following codon position can be suitably as target (using single-letter amino acid abbreviations and the numbering of amino-acid residue in said sequence):

In the aminoacid sequence shown in the SEQ ID No.1:

120-140＝VEVNRSNRNQETSGEYAIEAW

178-187＝YKFRGTGKAW

264-277＝VAEFWKNDLGAIEN

In the aminoacid sequence shown in the SEQ ID No.2:

120-140＝VEVNPNNRNQEISGDYTIEAW

178-187＝YKFRGDGKAW

264-277＝VAEFWKNDLGALEN

In the aminoacid sequence shown in the SEQ ID No.3:

119-139＝VEVNPSDRNQEISGTYQIQAW

176-185＝YKFRGIGKAW

262-275＝VGEYWSYDINKLHN

In the aminoacid sequence shown in the SEQ ID No.7:

120-140＝VEVNPNRNQEVTGEYTIEAW

178-187＝YKFRGHGKAW

264-277＝VAEFWKNDLGAIEN

To reach the Ca of reduction²⁺When dependency was purpose, specifically, following codon position can be suitably as target:

In the aminoacid sequence shown in the SEQ ID No.1:

178-209＝YKFRGTGKAWDWEVDTENGNYDYLMYADVDMD

237-246＝AVKHIKYSFT

In the aminoacid sequence shown in the SEQ ID No.2:

178-209＝YKFRGDGKAWDWEVDSENGNYDYLMYADVDMD

237-246＝AVKHIKYSFT

In the aminoacid sequence shown in the SEQ ID No.7:

178-209＝YKFRGHGKAWDWEVDTENGNYDYLMYADIDMD

237-246＝AVKHIKYSFT

With reach substrate that alpha-amylase variants increases in conjunction with (i.e. carbohydrate class of Zeng Jiaing-as the combination of the substrate of amylose starch or these Alpha-starch lytic enzymes of amylopectin), improve (for example, higher) substrate specificity and/or improvement (for example, higher) when the specificity of substrate cracking (hydrolysis) was purpose, seeming especially was suitable as target at the following codon position of the aminoacid sequence shown in the SEQ ID No.1 (or the suitable codon position of another parent's α-Dian Fenmei) in the present invention:

In the aminoacid sequence shown in the SEQ ID No.1:

15-20＝WYLPND

52-58＝SQNDVGY

72-78＝KGTVRTK

104-111＝VMNHKGGA

165-174＝TDWDQSRQLQ

194-204＝ENGNYDYLMYA

234-240＝RIDAVKH

332-340＝HDSQPGEAL

The random mutagenesis of the dna sequence dna of the coding parent α-Dian Fenmei of carrying out according to the step a) of aforesaid method of the present invention can use any known method in this area to carry out easily.For example, can carry out random mutagenesis by using the suitable oligonucleotide of suitable physics or chemical mutagen, use or making dna sequence dna stand the mutagenesis that PCR produces.And, can carry out random mutagenesis by any combination of using these mutagenic compound.

Mutagenic compound can be a kind of, for example, and the mutagenic compound of inducing conversion, transversion, inversion, out of order (scrambling), disappearance and/or inserting.

Being suitable for the physics of this purpose or the example of chemical mutagen comprises: ultraviolet (UV) irradiation, azanol, N-methyl-N '-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine, nitrous acid, ethyl methane sulfonate (EMS), sodium bisulfite, formic acid and nucleotide analog.

When using such reagent, mutagenesis is typically by when the mutagenic compound of selecting exist, and in being fit under the condition that mutagenesis produces, incubation needs the dna sequence dna of coding parent enzyme of mutagenesis and the mutant DNA of selecting to have required character to carry out.

When mutagenesis was undertaken by using oligonucleotide, between synthesis phase, in the position that will change, oligonucleotide can mix with three non-parents' Nucleotide or admixture at oligonucleotide.Mix or admixture to avoid undesirable amino acid whose codon.Mix or the oligonucleotide of admixture can use by any technology of having delivered, for example, PCR, LCR or any archaeal dna polymerase and ligase enzyme are incorporated among the DNA of coding amylolytic enzyme.

When using the mutagenesis of PCR generation, under the condition that the mistake that increases Nucleotide is mixed, make the gene of the coding parent α-Dian Fenmei of chemically treated or non-processing stand PCR (Deshler, 1992; Leung etc., technology, the 1st volume, 1989,11-15).

Intestinal bacteria (Fowler etc., molecular gene genetics, 133,1974,179-191), the mutator strain of yeast saccharomyces cerevisiae or any other microorganism (can pass through, for example, the plasmid that comprises parent enzyme is transformed into mutator strain, cultivates the plasmid contain the mutator strain of plasmid and from mutator strain, to separate sudden change) random mutagenesis of the DNA of the amylolytic enzyme that is used to encode.Thereafter, the plasmid of sudden change can transform into expression organism.

Want the dna sequence dna of mutagenesis can be present in easily in the genome or cDNA library that from the organism of expressing parent's amylolytic enzyme, prepares.Perhaps, dna sequence dna may reside in suitable carriers such as plasmid or the phage, and these carriers can or otherwise be exposed to mutagenic compound with the mutagenic compound incubation.The DNA that needs mutagenesis also can be by being integrated into said cell genome or be present in the contained carrier of cell be present in the host cell.At last, need the DNA of mutagenesis to exist with isolated form.Be appreciated that the dna sequence dna preferably cDNA or the genomic dna sequence that will stand random mutagenesis.

In some cases, expressing the dna sequence dna that step (b) or screening step (c) can increase easily before and suddenly change.This amplification can be carried out according to methods known in the art, and the Oligonucleolide primers for preparing on the DNA that present preferable methods is to use in parent enzyme or the basis of aminoacid sequence carries out the amplification that PCR produces.

With the mutagenic compound incubation or after being exposed to mutagenic compound, the DNA of sudden change cultivates the suitable host cell expression that carries dna sequence dna by making under the condition that can produce expression.The host cell that is used for this purpose can be the dna sequence dna cell transformed with the sudden change that is in or be not in carrier, or carries the cell of the dna sequence dna of coding parent enzyme during mutagenic treatment.The example of the host cell that is fit to is as follows: gram positive bacterium such as subtilis, Bacillus licheniformis, bacillus lentus, bacillus brevis, bacstearothermophilus, Alkaliphilic bacillus, bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, bacillus lautus, bacillus megaterium, bacillus thuringiensis, shallow Streptomyces glaucoviolaceus or mouse ash streptomycete; And gram negative bacterium such as intestinal bacteria.

The dna sequence dna of sudden change comprises that also coding allows the dna sequence dna of the function of mutant DNA sequence expression.

The location random mutagenesis: random mutagenesis advantageously is positioned at the part of said parent's α-Dian Fenmei.This, for example, when some zone that identifies enzyme for the given character particularly important of enzyme and when the expectation improvement can produce the variant with improved properties, may be favourable.When the tertiary structure of parent enzyme being illustrated and when relevant, can being differentiated such zone usually with the function of enzyme.

Can conveniently positioned random mutagenesis by the induced-mutation technique or any appropriate technology that other is known in the art that use above-mentioned PCR to produce.

Perhaps, can pass through, for example, be inserted into the dna sequence dna of the dna sequence dna part that will improve with the separation coding in the suitable carriers, thereafter, said part stands mutagenesis by using above-mentioned any mutafacient system.

About the screening step in above-mentioned method of the present invention, can use by measuring and carry out easily based on the filter membrane of following principle:

Can express the microorganism of the purpose amylolytic enzyme of sudden change and cultivate on suitable substratum under the condition of enzyme secretion being suitable for, substratum has filter membrane that comprises the first layer conjugated protein and the double-deck filter membrane that shows the second layer filter membrane of low protein bound ability on it.Microorganism is positioned on the second layer filter membrane.When cultivating thereafter, the first layer filter membrane of the enzyme that comprises microorganism secretion is separated from the second layer filter membrane that comprises microorganism.The first layer filter membrane carries out required enzymatic activity screening, differentiates the corresponding microorganism bacterium colony that is present on the second layer filter membrane simultaneously.

Being used in conjunction with the active filter membrane of enzymatic can be the filter membrane of any conjugated protein, for example, and nylon or nitrocellulose membrane.Carrying the upper strata filter membrane of expressing organic bacterium colony can be that conjugated protein is not had avidity or the low any filter membrane of avidity, for example, and rhodia or Durapore^TMFilter membrane can or be handled between the enzymatic activity detection period with any condition pre-treatment that is used to screen.

Enzymatic activity can detect by dyestuff, fluorescence, precipitation, pH indicator, IR-specific absorption or any other known technology that is used to detect enzymatic activity.

Detection compound can be fixed by any fixing agent, for example, and agarose, agar, gelatin, polyacrylamide, starch, filter paper, cloth; Or any combination of fixing agent.

Alpha-amylase activity can detect by the amylopectin of fixed Cibacron Red mark on agarose.In order to screen the heat with increase and the variant of high pH value stabilization, the filter membrane that is combined with alpha-amylase variants is the specified time of incubation in the damping fluid under 10.5 and 60 ℃ or 65 ℃ at pH, brief rinsing and place and carry out activity detection on amylopectin-agarose matrix in deionized water.Remaining activity is come as can be seen by the Cibacron Red cracking that the amylopectin degraded causes.Select such condition to be because the activity that has in the α-Dian Fenmei of the aminoacid sequence shown in the SEQ ID No.1 almost can not detect.Because the release of Cibacron Red increases, and stablizes variant and show higher colour intensity under identical condition.

In order to screen under lower temperature and/or active optimum variant in the wideer temperature range, the filter membrane that is combined with variant directly is placed on the substrate flat board of amylopectin-Cibacron Red and under required temperature (for example, 4 ℃, 10 ℃ or 30 ℃) the specified time of incubation.After this time,, and under lower temperature, there is optimum active variant to demonstrate the cracking that has improved amylopectin because the activity that has in the α-Dian Fenmei of the aminoacid sequence shown in the SEQ ID No.1 almost can not detect.In order to screen dependency or the reactivity that said variant changes described additive, can be before incubation on the amylopectin matrix at the required substratum of all kinds-for example, comprise Ca²⁺, scale remover, EDTA or other relevant additive solution-middle incubation.

The method for preparing the hybrid α-Dian Fenmei

As the alternative method of site-specific mutagenesis, can be by being the alpha-amylase variants of the hybrid of at least two kinds of composition α-Dian Fenmei in conjunction with said each the relative section preparation of gene.

The enzyme of natural generation can by above-mentioned at random or the site-directed mutagenesis genetic improvement.Perhaps, an a kind of part of enzyme can be replaced to obtain chimaeric enzyme by an alternative part.This replacement can be by routine outer-gene montage technology or body in the combination of reorganization or these two kinds of technology reach.When using conventional outer-gene montage technology, the required part of the encoding sequence of alpha-amylase gene is used suitable locus specificity restriction enzyme disappearance; The insertion replacement of the required part of the encoding sequence that the part of encoding sequence disappearance can be by different α-Dian Fenmei has then so just produced the chimeric nucleotide sequence of the new α-Dian Fenmei of encoding.Perhaps, can pass through, for example, use the overlapping extension method of the PCR that describes by (1988) such as Higuchi to merge alpha-amylase gene.

Recombinant technology depends on the following fact in the body: the different dna segments with height homologous region (dna sequence dna is identical) can be recombinated, and promptly cut off and exchange DNA, set up new key at homologous region then.Therefore, when two kinds of differences but the diastatic encoding sequence of homologous when being used for transformed host cell, the reorganization of homologous sequence just causes the generation of chimeric gene sequence in vivo.These encoding sequences will cause the generation of chimeric amylase gene product by the translation of host cell.Recombinant technology is described in the U.S. 5,093,257 and EP 252 666 in the special gonosome.

Perhaps, can be by the synthetic hybrid enzyme of standard chemical process known in the art.For example, referring to (1984) such as Hunkapiller.Therefore, the peptide with suitable aminoacid sequence can all or part ofly synthesize and connect to form hybrid enzyme of the present invention (variant).

The expression of alpha-amylase variants

According to the present invention, the dna sequence dna of the α-Dian Fenmei of the encoding mutant that produces by aforesaid method or any alternative method known in the art can use the formal representation of expression vector with enzyme, and this expression vector typically comprises control sequence and the dispensable repressor gene or the various activated gene of coding promotor, operon, ribosome bind site, translation initiation signal.

The recombinant expression vector that carries the dna sequence dna of coding alpha-amylase variants of the present invention can be any carrier that can stand the recombinant DNA process easily, and the host cell that it will import is often depended in the selection of carrier.Like this, carrier can be autonomously replicationg vector (promptly as the carrier of the outer entity existence of karyomit(e), duplicating of it is independent of THE REPLICATION OF CHROMOSOME), for example, and plasmid, phage or extrachromosomal element, minichromosome or artificial chromosome.Perhaps, carrier can be to be integrated into the host cell gene group and carrier that the karyomit(e) integrated with it duplicates when importing host cell.

In carrier, dna sequence dna should be operably connected on the suitable promoter sequence.Promotor can be any at transcriptional activity shown in the host cell of selecting and can be the dna sequence dna of homologous or allogenic proteinic gene to host cell derived from coding.The example (especially in host bacterium) of the promotor that is fit to of transcribing of the dna sequence dna of guidance coding alpha-amylase variants of the present invention is the promotor of intestinal bacteria lactose operon, the dagA promotor of streptomyces coelicolor gelase gene, the promotor of bacillus licheniformis alpha-amylase gene (amyL), bacstearothermophilus produces the promotor (amyM) of maltogenic amylase gene, the promotor of the α-Dian Fenmei of bacillus amyloliquefaciens (amyQ), the promotor of subtilis xylA and xylB gene etc.For transcribing in the fungal host, the example of useful promotor is those promotors derived from the gene of the following enzyme of coding: aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartate protease, the neutral α-Dian Fenmei of aspergillus niger, aspergillus niger acid stable alpha-amylase, aspergillus niger glucoamylase, Rhizomucor miehei lipase, aspergillus oryzae Sumizyme MP, aspergillus oryzae triosephosphate isomerase or Aspergillus nidulans acetamidase.

Expression vector of the present invention also can comprise suitable transcription terminator, also comprises the polyadenylation sequence on the dna sequence dna that is operably connected to coding alpha-amylase variants of the present invention in eukaryote.Stop suitably to derive from the source identical with promotor with the polyadenylation sequence.

Carrier can also comprise the dna sequence dna that carrier can be duplicated in said host cell.The example of such sequence is the replication orgin of plasmid pUC19, pACYC177, pUB110, pE194, pAMB1 and plJ702.

Carrier also can comprise selective marker, for example, its product has replenished the gene of the defective in the host cell, as from the dal gene of subtilis or Bacillus licheniformis or give antibiotics resistance such as the gene of penbritin, kantlex, paraxin or tetracyclin resistance.And then carrier can comprise selective marker such as amdS, argB, the niaD of Aspergillus and cause the mark sC of hygromycin resistance, selects also can pass through, and for example, the cotransformation of describing in WO 91/17243 is finished.

Have superiority although express in the born of the same parents in some aspects, for example, when using some bacterium as host cell, preferred usually the expression is that born of the same parents express outward.

The method that is suitable for making up coding alpha-amylase variants of the present invention and comprises the carrier of promotor, terminator and other composition respectively is [referring to, for example, Sambrook etc. (1989)] well known to those skilled in the art.

The cell of the present invention that comprises the DNA construct of the invention described above or expression vector is used as host cell and has superiority in the reorganization of alpha-amylase variants of the present invention produces.Cell can transform with the DNA construct with coding variant of the present invention by DNA construct (one or more copy) being integrated in the host chromosome easily.Because dna sequence dna more may be stablized maintenance in cell, this integration has been considered to an advantage usually.DNA construct is integrated into host chromosome and can carries out according to ordinary method (for example, by homology or allos reorganization).Perhaps, cell can transform with the expression vector that above-mentioned and dissimilar host cells is got in touch.

Cell of the present invention can be high organic cell, as the cell of Mammals or insect, but microorganism cells preferably, for example, bacterium or fungi (comprising yeast) cell.

The example of suitable bacterium is as follows: gram positive bacterium, as subtilis, Bacillus licheniformis, bacillus lentus, bacillus brevis, bacstearothermophilus, Alkaliphilic bacillus, bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, bacillus lautus, bacillus megaterium, bacillus thuringiensis, shallow Streptomyces glaucoviolaceus or mouse ash streptomycete; And gram negative bacterium such as intestinal bacteria.The conversion of bacterium can be passed through, for example, and the conversion of protoplastis or finish with original known mode use experience attitude cell.

The species that are selected from yeast belong or Schizosaccharomyces that the yeast organism is favourable, for example, yeast saccharomyces cerevisiae.Filamentous fungus can advantageously belong to the species of Aspergillus, for example, and aspergillus oryzae or aspergillus niger.Fungal cell's method for transformation comprises by original known mode and forms and transform the regeneration of protoplastis and cell walls then.The proper method that is used for the conversion of Aspergillus host cell is described atEP 238 023.

In yet another aspect, the present invention relates to produce the method for alpha-amylase variants of the present invention, this method comprises aforesaidly cultivates host cell and reclaim variant from cell and/or substratum under helping the condition that variant produces.

The substratum that is used for culturing cell can be any conventional substratum that is suitable for cultivating said host cell and obtains alpha-amylase variants expression of the present invention.Suitable culture medium can obtain from commercial provider, or according to the preparation of the prescription delivered (for example, the catalogue at U.S. typical case's culture collection center described in).

Secretion can be reclaimed from substratum by well-known method easily from the alpha-amylase variants of host cell, this method comprises by centrifugal or filter isolated cell from substratum, and the mode by salt (as ammonium sulfate) precipitates the protein component in the substratum and then uses chromatography (as ion exchange chromatography, affinity chromatography or the like).

Industrial application

Because the activity of alpha-amylase variants of the present invention under the alkaline pH value, they are particularly suitable for using in various industrial processes.Specifically, they all have potential to use in washing, dishwashing detergent and hard-surface cleaning de-sludging composition (as follows), also are useful at generation sweetener and ethanol from starch still.The condition that conventional starch transforms processing and liquefaction and/or saccharification processing exists, for example, and the U.S. 3,912,590, describe among EP 252,730 and the EP 63,909.

Some field of the application of alpha-amylase variants of the present invention is summarized as follows.

Application about paper: alpha-amylase variants of the present invention has from starch reinforces the waste paper of (starch-reinforced) and the valuable character that waste paperboard produces ligno-cellulosic materials (as paper pulp, paper and cardboard), and paper pulpization occurs in pH value and is higher than 7 place and amylase help the waste materials decomposition by the degraded of reinforcing starch place especially again.Alpha-amylase variants of the present invention is particularly suitable for from the deinking of the printed waste papermaking starch dressing or that comprise starch/utilization processing again.In order to produce the new paper of high brightness, it is normally desirable to remove printing ink; The example how variant of the present invention in this way uses is described in PCT/DK94/00437.

Alpha-amylase variants of the present invention also is very useful in modified starch, and the starch of enzymatic improvement is used for and basic filler (as lime carbonate, kaolin and clay) papermaking together.For alkali alpha amylase variant of the present invention, modified starch is practicable in the presence of filler, can carry out simpler, more complete processing with regard to making like this.

Textiles removes slurry (desizing): alpha-amylase variants of the present invention also is particularly suitable for textiles and removes slurry.In textiles processing industry, use α-Dian Fenmei to promote to remove the paste (size) that comprises starch in the processing traditionally going to starch as auxiliary agent, this paste during weaving as protectiveness dressing to fabric yarn.

Wherein said fabric is washed, bleaches and dyes being important for guaranteeing removing paste after the weaving fully with the optimal effectiveness in the post-treatment.The enzymatic starch degradation is preferred, because it does not damage the fiber of textiles or fabric.

In order to reduce tooling cost and to increase makers' turnout, go slurry to handle and be used in combination with washing and blanching step sometimes.In this case, non-enzyme auxiliary agent (as alkali or oxygenant) typically is used for starch-splitting, because traditional α-Dian Fenmei is not ten minutes and high pH level and bleach-compatible.The non-enzymatic decomposition of starch paste is because the destruction that employed quite aggressive (aggressive) pharmaceutical chemicals has caused some fibre really.

Of the present invention in higher pH level and there is the alpha-amylase variants that demonstrates the starch degradation performance of increase in the presence of oxidation (bleaching) agent to be particularly suitable for the above-mentioned slurry processing of going, especially be suitable for replacing the non-enzyme of current use to go to starch agent.Alpha-amylase variants can use separately or be used in combination with cellulase when going slurry to comprise cellulosic fabric or textiles.

Beer production: alpha-amylase variants of the present invention also has been considered to very useful in the brewage process; In this process, when smashing processing to pieces, add variant typically.

Wash or the washing agent additive of dishwashing detergent and the application of de-sludging composition being used for: because the washing of improvement and/or dishwashing detergent performance often are the results of above-mentioned character improvement, many alpha-amylase variants of the present invention (comprising hybrid) are particularly suited for mixing the de-sludging composition, for example, having in the scope that is intended to pH value 7-13, is the de-sludging composition that uses in the scope of pH value 8-11 specifically.According to the present invention, alpha-amylase variants can be used as the component of de-sludging composition to be added.Like this, it can be included in the form of washing agent additive in the de-sludging composition.

Like this, another aspect of the present invention relates to the washing agent additive that comprises according to alpha-amylase variants of the present invention.Enzyme can comprise the additive that separates of one or more enzymes or combined additive that interpolation comprises all these enzymes is included in the de-sludging composition by interpolation.Washing agent additive of the present invention (being the additive of a kind of isolating additive or combination) for example can be made, granular, liquid, slurry etc.The preferred zymin of washing agent additive is granula (being non-powdery granula specifically), liquid (being stable liquid specifically), slurry or protected enzyme (as follows).

De-sludging composition and washing agent additive can also comprise one or more other enzymes that uses usually in scale remover, as proteolytic enzyme, lipase, amylolytic enzyme, oxydase (comprising peroxidase) or cellulase.

Have been found that: when α-Dian Fenmei can obtain substantial improvement with another kind of amylolytic enzyme (as Starch debranching enzyme, isoamylase, beta-amylase, starch glucosidase and CTG enzyme) when combining in washing and/or dishwashing detergent performance.The example that is suitable for the commercially available amylolytic enzyme of given purpose is AMG^TM, Novamyl^TMAnd Promozyme^TM, they all can be from Novo Nordisk A/S, Bagsvaerd, and Demark obtains.Therefore, a particular of the present invention relates to and comprise alpha-amylase variants of the present invention and at least a other amylolytic enzyme (for example, selecting) bonded washing agent additive in those above-mentioned enzyme.

Non-powdery granula can as, for example disclosed method produces in the U.S. 4,106,991 and the U.S. 4,661,452, not essentially by the methods known in the art dressing; The details of relevant dressing is as described below.When being used in combination of different washing agent enzymes, enzyme can mix before or after granular becoming.

Liquid enzyme preparation can pass through according to known technology, for example, adds polyvalent alcohol such as propylene glycol, sugar or sugar alcohol, lactic acid or boric acid and makes it stable.Other enzyme stabilizers is well known in the art.Protected enzyme can be according to disclosed method preparation among theEP 238 216.

As mentioned above, another aspect of the present invention relates to de-sludging composition (for example, washing, dishwashing detergent or hard-surface cleaning are used to do washing), and said composition comprises alpha-amylase variants of the present invention (comprising hybrid) and tensio-active agent.

De-sludging composition of the present invention can be any form easily, for example, and pulvis, granula or liquid.Liquid washing agent can be aqueous (typically comprising nearly 90% water and the organic solvent of 0-20%) or water-free (as described at EP 120,659).

The de-sludging composition

When alpha-amylase variants of the present invention (for example is used as the de-sludging composition; laundry washing de-sludging composition or dishwashing detergent de-sludging composition) component the time; it can, for example, be included in the de-sludging composition with the form of non-powdery granula, stabilising liq or protected enzyme.As mentioned above, non-powdery granula can as, for example, the U.S. 4,106,991 and 4,661,452 (both belongs to Novo Industri A/S) disclosed method produces, and not essential the methods known in the art dressing of using.The example of wax coating material be poly-(oxyethane) product (polyoxyethylene glycol, PEG), molecular-weight average is 1000 to 20000; Nonyl phenol with ethoxylation of 16 to 50 ethylene oxide unit(s)s; The Fatty Alcohol(C12-C14 and C12-C18) of ethoxylation (wherein alcohol comprises 12 to 20 carbon atoms) wherein has 15 to 80 ethylene oxide unit(s)s; Fatty Alcohol(C12-C14 and C12-C18); Lipid acid; Single, double, three esters of fatty acid glycerine.The film that is suitable for using by the liquefied bed technology forms coating material and provides in GB 1483591.

The enzyme that adds with the liquid enzyme preparation form can pass through according to known technology as mentioned above, and for example, it is stable to add polyvalent alcohol such as propylene glycol, sugar or sugar alcohol, lactic acid or boric acid.Other enzyme stabilizers is well-known in the art.

Be included in protected enzyme in the de-sludging composition of the present invention can be as mentioned above according at EP238, disclosed method preparation in 216.

De-sludging composition of the present invention can be any form easily, for example, and pulvis, granula, paste or liquid.Liquid washing agent can be aqueous (typically comprising nearly 70% water and the organic solvent of 0-30%) or water-free.

The de-sludging composition comprises one or more tensio-active agents, and every kind can be anionic, non-ionic, cationic or amphoteric (zwitterion).Washing agent comprise usually the anion surfactant of 0-50% such as linear alkyl benzene sulfonate (LAS), sulfonated (AOS), alkyl-sulphate (aliphatic alcohol sulfate) (AS), alcohol ethoxy vitriol (AEOS or AES), secondary paraffin sulfonate (SAS), alpha-sulfo-fatty acid methyl ester, alkyl or alkenyl succsinic acid or soap.It also can comprise the nonionogenic tenside of 0-40% such as alcohol ethoxylate (AEO or AE), pure propoxylated glycerine, carboxylated alcohol ethoxylate, nonyl phenol ethoxylate, alkyl polysaccharide glucoside, alkyl-dimethyl amine oxide, ethoxylated fatty acid single ethanol amide, fatty monoethanol amide or poly-hydroxyalkyl fatty acid amide (for example, described in WO 92/06154).

The de-sludging composition also comprises one or more other enzymes, as Starch debranching enzyme, esterase, lipase, at, proteolytic enzyme, cellulase, peroxidase or oxydase, for example, laccase.

Usually washing agent comprises the material that 1-65% strengthens the washing agent detergent action (though some dishwashing detergent washing agent can comprise nearly the material of 90% enhancing washing agent detergent action) or complexing agent such as zeolite, diphosphate, triphosphate, phosphonate, Citrate trianion, complexon I (NTA), ethylenediamine tetraacetic acid (EDTA) (EDTA), diethylene triaminepentaacetic acid(DTPA) (DTMPA), alkyl or alkenyl succsinic acid, soluble silicate or layered silicate (for example, deriving from the SKS-6 of Hoechst).

The material of enhancing washing agent detergent action can be subdivided into phosphorous or not contain phosphor type.The examples of materials that the phosphorated inorganic alkaline strengthens the washing agent detergent action comprises: water-soluble salt, especially alkali metal pyrophosphate, orthophosphoric acid salt, polyphosphate and phosphonate.The examples of substances of the inorganic enhancing washing agent detergent action of phosphorated does not comprise water soluble alkali metal carbonate, borate and silicate, and layering bisilicate and various types of water-insoluble crystallization or unbodied aluminosilicate, its mesolite is the representative of knowing most.

The examples of substances of suitable organic enhancing washing agent detergent action comprises the ammonium salt of basic metal, ammonium or the replacement of succsinic acid, propanedioic acid, lipid acid propanedioic acid, lipid acid sulfonic acid, carboxyl methoxyl group succsinic acid, poly-acetic acid, carboxylic acid, poly carboxylic acid, aminopolycarboxylic and poly-ethanoyl carboxylic acid.

Washing agent also can not strengthen detergent action, promptly is substantially free of the material that strengthens the washing agent detergent action.

Washing agent can comprise one or more polymkeric substance.Example is carboxymethyl cellulose (CMC; Typically with sodium-salt form), poly-(V-Pyrol RC) (PVP), polyoxyethylene glycol (PEG), poly-(vinyl alcohol) (PVA), polycarboxylate such as polyacrylic ester, polymaleic acid ester, toxilic acid/acrylic acid multipolymer and methylacrylic acid lauryl/acrylic copolymer.

The de-sludging composition also can comprise the SYNTHETIC OPTICAL WHITNER of chlorine/bromine type or oxygen type.SYNTHETIC OPTICAL WHITNER can be dressing or encapsulated.The example of the SYNTHETIC OPTICAL WHITNER of inorganic chlorine/bromine type is hypochlorite or the hypobromite and the Efficacious Disinfeitant of lithium, sodium or calcium.Bleaching system also can comprise H₂O₂Source such as perborate or percarbonate, they can with the bleach activator of peracid form such as tetraacetyl ethylene diamine (TAED) or nonanoyl hydroxy benzene sulfonate (NOBS) combination.

The example of organochlorine/bromine type SYNTHETIC OPTICAL WHITNER be heterocycle N-bromination and N-chlorimide such as trichloroisocyanuric acid, tribromo tricarbimide, dibromo isocyanurate, DICHLOROISOCYANURIC ACID and with the salt of water lyotropy positively charged ion such as potassium and sodium.Hydantoin compound also is suitable.Bleaching system also can comprise, for example, and acid amides, imines or sulfone type peroxy acid (peroxyacids).

In the dishwashing detergent washing agent, oxygen bleaching agent is preferred, and inorganic peracid salt form for example preferably has bleach precursor or as peracetic acid compound.The exemplary of the peroxy bleaching agent compound that is fit to is alkali metal perborate, tetrahydrochysene salt and single hydrogen salt, alkali metal percarbonate, persilicate and superphosphate.Preferred activator material is TAED or NOBS.

The enzyme of de-sludging composition of the present invention can use conventional stablizer stable, for example, polyvalent alcohol such as propylene glycol or glycerine, sugar or sugar alcohol, lactic acid, boric acid or boric acid derivatives as, for example, the aromatics borate, and composition can be as preparing in the method described in WO 92/19709 and the WO 92/19708.Enzyme of the present invention also can be stable by adding the reversible enzyme inhibitors, for example, and protein type (as described in) or borate type enzyme inhibitors at EP 0,544 777 B1.

The conventional washing agent composition that washing agent also can comprise other as, for example, fabric instrumentality (fabricconditioners) the foam depressor, corrosion inhibitor, dirt suspension agent, anti-soil thing deposition agent, dyestuff, dewatering agent, sterilant, optical whitening agent or the spices again that comprise clay, deflocculation agent material, foam exciting agent/foam depressor (in dishwashing detergent washing agent foam depressor), foaming suds.

PH value (in the aqueous solution so that the measurement of concetration of time spent) is normally neutral or alkaline, for example, and between 7-11.

The specific forms of the laundry de-sludging composition within the scope of the present invention comprises:

1) be mixed with and have the de-sludging composition of the granula of the bulk density of 600g/l at least, it comprises

Linear alkyl phenyl sulfonate (calculating) with acid	7-12％
		Alcohol ethoxy vitriol (for example, C12-18Alcohol, 1-2EO) or alkyl-sulphate (C for example16-18)	1-4％
Alcohol ethoxylate (for example, C14-15Alcohol, 7EO)	5-9％
		Yellow soda ash is (as Na2CO3)	14-20％
Soluble silicate is (as Na2O，2SiO2)	2-6％
		Zeolite is (as NaAlSiO4)	15-22％
Sodium sulfate is (as Na2SO4)	0-6％
		Trisodium Citrate/citric acid is (as (C6H5Na3O7/C6H8O7)	0-15％
Sodium peroxoborate is (as NaBO3.H2O)	11-18％
		TAED	2-6％
Carboxymethyl cellulose	0-2％
		Polymkeric substance (for example, toxilic acid/acrylic copolymer, PVP, PEG)	0-3％

Enzyme (calculating) with pure enzyme protein matter	0.0001-0.1％
		Accessory integral part (for example, the froth suppressor of foaming suds, spices, optical whitening agent, optical white)	0-5％

2) be mixed with and have the de-sludging composition of the granula of the bulk density of 600g/l at least, it comprises

Linear alkyl phenyl sulfonate (calculating) with acid	6-11％
		Alcohol ethoxy vitriol (for example, C12-18Alcohol, 1-2EO) or alkyl-sulphate (C for example16-18)	1-3％
Alcohol ethoxylate (for example, C14-15Alcohol, 7EO)	5-9％
		Yellow soda ash is (as Na2CO3)	15-21％
Soluble silicate is (as Na2O，2SiO2)	1-4％
		Zeolite is (as NaAlSiO4)	24-34％
Sodium sulfate is (as Na2SO4)	4-10％
		Trisodium Citrate/citric acid is (as C6H5Na3O7/C6H8O7)	0-15％
Carboxymethyl cellulose	0-2％
		Polymkeric substance (for example, toxilic acid/acrylic copolymer, PVP, PEG)	1-6％
Enzyme (calculating) with pure enzyme protein matter	0.0001-0.1％
		Accessory integral part (for example, the froth suppressor of foaming suds, spices)	0-5％

3) be mixed with and have the de-sludging composition of the granula of the bulk density of 600g/l at least, it comprises

Linear alkyl phenyl sulfonate (calculating) with acid	5-9％
		Alcohol ethoxylate (for example, C12-15Alcohol, 7EO)	7-14％
Soap is as lipid acid (for example, C16-22Lipid acid)	1-3％
		Yellow soda ash is (as Na2CO3)	10-17％
Soluble silicate is (as Na2O，2SiO2)	3-9％
		Zeolite is (as NaAlSiO4)	23-33％

Sodium sulfate is (as Na2SO4)	0-4％
		Sodium peroxoborate is (as NaBO3.H2O)	8-16％
TAED	2-8％
		Phosphonate (for example, EDTMPA)	0-1％
Carboxymethyl cellulose	0-2％
		Polymkeric substance (for example, toxilic acid/acrylic copolymer, PVP, PEG)	0-3％
Enzyme (calculating) with pure enzyme protein matter	0.0001-0.1％
		Accessory integral part (for example, the froth suppressor of foaming suds, spices, optical whitening agent)	0-5％

4) system is mixed with and has the de-sludging composition of the granula of the bulk density of 600g/l at least, and it comprises

Linear alkyl phenyl sulfonate (calculating) with acid	8-12％
		Alcohol ethoxylate (for example, C12-15Alcohol, 7EO)	10-25％
Yellow soda ash is (as Na2CO3)	14-22％
		Soluble silicate is (as Na2O，2SiO2)	1-5％
Zeolite is (as NaAlSiO4)	25-35％
		Sodium sulfate is (as Na2SO4)	0-10％
Carboxymethyl cellulose	0-2％
		Polymkeric substance (for example, toxilic acid/acrylic copolymer, PVP, PEG)	1-3％
Enzyme (calculating) with pure enzyme protein matter	0.0001-0.1％
		Accessory integral part (for example, the froth suppressor of foaming suds, spices)	0-5％

5) aqueous liquid de-sludging composition, it comprises

Linear alkyl phenyl sulfonate (calculating) with acid

15-21％

Alcohol ethoxylate (for example, C12-15Alcohol, 7EO or C12-15Alcohol, 5EO)	12-18％
		Soap is as lipid acid (for example, oleic acid)	3-13％
Alkenyl succinic acid (C12-14)	0-13％
		Monoethanolamine	8-18％
Citric acid	2-8％
		Phosphonate	0-3％
Polymkeric substance (for example, PVP, PEG)	0-3％
		Borate is (as B4O72-)	0-2％
Ethanol	0-3％
		Propylene glycol	8-14％
Enzyme (calculating) with pure enzyme protein matter	0.0001-0.1％
		Accessory integral part (for example, dispersion agent, the froth suppressor of foaming suds, spices, optical whitening agent)	0-5％

6) aqueous (structured) liquid de-sludging composition that structure is arranged, it comprises

Linear alkyl phenyl sulfonate (calculating) with acid	15-21％
		Alcohol ethoxylate (for example, C12-15Alcohol, 7EO or C12-15Alcohol, 5EO)	3-9％
Soap is as lipid acid (for example, oleic acid)	3-10％
		Zeolite is (as NaAlSiO4)	14-22％
Tripotassium Citrate	9-18％
		Borate is (as B4O72-)	0-2％
Carboxymethyl cellulose	0-2％
		Polymkeric substance (for example, PVP, PEG)	0-3％
The grappling polymkeric substance is as, lauryl methylacrylic acid/acrylic copolymer for example; Molar ratio 25:1; Molecular weight 3800	0-3％
		Glycerine	0-5％
Enzyme (calculating) with pure enzyme protein matter	0.0001-0.1％

Accessory integral part (for example, dispersion agent, the froth suppressor of foaming suds, spices, optical whitening agent)

0-5％

7) be mixed with and have the de-sludging composition of the granula of the bulk density of 600g/l at least, it comprises

Aliphatic alcohol sulfate	5-10％
		The fatty monoethanol amide of ethoxylation	3-9％
Soap is as lipid acid	0-3％
		Yellow soda ash is (as Na2CO3)	5-10％
Soluble silicate is (as Na2O，2SiO2)	1-4％
		Zeolite is (as NaAlSiO4)	20-40％
Sodium sulfate is (as Na2SO4)	2-8％
		Sodium peroxoborate is (as NaBO3.H2O)	12-18％
TAED	2-7％
		Polymkeric substance (for example, toxilic acid/acrylic copolymer, PEG)	1-5％
Enzyme (calculating) with pure enzyme protein matter	0.0001-0.1％
		Accessory integral part (for example, optical whitening agent, the froth suppressor of foaming suds, spices)	0-5％

8) be mixed with the de-sludging composition of granula, it comprises

Linear alkyl phenyl sulfonate (calculating) with acid	8-14％
		The fatty monoethanol amide of ethoxylation	5-11％
Soap is as lipid acid	0-3％
		Yellow soda ash is (as Na2CO3)	4-10％
Soluble silicate is (as Na2O，2SiO2)	1-4％
		Zeolite is (as NaAlSiO4)	30-50％
Sodium sulfate is (as Na2SO4)	3-11％
		Trisodium Citrate is (as C6H5Na3O7)	5-12％

Polymkeric substance (for example, PVP, toxilic acid/acrylic copolymer, PEG)	1-5％
		Enzyme (calculating) with pure enzyme protein matter	0.0001-0.1％
Accessory integral part (for example, the froth suppressor of foaming suds, spices)	0-5％

9) be mixed with the de-sludging composition of granula, it comprises

Linear alkyl phenyl sulfonate (calculating) with acid	6-12％
		Nonionogenic tenside	1-4％
Soap is as lipid acid	2-6％
		Yellow soda ash is (as Na2CO3)	14-22％
Zeolite is (as NaAlSiO4)	18-32％
		Sodium sulfate is (as Na2SO4)	5-20％
Trisodium Citrate is (as C6H5Na3O7)	3-8％
		Sodium peroxoborate is (as NaBO3.H2O)	4-9％
Bleach-activating agent (as NOBS or TAED)	1-5％
		Carboxymethyl cellulose	0-2％
Polymkeric substance (for example, polycarboxylate or PEG)	1-5％
		Enzyme (calculating) with pure enzyme protein matter	0.0001-0.1％
Accessory integral part (for example, optical whitening agent, spices)	0-5％

10) aqueous liquid de-sludging composition, it comprises

Linear alkyl phenyl sulfonate (calculating) with acid	15-23％
		Alcohol ethoxy vitriol (for example, C12-15Alcohol, 2-3EO)	8-15％
Alcohol ethoxylate (for example, C12-15Alcohol, 7EO or C12-15Alcohol, 5EO)	3-9％
		Soap is as lipid acid (for example, lauric acid)	0-3％
Monoethanolamine	1-5％

Trisodium Citrate	5-10％
		Hydrotropic solvent (for example, toluenesulfonic acid sodium salt)	2-6％
Borate is (as B4O72-)	0-2％
		Carboxymethyl cellulose	0-1％
Ethanol	1-3％
		Propylene glycol	2-5％
Enzyme (calculating) with pure enzyme protein matter	0.0001-0.1％
		Accessory integral part (for example, polymkeric substance, dispersion agent, spices, optical whitening agent)	0-5％

11) aqueous liquid de-sludging composition, it comprises

Linear alkyl phenyl sulfonate (calculating) with acid	20-32％
		Alcohol ethoxylate (for example, C12-15Alcohol, 7EO or C12-15Alcohol, 5EO)	6-12％
Monoethanolamine	2-6％
		Citric acid	8-14％
Borate is (as B4O72-)	1-3％
		Polymkeric substance (as toxilic acid/acrylic copolymer, the grappling polymkeric substance is as, methylacrylic acid lauryl/acrylic copolymer for example)	0-3％
Glycerine	3-8％
		Enzyme (calculating) with pure enzyme protein matter	0.0001-0.1％
Accessory integral part (for example, hydrotropic solvent, dispersion agent, spices, optical whitening agent)	0-5％

12) be mixed with and have the de-sludging composition of the granula of the bulk density of 600g/l at least, it comprises

Anion surfactant (linear alkyl benzene sulfonate, alkyl-sulphate, sulfonated, alpha-sulfo-fatty acid methyl ester, chain alkyl sulfonate, soap)	25-40％
		Nonionogenic tenside (for example, alcohol ethoxylate)	1-10％

Yellow soda ash is (as Na2CO3)	8-25％
		Soluble silicate is (as Na2O，2SiO2)	5-15％
Sodium sulfate is (as Na2SO4)	0-5％
		Zeolite is (as NaAlSiO4)	15-28％
Sodium peroxoborate is (as NaBO3.4H2O)	0-20％
		Activator of bleaching agent (as TAED or NOBS)	0-5％
Enzyme (calculating) with pure enzyme protein matter	0.0001-0.1％
		Accessory integral part (for example, spices, optical whitening agent)	0-3％

13) as 1)-12) described in the washing agent preparation, wherein all or part of linear alkyl phenyl sulfonate is with (C₁₂-C₁₈) alkyl-sulphate substitutes.

14) be mixed with and have the de-sludging composition of the granula of the bulk density of 600g/l at least, it comprises

(C12-C18) alkyl-sulphate	9-15％
		Alcohol ethoxylate	3-6％
Poly-hydroxy alkyl fatty acid acid amides	1-5％
		Zeolite is (as NaAlSiO4)	10-20％
Layering bisilicate (for example deriving from the SK56 of Hoechst)	10-20％
		Yellow soda ash is (as Na2CO3)	3-12％
Soluble silicate is (as Na2O，2SiO2)	0-6％
		Trisodium Citrate	4-8％
SPC-D	13-22％
		TAED	3-8％
Polymkeric substance (for example polycarboxylate and PVP)	0-5％
		Enzyme (calculating) with pure enzyme protein matter	0.0001-0.1％
Accessory integral part (for example, the froth suppressor of optical whitening agent, optical bleaching agent, spices, foaming suds)	0-5％

15) be mixed with and have the de-sludging composition of the granula of the bulk density of 600g/l at least, it comprises

(C12-C18) alkyl-sulphate	4-8％
		Alcohol ethoxylate	11-15％
Soap	1-4％
		Zeolite MAP or zeolite A	35-45％
Yellow soda ash is (as Na2CO3)	2-8％
		Soluble silicate is (as Na2O，2SiO2)	0-4％
SPC-D	13-22％
		TAED	1-8％
Carboxymethyl cellulose	0-3％
		Polymkeric substance (as polycarboxylate and PVP)	0-3％
Enzyme (calculating) with pure enzyme protein matter	0.0001-0.1％
		Accessory integral part (for example, optical whitening agent, phosphonate, spices)	0-3％

16) as 1)-15) described washing agent preparation, said preparation comprises the stable or encapsulated peracid as the surrogate of component of adding or the specified bleaching system of conduct.

17) as 1), 3), 7), 9) and 12) described de-sludging composition, wherein perborate is substituted by percarbonate.

18) as 1), 3), 7), 9), 12), 14) and 15) described de-sludging composition, said composition also comprises Mn catalyst.Mn catalyst can be, for example, at " effective Mn catalyst of cold bleaching ", nature, 369,1994, one of compound of describing among the 637-639.

19) be mixed with the de-sludging composition of water-free washing agent liquid, said composition comprise liquid non-ionic surfactant as, for example, linear oxyalkylated primary alconol, strengthen system's (for example, phosphoric acid salt), enzyme and the alkali of washing agent detergent action.Washing agent also can comprise anion surfactant and/or bleaching system.

The specific form of dish washing compositions within the scope of the present invention comprises:

1) pulvis machine (automatic) dish washing compositions

Nonionogenic tenside	0.4-2.5％
		Sodium metasilicate	0-20％

Sodium disilicate	3-20％
		Tri sodium Phosphate	20-40％
Yellow soda ash	0-20％
		Sodium peroxoborate	2-9％
Tetraacetyl ethylene diamine (TAED)	1-4％
		Sodium sulfate	5-33％
Enzyme	0.0001-0.1％

2) pulvis dishware cleaning composition

Nonionogenic tenside (for example, alcohol ethoxylate)	1-2％
		Sodium disilicate	2-30％
Yellow soda ash	10-50％
		Alendronate	0-5％
Two hydration trisodium citrates	9-30％
		B,EDTA (NTA)	0-20％
Single hydrated sodium perborate	5-10％
		Tetraacetyl ethylene diamine (TAED)	1-2％
Polyacrylate polymers (as toxilic acid/acrylic copolymer)	6-25％
		Enzyme	0.0001-0.1％
Spices	0.1-0.5％
		Water	5-10

3) pulvis dishware cleaning composition

Nonionogenic tenside	0.5-2.0％
		Sodium disilicate	25-40％
Trisodium Citrate	30-55％
		Yellow soda ash	0-29％
Sodium bicarbonate	0-20％

Single hydrated sodium perborate	0-15％
		Tetraacetyl ethylene diamine (TAED)	0-6％
Toxilic acid/acrylic copolymer	0-5％
		Clay	1-3％
Poly-(amino acid)	0-20％
		Sodium polyacrylate	0-8％
Enzyme	0.0001-0.1％

4) pulvis dishware cleaning composition

Nonionogenic tenside	1-2％
		Zeolite MAP	15-42％
Sodium disilicate	30-34％
		Trisodium Citrate	0-12％
Yellow soda ash	0-20％
		Single hydrogen Sodium peroxoborate	7-15％
Tetraacetyl ethylene diamine (TAED)	0-3％
		Polymkeric substance	0-4％
Toxilic acid/acrylic copolymer	0-5％
		Organophosphonate	0-4％
Clay	1-2％
		Enzyme	0.0001-0.1％
Sodium sulfate	Balance

5) pulvis dishware cleaning composition

Nonionogenic tenside	1-7％
		Heavy water glass	18-30％
Trisodium citrate	10-24％
		Yellow soda ash	12-20％

Single persulphate (2KHSO5.KHSO4.K2SO4)	15-21％
		The stablizer of SYNTHETIC OPTICAL WHITNER	0.1-2％
Toxilic acid/acrylic copolymer	0-6％
		Diethylenetriamine five acetate, five sodium-salt	0-2.5％
Enzyme	0.0001-0.1％
		Sodium sulfate, water	Balance

6) have the pulvis and the liquid tableware cleaning composition of cleansing surfactants system

Nonionogenic tenside	0-1.5％
		Two hydration octadecyl diformazan amine n-oxides	0-5％
(80:20 weight C18/C16) mixture of two hydration octadecyl diformazan amine n-oxides and two hydration hexadecyl diformazan amine n-oxides	0-4％
		(70:30 weight C18/C16) mixture of two (hydroxyethyl) amine n-oxides of anhydrous octadecyl and two (hydroxyethyl) amine n-oxides of anhydrous hexadecyl	0-5％
Average degree of ethoxylation is 3 C13-C15Alkyl ethoxy sulfate	0-10％
		Average degree of ethoxylation is 3 C12-C15Alkyl ethoxy sulfate	0-5％
Average degree of ethoxylation is 12 C13-C15Ethoxylated alcohol	0-5％
		Average degree of ethoxylation is 9 C12-C15The mixture of ethoxylated alcohol	0-6.5％
Average degree of ethoxylation is 30 C13-C15The mixture of ethoxylated alcohol	0-4％
		Sodium disilicate	0-33％
Tripoly phosphate sodium STPP	0-46％
		Trisodium Citrate	0-28％
Citric acid	0-29％

Yellow soda ash	0-20％
		Single hydrated sodium perborate	0-11.5％
Tetraacetyl ethylene diamine (TAED)	0-4％
		Toxilic acid/acrylic copolymer	0-7.5％
Sodium sulfate	0-12.5％
		Enzyme	0.0001-0.1％

7) water-free liquid dishware cleaning composition

Liquid non-ionic surfactant (as alcohol ethoxylate)	2.0-10.0％
		Alkalimetal silicate	3.0-15.0％
Alkali metal phosphate	20.0-40.0％
		Be selected from the liquid vehicle of higher glycols, polyglycol, poly oxide, glycol ethers	25.0-45.0％
Stablizer (for example, phosphoric acid and C16-C18The part ester of alkanol)	0.5-7.0％
		Froth suppressor (as siloxanes)	0-1.5％
Enzyme	0.0001-0.1％

8) water-free liquid tableware cleaning composition

Liquid non-ionic surfactant (as alcohol ethoxylate)	2.0-10.0％
		Water glass	3.0-15.0％
Alkaline carbonate	7.0-20.0％
		Trisodium Citrate	0.0-1.5％
Stable system (for example, the mixture of the siloxanes of segmentation and lower molecular weight dialkyl group polyglycol ether)	0.5-7.0％
		The low molecular weight polyacrylate polymkeric substance	5.0-15.0％
Clay gel thickening material (for example, wilkinite)	0.0-10.0％
		Hydroxypropyl cellulose polymer	0.0-0.6％
Enzyme	0.0001-0.1％

Be selected from the liquid vehicle of higher glycols, polyglycol, poly oxide, glycol ethers

Balance

9) the liquid dishware cleaning composition of thixotropic (thixotropic)

C12-C14Lipid acid	0-0.5％
		Block copolymer surfactant	1.5-15.0％
Trisodium Citrate	0-12％
		Tripoly phosphate sodium STPP	0-15％
Yellow soda ash	0-8％
		Aluminium Tristearate Micronized sterile	0-0.1％
The isopropyl benzene sodium sulfonate	0-1.7％
		Polyacrylate thickeners	1.32-2.5％
One of sodium polyacrylate	2.4-6.0％
		Boric acid	0-4.0％
Sodium formiate	0-0.45％
		Calcium formiate	0-0.2％
N-biphenyl in last of the ten Heavenly stems oxide compound stilbene-4,4'-bis-(1-azo-3, 4-dihydroxy-benzene)-2,2'-disulfonate	0-4.0％
		Monoethanolamine MEA BASF (MEA)	0-1.86％
Sodium hydroxide (50%)	1.9-9.3％
		1, the 2-propylene glycol	0-9.4％
Enzyme	0.0001-0.1％
		The froth suppressor of foaming suds, dyestuff, spices, water	Balance

10) liquid dishware cleaning composition

Alcohol ethoxylate	0-20％
		Fatty sulfonate	0-30％
Sodium lauryl sulphate	0-20％
		The alkyl polysaccharide glucoside	0-21％

Oleic acid	0-10％
		Single hydration sodium disilicate	18-33％
Two hydration Trisodium Citrates	18-33％
		Sodium stearate	0-2.5％
Single hydrated sodium perborate	0-13％
		Tetraacetyl ethylene diamine (TAED)	0-8％
Toxilic acid/acrylic copolymer	4-8％
		Enzyme	0.0001-0.1％

11) comprise the liquid dishware cleaning composition of shielded bleaching particle

Water glass	5-10％
		Tetrapotassium pyrophosphate	15-25％
Tri sodium Phosphate	0-2％
		Salt of wormwood	4-8％
Shielded SYNTHETIC OPTICAL WHITNER particle is as chlorine	5-10％
		Polymeric viscosifier	0.7-1.5％
Potassium hydroxide	0-2％
		Enzyme	0.0001-0.1％
Water	Balance

11) as 1), 2), 3), 4), 6) and 10) the dishware cleaning composition described, wherein perborate is substituted by percarbonate.

12) as 1)-6) described dishware cleaning composition, said composition also comprises Mn catalyst.Mn catalyst can be, for example, at " effective Mn catalyst of cold bleaching ", nature, 369,1994, one of compound of describing among the 637-639.

Alpha-amylase variants of the present invention can mix with the concentration that is generally used for washing agent.Imagination is in de-sludging composition of the present invention now, and alpha-amylase variants can add with the amount that is equivalent to every liter of washing/dishwashing detergent liquid 0.00001-1mg (calculating with pure enzyme protein matter).

In addition, the present invention also can be further by being described with reference to the drawings, wherein:

Fig. 1 is the sequence contrast of the aminoacid sequence of four kinds of parent's α-Dian Fenmei of the present invention.The aminoacid sequence that leftmost numeral refers to respectively is as follows:

1: at the aminoacid sequence shown in the SEQ ID No.1;

2: at the aminoacid sequence shown in the SEQ ID No.2;

3: at the aminoacid sequence shown in the SEQ ID No.3;

4: at the aminoacid sequence shown in the SEQ ID No.7.

Provided the successive amino acid sum of said every kind of sequence in the rightmost numeral of figure.It should be noted and be numbered 3 sequence (corresponding at the aminoacid sequence shown in the SEQ ID No.3), in sequence contrast, produce " gap " on its corresponding the 1st amino acid in the sequence that is numbered 1 (SEQ ID No.1), 2 (SEQ ID No.), 4 (SEQ ID No.7) respectively and the 175th amino acid whose position.

Fig. 2 is the restricted figure of plasmid pTVB106.

Fig. 3 is the restricted figure of plasmid pPM103.

Fig. 4 is the restricted figure of plasmid pTVB112.

Fig. 5 is the restricted figure of plasmid pTVB114.

Experimental section

Have in preparation, purifying and the order-checking of parent's α-Dian Fenmei of the aminoacid sequence (deriving from Bacillus strain NCIB 12512 and NCIB 12513 respectively) shown in SEQ ID No.1 and the SEQ ID No.2 and in WO 95/26397, describe.The pI value of these two kinds of parent's α-Dian Fenmei and molecular weight (providing in WO95/26397) are as follows:

SEQ ID No.1:pI is approximately 8.8-9.0 (at LKB Ampholine^TMMeasure by isoelectrofocusing on the PAG flat board); Molecular weight is approximately 55kD (measuring by SDS-PAGE).

SEQ ID No.2:pI is approximately 5.8 (at LKBAmpholine^TMMeasure by isoelectrofocusing on the PAG flat board); Molecular weight is approximately 55kD (measuring by SDS-PAGE).

The purifying of alpha-amylase variants of the present invention

According to the structure of variant of the present invention be expressed in the following examples 2 and describe.The purifying of variant of the present invention reference in this article illustrates at the variant of the aminoacid sequence shown in SEQ ID No.1 and the SEQ ID No.2 respectively:

The purifying of SEQ ID No.1 variant (pI is approximately 9.0): filter the fermented liquid of the alpha-amylase variants that comprises expression, add ammonium sulfate to 15% of its concentration degree of reaching capacity.Then liquid is added in the drainage column (Toyopearl butyl/TOSOH).20mM dimethylated pentanedioic acid damping fluid washing column with pH7.0.α-Dian Fenmei in conjunction with very firm, be used in the 25%w/w 2-propyl alcohol wash-out in the 20mM dimethylated pentanedioic acid damping fluid of pH7.0 then.After wash-out, the 2-propyl alcohol is removed by evaporation, and enriched material is administered to the 20mM dimethylated pentanedioic acid damping fluid equilibrated cationic exchange coloum (S-Sepharose with pH6.0^TMFF, Pharmacia, Sweden) in.

In identical damping fluid, use the linear gradient elution amylase of 0-250mM NaCl.After 10mM borate/KCl damping fluid dialysis to pH8.0, the pH value of sample is adjusted to 9.6, be administered to 10mM borate/KCl damping fluid equilibrated anion-exchange column (Q-Sepharose then with pH9.6^TMFF, Pharmacia) in.Linear gradient elution amylase with 0-250mM NaCl.PH is adjusted to 7.5.α-Dian Fenmei judges it is pure by rSDS-PAGE.For the stabilizing starch enzyme, all damping fluids all comprise 2mM CaCl₂

The purifying of SEQ ID No.2 variant (pI is approximately 5.8): filter the fermented liquid of the alpha-amylase variants that comprises expression, add ammonium sulfate to 15% of its concentration degree of reaching capacity.Then liquid is added in the drainage column (Toyopearl butyl/TOSOH).Be used in the linear gradient elution bonded amylase of the ammonium sulfate of the 15%-0%w/w in the 10mM Tris damping fluid of pH8.0.After the 10mM borate/KCl damping fluid dialysis of eluate to pH8.0, the pH value of liquid is adjusted to 9.6, be administered to then with same buffer equilibrated anion-exchange column (Q-Sepharose^TMFF, Pharmacia) in.With 150mMNaCl stepwise elution amylase.

After wash-out, in order to remove NaCl, the amylase sample is to the same buffer dialysis of pH8.0.After the dialysis, pH is adjusted to 9.6, amylase is attached on the anion-exchange column once more.Linear gradient elution amylase with 0-250mMNaCl.PH is adjusted to 7.5.Amylase judges it is pure by rSDS-PAGE.For the stabilizing starch enzyme, all damping fluids all comprise 2mM CaCl₂

Alpha-amylase activity is measured

Alpha-amylase activity is by using

Tablet is measured as the method for substrate.The Phadebas tablet (

Amylase test is by Pharmacia Diagnostic supply) comprise crosslinked insoluble blue starch polymer, this polymkeric substance and bovine serum albumin(BSA) and cushion material and mix and make tablet.

For the mensuration of measuring each time, a slice medicament is suspended in comprises 5ml 50mMBritton-Robinson damping fluid (50mM acetic acid, 50mM phosphoric acid, 50mM boric acid, 0.1mMCaCl₂, pH adjusts to the purpose value with NaOH) test tube in.Test is carried out in the water-bath of purpose temperature.The α-Dian Fenmei of test is diluted in the 50mM Britton-Robinson damping fluid of * ml.This α-Dian Fenmei solution of 1ml is added in the Britton-Robinson damping fluid of 5ml 50mM.Starch provides the blue fragment of solubility by α-Dian Fenmeishuixie.The absorbancy of the blue solution that produces is at 620nm place metric measurement, and it is the function of alpha-amylase activity.

Importantly: the absorbancy at the 620nm place that 15 minutes incubations (test duration) record afterwards is between 0.2 to 2.0 absorbance unit at 620nm.Linear relationship (Law of Lambert-Beer) is arranged between this absorbancy scope intrinsic activity and absorbancy.Therefore must be adjusted to be fit to this standard the dilution of enzyme.

Under specified condition (temperature, pH value, reaction times, buffer conditions), a certain amount of substrate of the α-Dian Fenmeishuixie that 1mg is given produces blue.Colour intensity is measured at the 620nm place.Measure absorbancy ratio direct and said α-Dian Fenmei under specified criteria proportional (the pure α-Dian Fenmei protein of activity/mg) alive.Like this, test different purpose α-Dian Fenmei under the same conditions and (comprised the reference α-Dian Fenmei, be said parent's α-Dian Fenmei in this case) afterwards, just can directly compare, can measure the ratio of the ratio work of every kind of purpose α-Dian Fenmei living under fixed temperature and the given pH ratio of every kind of α-Dian Fenmei with respect to the ratio work of reference α-Dian Fenmei.

Small-sized dishwashing detergent is measured

Used following small-sized dishwashing detergent to measure: to boil the suspension of starch-containing material, be cooled to 20 ℃ then.Cooled starch suspension be applied to each evaluation little glass plate (on about 2 * 2cm), then in desiccator cabinet with about 140 ℃ temperature drying.Then single flat board is weighed.In order to measure, prepared standard Europe class dishware washing washing agent solution (5g/l) with 55 ℃ of temperature.Make washing agent dissolving 1 minute, in washing agent solution (being included in the beaker that is equipped with the magnetic stirring), add said α-Dian Fenmei thereafter so that enzyme concn reaches 0.5mg/l.Simultaneously, the glass plate of weighing that places little supporting rack is immersed in α-Dian Fenmei/washing agent solution with the perpendicular position, stirred 15 minutes down at 55 ℃ then.Then glass plate is taken out from α-Dian Fenmei/washing agent solution, with the distilled water rinsing, drying is also weighed once more in desiccator cabinet under 60 ℃.Then the performance of said α-Dian Fenmei [with among the embodiment with respect to the reference α-Dian Fenmei of selecting (index is 100)-below (embodiment 1) be have parent's α-Dian Fenmei at the aminoacid sequence shown in the SEQ ID No.1-index express] measure the weight difference of glass plate before and after handling, the result is as follows:

Following embodiment further specifies the present invention.They have no intention to limit by any way scope of the presently claimed invention.

Embodiment 1

Has small-sized dishwashing detergent test at the variant of parent's α-Dian Fenmei of the aminoacid sequence shown in the SEQ ID No.1

Above-mentioned small-sized dishwashing detergent test is carried out (their structure and purifying are described below): T183 with the parent's α-Dian Fenmei and the following variant thereof that have at the aminoacid sequence shown in the SEQ ID No.1 under pH10.5^*+ G184^*Y243F; K269R.Test result is as follows:

Parent (SEQ ID No.1) index: 100

T183^*+ G184^*Index: 120

Y243F index: 120

K269R index: 131

Clearly, the variant T183 of test^*+ G184^*(it shows lower Ca than parent α-Dian Fenmei for (it shows extra high thermostability than parent α-Dian Fenmei), Y243F²⁺Ionic dependent) and K269R (it shows lower Ca than parent α-Dian Fenmei²⁺Ionic dependent and under high pH higher stability) each show the dishwashing detergent performance of obvious improvement with respect to parent's α-Dian Fenmei.

Embodiment 2

Has respectively structure at the variant of parent's α-Dian Fenmei of the aminoacid sequence shown in SEQ ID No.1 and the SEQ ID No.2

Primer: the dna primer that uses during following variant makes up comprises that [all dna primers provide with from 5 ' to 3 ' direction (from left to right) following primer; P refers to 5 '-phosphoric acid salt]:

#7113:

GCT GCG GTG ACC TCT TTA AAA AAT AAC GGC

Y296:

CC ACC GCT ATT AGA TGC ATT GTA C

#6779:

CTT ACG TAT GCA GAC GTC GAT ATG GAT CAC CC

#6778:

G ATC CAT ATC GAC GTC TGC ATA CGT AAG ATA GTC

#3811:

TT A(C/G)G GGC AAG GCC TGG GAC TGG

#7449:

C CCA GGC CTT GCC C(C/G)T AAA TTT ATA TAT TTT GTT TTG

#3810:

G GTT TCG GTT CGA AGG ATT CAC TTC TAC CGC

#7450:

GCG GTA GAA GTG AAT CCT TCG AAC CGA AAC CAG

B1:

GGT ACT ATC GTA ACA ATG GCC GAT TGC TGA CGC TGT TAT TTG C

#6616:

P CTG TGA CTG GTG AGT ACT CAA CCA AGT C

#8573:

CTA CTT CCC AAT CCC AAG CTT TAC CTC GGA ATT TG

#8569:

CAA ATT CCG AGG TAA AGC TTG GGA TTG GGA AGT AG

#8570:

TTG AAC AAC CGT TCC ATT AAG AAG

A: have structure at parent's alpha-amylase variants of the aminoacid sequence shown in the SEQ ID No.1

The description of plasmid pTVB106: have at the parent's α-Dian Fenmei and the variant thereof of the aminoacid sequence shown in the SEQ ID No.1 and express from the gene SF16 that carries at the plasmid shown in Fig. 2.Plasmid pTVB106 comprises from the replication orgin (Gryczan etc., 1978) of plasmid pUB110 acquisition and the cat gene of giving chlorampenicol resistant.Amylase secretion obtains Termamyl^TMThe help of signal sequence, this signal sequence are fused to (promptly ripe proteinic first codon) coding exactly to have respectively in parent's alpha-amylase gene of Nucleotide shown in SEQID No.4 and the SEQ ID No.1 and aminoacid sequence (mature protein).Transcribing of Termamyl promotor promotor gene.

Plasmid pTVB106 is similar to pDN1528 (referring to Denmark's publication application 1155/94).On the plasmid map of Fig. 2, point out some single restriction sites, comprised BstBI, BamHI, BstEII, EcoNI, DrdI, AflIII, DraIII, XmaI, SalI and BglII.

The structure of variant M202T: used the overlapping extension mutagenesis of PCR (overlap extensionmutagenesis) to make up this variant (Higuchi etc., 1988).Use the dna fragmentation of primer #7113 and mutagenic primer #6778 about 350bp of amplification pTVB106 in PCR reaction A.In a similar PCR reaction B, use the increase dna fragmentation of about 300bp of primer Y296 and #6779.Use these primers and in reaction A and B the dna fragmentation of purifying, in PCR reaction C, increased and striden the full dna fragmentation in mutational site (M202) from primer #7113 to primer Y296.

With restriction endonuclease BstEII and AflIII digestion PCR C DNA, use in the bacillus subtilis strain that advances to hang down proteolytic enzyme and low amylase with the plasmid pTVB106 connection 480bp fragment and the conversion of identical enzymic digestion (the SHA273 bacterial strain of for example, in WO 92/11357, mentioning).

Other M202 variant makes up in a similar fashion.

Variant T183^*+ G184^*And R181^*+ G182^*Structure: used the overlapping extension mutafacient system of PCR to make up these variants (Higuchi etc., 1988).Use has been synthesized the oligonucleotide of mutagenesis at the C of a position and the mixture of G (moiety), therefore can make up two kinds of different sudden changes by this method.Use the dna fragmentation of primer #7113 and mutagenic primer #7449 about 300bp of amplification pTVB106 in the PCR reaction.In similar PCR reaction B, use the increased dna fragmentation of about 400bp of primer Y296 and #3811.Use these primers and in reaction A and B the dna fragmentation of purifying, in PCR reaction C, increased and striden the full dna fragmentation in mutational site (amino acid/11 81-184) from primer #7113 to primer Y296.

With restriction endonuclease BstEII and AflIII digestion PCR C DNA, use in the bacillus subtilis strain that advances to hang down proteolytic enzyme and low amylase with the plasmid pTVB106 connection 480bp fragment and the conversion of identical enzymic digestion (the SHA273 bacterial strain of for example, in WO 92/11357, mentioning).The order-checking that derives from the plasmid DNA of these transformant identifies two correct sudden changes: i.e. R181^*+ G182^*And T183^*+ G184^*

The structure of variant R124P: used the overlapping extension mutafacient system of PCR to make up this variant in the mode that is similar to variant M202T structure (on seeing).PCR reaction A (with primer #3810 and B1) produces the fragment of about 500bp, and PCR reaction B (with primer 7450 and Y296) produces the fragment of about 550bp.React the PCR reaction C of product, primer B1 and the Y296 of A and B with restriction endonuclease BstEII and AflIII digestion PCR-based, and the fragment subclone of the 480bp that strides amino acid position 124 of gained advanced among the pTVB106 with identical enzymic digestion, and transform in the above subtilis.

Variant R124P+T183^*+ G184^*Structure: in order to make up in conjunction with R124P and T183^*+ G184^*The variant of sudden change, (one is positioned at position 1.774kb, promptly at R124P sudden change and T183 to have used two EcoNI restriction sites^*+ G184^*Between the sudden change, one is positioned at position 0.146kb).Comprising T183^*+ G184^*The EcoNI fragment subclone of about 1630bp of the pTVB106 sample plasmid of sudden change advances the carrier part (dna fragmentation that comprises about 3810bp of replication orgin) that comprises the pTVB106 sample plasmid of R124P sudden change with the another kind of identical enzymic digestion.Transform subtilis as mentioned above.

Variant G182^*+ G184^*R181^*+ T183^*Y243F; The structure of K269R and L351C+M430C: these variants make up as follows:

The specificity mutagenesis carrier that has prepared the major portion that is included in the amino acid sequence encode district shown in the SEQ ID No.1.The key character of this carrier (name and be pPM103) comprises replication orgin derived from the pUC plasmid, gives the cat gene of chlorampenicol resistant, the wild-type (amp that comprises the bla gene of phase shift mutation and give amicillin resistance usually^RPhenotype).The bla gene of sudden change has caused amp^SPhenotype.Plasmid pPM103 has pointed out the restriction site of SF16 amylase gene, ori, bla, cat and the selection of intestinal bacteria replication orgin, 5 '-brachymemma as shown in Figure 3 on plasmid.

Except having " selectivity primer " plasmid (#6616) of mixing amp based on the transformed into escherichia coli cell of the plasmid that carries bla gene with reparation^RPhenotype is selected, but not use Deng and Nickoloff general introduction pass through restriction enzyme digest select outside, according to Deng and the described method of Nickoloff [analytical biochemistry, 200 (1992), 81-88] sudden change is imported goal gene.Be used for the pharmaceutical chemicals of mutagenesis and enzyme Chameleon from Stratagene^TMMutagenesis kit obtains (catalog number (Cat.No.) 200509).

After to the dna sequence dna verification in the variant plasmid, the gene that comprises the brachymemma of desired variation advances pTVB106 as the BstBI-SalI fragment of about 1440bp from pPM103 sample plasmid subclones, and transforms subtilis to express variant enzyme.

Be built into disappearance variant G182^*+ G184^*Used following mutagenic primer:

P CTCTGTATC GACTTC CCA GTC CCA AGC TTT TGT CCT GAA TTT

ATA TAT TTT GTT TTG AAG

Be built into disappearance variant R181^*+ T183^*Used following mutagenic primer:

P CTC TGT ATC GAC TTC CCA GTC CCA AGC TTT GCC TCC GAA TTT

ATA TAT TTT GTT TTG AAG

Make up replacement variant Y243F and used following mutagenic primer:

P ATG TGT AAG CCA ATC GCG AGT AAA GCT AAA TTT TAT ATG TTT

CAC TGC ATC

Make up replacement variant K269R and used following mutagenic primer:

P GC ACC AAG GTC ATT TCG CCA GAA TTC AGC CAC TG

Be built into replacing variant L351C+M430C, used following mutagenic primer simultaneously:

1)P TGT CAG AAC CAA CGC GTA TGC ACA TGG TTT AAA CCA TTG

2)P ACC ACC TGG ACC ATC GCT GCA GAT GGT GGC AAG GCC TGA

ATT

Variant L351C+M430C+T183^*+ G184^*Structure: this variant by L351C+M430C is replaced in pairs the sudden change and T183^*+ G184^*Deletion mutantion is in conjunction with structure in pairs, and the combination of the two (has T183 by the pTVB106 sample plasmid that the HindIII-AflIII fragment subclone that comprises about 1430bp of L351C+M430C is advanced with identical enzymic digestion^*+ G184^*Sudden change) finishes.

Variant Y243F+T183^*+ G184^*Structure: this variant is by Y243F sudden change and T183^*+ G184^*Sudden change is in conjunction with making up, and the combination of the two comprises T183 by handle^*+ G184^*The pTVB106 sample plasmid (having the Y243 sudden change) that advances with identical enzymic digestion of the DrdI fragment subclone of about 1148bp finish.

Screening has the subtilis transformant of alpha-amylase activity on amyloid agar plate, by the existence of the correct sudden change of dna sequencing inspection.

Variant Y243F+T183^*+ G184^*The structure of+L351C+M430C: L351C+M430C is replaced sudden change in pairs (comprise Y243F+T183 as the pTVB106 sample carrier that the Xmal-SalI fragment subclone of about 470bp advances with identical enzymic digestion^*+ G184^*).

Variant Y243F+T183^*+ G184^*The structure of+L351C+M430C+Q391E+K444Q: replace the SF16 of brachymemma among the pPM103 by BstB1-SalI fragment, made up and comprise sudden change Y243F+T183 with about 1440bp of the pTVB106 sample carrier that comprises said five kinds of sudden changes^*+ G184^*The pPM103 sample carrier of+L351C+M430C.By using following two kinds of mutagenic primers simultaneously Q391E and K444Q sudden change importing pPM103 sample carrier (to be comprised Y243F+T183 to be similar to above-mentioned mode to pPM103 mutagenesis^*+ G184^*+ L351C+M430C) in: P GGC AAA AGT TTG ACG TGC CTC GAG AAG AGG GTC TATP TTG TCC CGC TTT ATT CTG GCC AAC ATA CAT CCA TTT

B: have structure at parent's alpha-amylase variants of aminoacid sequence shown in the SEQ ID No.2

The description of plasmid pTVB112: made up to be used for having and named carrier for pTVB112 what subtilis was expressed in the aminoacid sequence α-Dian Fenmei shown in the SEQ ID No.2.The gene of the ripe α-Dian Fenmei of this carrier in coding SEQ ID No.2 is inserted between the Pstl of pTVB106 and the HindIII site, similar with pTVB106 ten minutes.Like this, the expression of this α-Dian Fenmei (SEQ IDNo.2) is instructed by amyL promotor and signal sequence also.Plasmid pTVB112 is shown in Fig. 4.

Modification D 183^*+ G184^*Structure: the structure of this variant uses the overlapping extension mutafacient system of PCR mentioned earlier (on seeing) to finish.Primer #8573 and B1 are used for PCR reaction A, and primer #8569 and #8570 are used for PCR reaction B.Be used for PCR reaction C from the fragment of reaction A and reaction B purifying and primer 1B and #8570, produced the dna fragmentation of about 1020bp.This this fragment is advanced expression vector and is transformed subtilis with restriction endonuclease Pstl and MluI digestion, subclone.

The structure of other variant: the plasmid pPM103 that produces by the mutant with the aminoacid sequence that is used for SEQ ID No.1 makes up similar method (opinion), has made up a kind of to modification D 183^*+ G184^*(SEQ ID No.2) continues the plasmid of mutagenesis and (names and be pTVB114; Shown in Fig. 5).In the mode that is similar to pPM103 (SEQ ID No.1) sudden change is imported (SEQ IDNo.2 among the pTVB114; D183^*+ G184^*).

Be built into disappearance variant R181^*+ D183^*And R181^*+ G182^*, selection be to change modification D 183^*+ G184^*In flanking amino acid, rather than the specific amino acids in the wild type gene of disappearance SEQ ID No.2.Following mutagenic primer and be used from mutagenesis as the pTVB114 one of template:

PCC CAA TCC CAA GCT TTA CCA(T/C)CG AAC TTG TAG ATA CG

The existence of the mixture of two kinds of bases (T/C) a position makes and can exist based on two kinds of a kind of mutagenic primer different disappearance flanking amino acids.The dna sequencing of the plasmid that forms proves the existence of one or another kind of sudden change.The goal gene of sudden change advances among the pTVB112 with identical enzymic digestion as Pstl-DraIII fragment subclone, transforms subtilis then.

Make up G182^*+ G184^*And R181^*+ G184^*Used following mutagenic primer and as the pTVB114 of template:

PCC CAA TCC CAA GCT TTA TCT C(C/G)G AAC TTG TAG ATA CG

As mentioned above, the existence of the mixture of two kinds of bases (C/G) a position makes and can exist based on two kinds of a kind of mutagenic primer different disappearance flanking amino acids.The dna sequencing of the plasmid that forms proves the existence of one or another kind of sudden change.The goal gene of sudden change advances among the pTVB112 with identical enzymic digestion as Pstl-DraIII fragment subclone, transforms subtilis then.

Make up D183^*+ G184^*+ M202L has used following mutagenic primer:

PGA TCC ATA TCG ACG TCT GCA TAC AGT AAA TAA TC

Make up D183^*+ G184^*+ M202I has used following mutagenic primer:

PGA TCC ATA TCG ACG TCT GCA TAA ATT AAA TAA TC

Embodiment 3

Having the oxidative stability that replaces variant at the M202 of parent's α-Dian Fenmei of the aminoacid sequence shown in SEQ ID No.1 and the SEQ ID No.2 measures

The oxidative stability of the variant of the sequence among the A:SEQ ID No.1

Measure use variant separately at the 50mM of pH9.0 Britton-Robinson damping fluid (50mM acetic acid, 50mM phosphoric acid, 50mM boric acid, 0.1mM CaCl₂, with NaOH pH is adjusted to the purpose value) in solution carry out, and in solution, add hydrogen peroxide (when time t=0) and make its ultimate density reach 200mM H₂O₂Solution incubation in 40 ℃ water-bath then.

Incubation is after 5,10,15 and 20 minutes after adding hydrogen peroxide, and remaining alpha-amylase activity uses above-mentioned Phadebas assay method to measure.Remaining activity uses the 50mMBritton-Robinson damping fluid of pH7.3 to measure (analyze publication AF207-1/1 referring to Novo, can obtain from Novo NordiskA/S) down at 37 ℃ in the sample.Measured with respect to not and the activity decline of (100% activity) 0 minute time of the corresponding reference solution of the same enzyme of hydrogen peroxide incubation.

The per-cent of initial activity is as shown in the table of said below parent enzyme of the function of time (SEQ ID No.1) and variant.

The M202 of all tests replaces variant and clearly shows the oxidative stability of obvious improvement with respect to parent's α-Dian Fenmei (SEQ ID No.1).

The oxidative stability of the variant of the sequence among the B:SEQ ID No.2

Use said parent's α-Dian Fenmei (SEQ ID No.2), variant M202L+D183^*+ G184^*(in following table, being called L), variant M202I+D183^*+ G184^*(being called I in following table) measures respectively as mentioned above.In this case, used 5,10,15 and 30 minutes incubation time (after adding hydrogen peroxide).As above show, the per-cent of initial activity is as shown in the table of said below parent enzyme of the function of time and variant.

Two " replacing+lack in pairs " variants (they all comprise M202 and replace) of test clearly show the oxidative stability of obvious improvement with respect to parent's α-Dian Fenmei (SEQ ID No.2).

Embodiment 4

Has thermal stability determination at parent's alpha-amylase variants of the aminoacid sequence shown in SEQ ID No.1 and the SEQ ID No.2

A: the thermostability of the paired disappearance variant in SEQ ID No.1 sequence

Measuring the solution of variant in the 50mM of pH9.0 Britton-Robinson damping fluid (on seeing) that uses separately carries out.Solution is incubation in 65 ℃ water-bath, and sample takes out after the specified time of incubation.Each residue alpha-amylase activity that takes out sample uses above-described Phadebas measuring method to measure.Measured with respect to the corresponding reference solution of the same enzyme that the does not have incubation activity of (100% activity) 0 minute the time and descended.

The per-cent of initial activity is as shown in the table of said below parent enzyme of the function of time (SEQ ID No.1) and following paired disappearance variant.

Variant 1:R181^*+ G182^*

Variant 2:R181^*+ T183^*

Variant 3:G182^*+ G184^*

Variant 4:T183^*+ G184^*

Variant 5:T183^*+ G184^*+ R124P

Clearly, the paired disappearance variant of all tests shows obvious improved thermal stability with respect to parent's α-Dian Fenmei (SEQ ID No.1), and the thermostability of variant 5 (also comprise except the paired deletion mutantion ofvariant 4 and replace R124P) is higher significantly than other variant.Show the thermostability that it is about 7 ℃ with respect to parent's α-Dian Fenmei because replace the calorimetric result of variant R124P (only comprise and replace R124P), seem to suddenly change R124P and the paired thermal stability result that lacks are strengthened respectively mutually.

B: the thermostability of the paired disappearance variant of the sequence in SEQ ID No.2

Parent enzyme (SEQ ID No.2) and following paired disappearance variant have been carried out corresponding mensuration:

Modification A: D183^*+ G184^*

Variant B:R181^*+ G182^*

Variant C:G182^*+ G184^*

Still clearly, said paired disappearance variant shows obvious improved thermal stability with respect to parent's α-Dian Fenmei (SEQ ID No.2).

The thermostability of many combinatory variants of sequence among the C:SEQ ID No.1

Following variant at the aminoacid sequence shown in the SEQ ID No.1 has been carried out comparing and measuring accordingly.

Variant 4:T183^*+ G184^*

Variant 6:L351C+M430C

Variant 7:Y243F

Variant 8:Q391E+K444Q

Variant 9:T183^*+ G184^*+ L351C+M430C+Y243F+Q391E+K444Q

Obviously explanation again, the thermostable effect of multiple mutation (each in the multiple mutation all has thermostable effect) is-is at least qualitatively-cumulative.

Embodiment 5

The Ca of alpha-amylase variants of the present invention²⁺Binding affinity

Amylase is exposed to folding the weakening of fluorescence that be accompanied by of separating of heat or denaturing agent example hydrochloric acid guanidine.Losing calcium ion causes separating folding, a series of α-Dian Fenmei can be by the calcium (as in the scope of 1 μ M to 100mM) or EGTA (as in the scope of 1-1000 μ the M) [EGTA=1 of every kind of α-Dian Fenmei (as the concentration with 10 μ g/ml) (as the 50mM HEPES of pH7) and different concns in damping fluid to the avidity of calcium, two (2-amino ethoxy) ethane-N of 2-, N, N ', N '-tetraacethyl] the sufficiently long time of incubation (as following 22 hours) at 55 ℃ before and fluorescence measurement afterwards measure.

Measured fluorescence F is made up of folding contribution of conciliating the enzyme of folded form.Can derive following equation to describe the dependence of F to calcium concn ([Ca]):

F＝[Ca]/(K_diss+[Ca])(α_N-β_Nlog([Ca]))+(K_diss/(K_diss+[Ca])(α_U-β_Ulog([Ca]))

α wherein_NBe the fluorescence of the enzyme of natural (folding) form, β_NBe α_NTo the logarithmic linear dependence of calcium concn (experimental observation obtains), α_UBe the fluorescence of separating folded form, β_UBe α_UTo the logarithmic linear dependence of calcium concn.K_DissBe the apparent calcium binding constant of equilibrium process, described equilibrium process is expressed as follows:

In fact, it is extremely slow and irreversible to separate folding process.Separate folding rate dependent in calcium concn, the dependency of given α-Dian Fenmei is provided the Ca-binding affinity measuring method of enzyme.By the reaction conditions (for example, 55 ℃ following 22 hours) that defines one group of standard, can carry out the K of different α-Dian Fenmei_DissSignificant comparison.But the above-mentioned equation of calcium dissociation curve match of α-Dian Fenmei makes and can carry out corresponding K usually_DissThe mensuration of value.

Following K_DissValue is to have in parent's α-Dian Fenmei of the aminoacid sequence shown in SEQ ID No.1 and the SEQ ID No.2 and the value (parent's α-Dian Fenmei is pointed out parenthetic) that obtains according to the alpha-amylase variants of pointing out of the present invention:

Can obviously see from above-mentioned: the calcium binding affinity of the latter's Alpha-starch lytic enzyme reduces with the direction that reduces above-mentioned table, promptly lacks modification D 183 in pairs^*+ G184^*(SEQ ID No.2) (promptly has minimum Ca in conjunction with the calcium ability is the strongest²⁺And parent's α-Dian Fenmei of SEQ ID No.1 (promptly has the highest Ca in conjunction with the calcium ability is the most weak dependency),²⁺Dependency).

The reference of quoting in the specification sheets

Suzuki etc., journal of biological chemistry, 264 volumes, 32, November 15 published, 18933-18938 (1989).

Hudson etc. put into practice immunology, the third edition (1989), Blackwell Science Press.

Lipman and Pearson (1985) science 227,1435.

Sambrook etc., molecular cloning: laboratory manual, second edition, cold spring port, 1989.

S.L Beaucage and M.H.Caruthers, Tetrahedron Letters 22,1981,1859-1869.

Matthes etc., EMBO magazine, 3,1984,801-805.

R.K.Saiki etc., science 239,1988,487-491.

Morinaga etc., 1984, biotechnology, 2,646-639.

Nelson and Long, analytical biochemistry, 180,1989,147-151.

Hunkapiller etc., 1984, nature, 310,105-111.

R.Higuchi, B.Krummel and R.K.Saiki (1988), the general method of external preparation of dna fragmentation and specificity mutagenesis: protein and DNA Study of Interaction, nucleic acids research, 16,7351-7367.

Dubnau etc., 1971, molecular biology magazine, 56,209-221.

Gryczan etc., 1978, bacteriology magazine, 134,318-329.

S.D.Erlich, 1977, Proc. Natl. Acad. Sci.USA, 74,1680-1682.

Bool etc., 1990, biological chemistry, 29,6244-6249.

Deng and Nickoloff, 1992, analytical biochemistry, 200,81-88.

Sequence table

(1) general information:

(i) applicant:

(a) name: NOVO NORDISK A/S

(b) street: NOVO Alle

(c) city: DK-2880 Bagsvaerd

(e) country: Denmark

(f) zip code (ZIP): DK-2880

(g) phone :+45 44 44 88 88

(h) fax :+45 44 49 32 56

(ii) invention exercise question: amylase variant

(iii) sequence number: 7

(iv) computer-reader form:

(A) media type: floppy disk

(B) computer: IBM PC compatible

(C) operating system: PC-DOS/MS-DOS

(D) software: PatentIn Release#1.0, version #1.25 (EPO)

(2) information of SEQ ID No.1:

(i) sequence signature:

(A) length: 485 amino acid

(B) type: amino acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: peptide

(xi) sequence description: SEQ ID No.1:

(2) information of SEQ ID No.2:

(i) sequence signature:

(A) length: 485 amino acid

(B) type: amino acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: peptide

(xi) sequence description: SEQ ID No.2:

(2) information of SEQ ID No.3:

(i) sequence signature:

(A) length: 514 amino acid

(B) type: amino acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: peptide

(xi) sequence description: SEQ ID No.3:

(2) information of SEQ ID No.4:

(i) sequence signature:

(A) length: 1455 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: DNA (genome)

(xi) sequence description: SEQ ID No.4:

(2) information of SEQ ID No.5:

(i) sequence signature:

(A) length: 1455 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: DNA (genome)

(xi) sequence description: SEQ ID No.5:

(2) information of SEQ ID No.6:

(i) sequence signature:

(A) length: 1548 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: DNA (genome)

(xi) sequence description: SEQ ID No.6:

(2) information of SEQ ID No.7:

(i) sequence signature:

(A) length: 485 amino acid

(B) type: amino acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: peptide

(xi) sequence description: SEQ ID No.7:

Claims (29)

Translated fromChinese

1.一种亲本α-淀粉酶的变体，该亲本α-淀粉酶:(i)具有选自分别在SEQID No.1、SEQ ID No.2、SEQ ID No.3和SEQ ID No.7中所示氨基酸序列的氨基酸序列；或(ii)与这些氨基酸序列中的一种或多种显示出至少80％的同源性；和/或显示出与如下抗体的免疫交叉反应性，所述抗体针对具有这些氨基酸序列之一的α-淀粉酶产生；和/或由如下DNA序列编码，所述DNA序列与编码具有这些氨基酸序列之一的α-淀粉酶之DNA序列与相同的探针杂交；在这种变体中:1. A variant of a parent alpha-amylase, the parent alpha-amylase: (i) having or (ii) exhibits at least 80% homology to one or more of these amino acid sequences; and/or exhibits immunological cross-reactivity with an antibody that The antibody is raised against an alpha-amylase having one of these amino acid sequences; and/or is encoded by a DNA sequence that hybridizes to the same probe as the DNA sequence encoding the alpha-amylase having one of these amino acid sequences ; in this variant:(a)所说的亲本α-淀粉酶的至少一个氨基酸残基已被缺失；和/或(a) at least one amino acid residue of said parent alpha-amylase has been deleted; and/or(b)所说的亲本α-淀粉酶的至少一个氨基酸残基已被不同的氨基酸残基取代；和/或(b) at least one amino acid residue of said parent alpha-amylase has been substituted by a different amino acid residue; and/or(c)相对所说的亲本α-淀粉酶而言至少有一个氨基酸残基已被插入；(c) at least one amino acid residue has been inserted relative to said parent alpha-amylase;所说的变体具有α-淀粉酶活性并且相对于所说的亲本α-淀粉酶表现出至少一种下列性质:增加的热稳定性；增加的氧化稳定性；和降低的Ca²⁺依赖性；Said variant has α-amylase activity and exhibits at least one of the following properties relative to said parent α-amylase: increased thermostability; increased oxidative stability; and reduced Ca^dependence ;其条件是所说的变体的氨基酸序列不与分别在SEQ ID No.1、SEQ IDNo.2、SEQ ID No.3和SEQ ID No.7中所示的氨基酸序列之任一相同。This is provided that the amino acid sequence of said variant is not identical to any of the amino acid sequences shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No. 7, respectively.2.按照权利要求1的变体，其中所说的亲本α-淀粉酶的至少一个可氧化的氨基酸残基已被缺失或已被不同的氨基酸残基取代，后者对氧化不如所说的可氧化的氨基酸残基敏感。2. The variant according to claim 1, wherein at least one oxidizable amino acid residue of said parent alpha-amylase has been deleted or has been replaced by a different amino acid residue which is less oxidizable than said oxidizable Sensitive to oxidized amino acid residues.3.按照权利要求2的变体，其中所说的可氧化的氨基酸残基选自由甲硫氨酸、色氨酸、半胱氨酸和酪氨酸组成的组。3. The variant according to claim 2, wherein said oxidizable amino acid residue is selected from the group consisting of methionine, tryptophan, cysteine and tyrosine.4.按照权利要求2或3的变体，其中所说的可氧化的氨基酸残基是甲硫氨酸，该甲硫氨酸是或等同于在SEQ ID No.1中所示氨基酸序列的M9、M10、M105、M202、M208、M261、M309、M382、M430或M440。4. The variant according to claim 2 or 3, wherein said oxidizable amino acid residue is methionine which is or is equivalent to M9 of the amino acid sequence shown in SEQ ID No.1 , M10, M105, M202, M208, M261, M309, M382, M430 or M440.5.按照权利要求4的变体，该变体包含甲硫氨酸取代，此甲硫氨酸取代是或等同于在SEQ ID No.1中所示氨基酸序列的下列取代之一:M9L；M10L；M105L；M202L、T、F、I、V；M208L；M261L；M309L；M382L；M430L；M440L。5. A variant according to claim 4 comprising a methionine substitution which is or is equivalent to one of the following substitutions of the amino acid sequence shown in SEQ ID No.1: M9L; M10L ;M105L;M202L,T,F,I,V;M208L;M261L;M309L;M382L;M430L;M440L.6.按照权利要求3-5任一之变体，其中所说的甲硫氨酸残基已被苏氨酸取代。6. A variant according to any one of claims 3-5, wherein said methionine residue has been substituted by threonine.7.根据以上权利要求任一之变体，其中至少一个氨基酸已被缺失，该氨基酸是或等同于在SEQ ID No.1中所示氨基酸序列的F180、R181、G182、T183、G184或K185。7. The variant according to any one of the preceding claims, wherein at least one amino acid which is or is identical to F180, R181 , G182, T183, G184 or K185 of the amino acid sequence shown in SEQ ID No. 1 has been deleted.8.按照权利要求7的变体，其中所说的缺失的氨基酸是或等同于所说的氨基酸残基中的任何两个。8. A variant according to claim 7, wherein said deleted amino acid is or is identical to any two of said amino acid residues.9.按照权利要求8的变体，其中所说的缺失是或等同于R181^*+G182^*或T183^*+G184^*。9. A variant according to claim 8, wherein said deletion is or is equivalent to R181^* +G182^* or T183^* +G184^* .10.根据以上权利要求任一之变体，该变体包含氨基酸取代，此取代是或等同于在SEQ ID No.1中所示氨基酸序列中的下列取代之一:K269R；P260E；R124P；M105F、I、L、V；M208F、W、Y；L217I；V206I、L、F。10. The variant according to any one of the preceding claims, comprising an amino acid substitution which is or is equivalent to one of the following substitutions in the amino acid sequence shown in SEQ ID No. 1: K269R; P260E; R124P; M105F , I, L, V; M208F, W, Y; L217I; V206I, L, F.11.按照以上权利要求任一之变体，该变体包含氨基酸取代，此取代是或等同于在SEQ ID No.1中所示氨基酸序列中的下列取代之一:Y243F、K108R、K179R、K239R、K242R、K269R、D163N、D188N、D192N、D199N、D205N、D207N、D209N、E190Q、E194Q、N106D。11. According to the variant according to any one of the preceding claims, the variant comprises an amino acid substitution which is or is equivalent to one of the following substitutions in the amino acid sequence shown in SEQ ID No.1: Y243F, K108R, K179R, K239R , K242R, K269R, D163N, D188N, D192N, D199N, D205N, D207N, D209N, E190Q, E194Q, N106D.12.一种包含编码按照权利要求1-11任一之α-淀粉酶变体的DNA序列的DNA构建体。12. A DNA construct comprising a DNA sequence encoding an alpha-amylase variant according to any one of claims 1-11.13.一种携带按照权利要求12的DNA构建体的重组表达载体。13. A recombinant expression vector carrying the DNA construct according to claim 12.14.一种用按照权利要求12的DNA构建体或按照权利要求13的载体转化的细胞。14. A cell transformed with a DNA construct according to claim 12 or a vector according to claim 13.15.按照权利要求14的细胞，该细胞是微生物。15. A cell according to claim 14 which is a microorganism.16.按照权利要求15的细胞，该细胞是细菌或真菌。16. A cell according to claim 15 which is a bacterium or a fungus.17.按照权利要求16的细胞，该细胞是革兰氏阳性细菌，如枯草芽孢杆菌、地衣芽孢杆菌、迟缓芽孢杆菌、短芽孢杆菌、嗜热脂肪芽孢杆菌、嗜碱芽孢杆菌、解淀粉芽孢杆菌、凝结芽孢杆菌、环状芽孢杆菌、灿烂芽孢杆菌、苏云金芽孢杆菌、浅青紫链霉菌、鼠灰链霉菌，或者是革兰氏阴性细菌，如大肠杆菌。17. The cell according to claim 16, which is a Gram-positive bacterium such as Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens , Bacillus coagulans, Bacillus circulans, Bacillus cannulae, Bacillus thuringiensis, Streptomyces lividans, Streptomyces grinotis, or Gram-negative bacteria such as Escherichia coli.18.一种产生按照权利要求1-11任一之α-淀粉酶变体的方法，其中在有利于α-淀粉酶变体生成的条件下培养按照权利要求14-17任一之细胞，随后从所用的培养基中回收所说的α-淀粉酶变体。18. A method for producing an α-amylase variant according to any one of claims 1-11, wherein cultivating a cell according to any one of claims 14-17 under conditions conducive to the production of the α-amylase variant, followed by The alpha-amylase variants are recovered from the culture medium used.19.按照权利要求1-11任一之α-淀粉酶变体在洗涤和/或餐具洗涤上的用途。19. Use of the alpha-amylase variant according to any one of claims 1-11 for washing and/or dishwashing.20.一种包含按照权利要求1-11任一之α-淀粉酶变体的去垢剂添加剂，其可以以或不以无粉尘粒状、稳定的液体或受保护酶形式存在。20. A detergent additive comprising an alpha-amylase variant according to any one of claims 1-11, which may or may not be present in dust-free granular, stable liquid or protected enzyme form.21.按照权利要求20的去垢剂添加剂，该添加剂每克包含0.02-200mg的酶蛋白质。21. A detergent additive according to claim 20, comprising 0.02-200 mg of enzyme protein per gram.22.按照权利要求20或21的去垢剂添加剂，该添加剂还包括另一种酶，如蛋白酶、脂酶、过氧化物酶、另一种淀粉分解酶和/或纤维素酶。22. A detergent additive according to claim 20 or 21 which additionally comprises a further enzyme, such as a protease, a lipase, a peroxidase, another amylolytic enzyme and/or a cellulase.23.一种包含按照权利要求1-11任一之α-淀粉酶变体和表面活性剂的去垢组合物。23. A detergent composition comprising an alpha-amylase variant according to any one of claims 1-11 and a surfactant.24.按照权利要求23的去垢组合物，该组合物还包含另一种酶如蛋白酶、脂酶、过氧化物酶、另一种淀粉分解酶和/或纤维素酶。24. A detergent composition according to claim 23 which further comprises another enzyme such as a protease, a lipase, a peroxidase, another amylolytic enzyme and/or a cellulase.25.一种手工或机用餐具洗涤剂组合物，该组合物包含按照权利要求1-11任一之α-淀粉酶变体和表面活性剂。25. A hand or machine dishwashing detergent composition comprising an alpha-amylase variant according to any one of claims 1-11 and a surfactant.26.按照权利要求25的餐具洗涤剂组合物，该组合物还包含另一种酶如蛋白酶、脂酶、过氧化物酶、另一种淀粉分解酶和/或纤维素酶。26. A dishwashing detergent composition according to claim 25 which further comprises another enzyme such as a protease, lipase, peroxidase, another amylolytic enzyme and/or a cellulase.27.一种手工或机用洗衣组合物，该组合物包含按照权利要求1-11任一之α-淀粉酶变体和表面活性剂。27. A hand or machine laundry composition comprising an alpha-amylase variant according to any one of claims 1-11 and a surfactant.28.按照权利要求27的洗衣组合物，该组合物还包含另一种酶如蛋白酶、脂酶、过氧化物酶、淀粉分解酶和/或纤维素酶。28. A laundry composition according to claim 27 which further comprises another enzyme such as a protease, lipase, peroxidase, amylolytic enzyme and/or cellulase.29.按照权利要求1-11任一之α-淀粉酶变体在纺织品去浆上的用途。29. Use of the alpha-amylase variant according to any one of claims 1-11 for desizing textiles.

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