CN109810961B - A- amylase mutant and its encoding gene and their application for high concentration starch liquefacation - Google Patents

Abstract

The invention belongs to enzyme engineering and genetic engineering field, the a- amylase mutant and its encoding gene for high concentration starch liquefacation and their application are disclosed.The a- amylase mutant has amino acid sequence shown in SEQ ID NO:2；Or by replacing, missing or adding one or several amino acid and the constant amino acid sequence as derived from SEQ ID NO:2 of enzymatic activity on the basis of with the amino acid sequence shown in SEQ ID NO:2.Artificial synthesized a- amylase mutant of the invention can carry out the hydrolysis of efficient stable to high-concentration starch milk initial concentration, reach the requirement of the rapid viscosity reduction of high concentration starch liquefying process in pH4.5-6.0,60-110 DEG C of temperature and without under calcium ion liquefaction system.Meanwhile the addition for reducing calcium ion is more advantageous to the Downstream processing application for realizing product.

Description

For the a- amylase mutant and its encoding gene of high concentration starch liquefacation and they Application

Technical field

The invention belongs to enzyme engineerings and genetic engineering field, and in particular, to a kind of a- for high concentration starch liquefacation Amylase mutant and its encoding gene and their applications in high concentration starch liquefacation.

Background technique

Starch is one of reserves carbohydrate the most abundant and the extremely important raw material of industry in nature, so And starch needs just to can be applied in the deep processings industry such as carbohydrate, amino acid, alcoholic fermentation after preliminary treatment, starch is preliminary Emulsion containing starch granules is warming up to 60-70 DEG C first by processing, so that starch granules water swelling, forms the liquid of paste Body (is gelatinized), and the starch after gelatinization is liquefied as the dextrin (liquefying) of short chain, liquid under the action of amylase by rapid viscosity reduction Change is a particularly significant step in Starch Hydrolysis, and wherein a- amylase has conclusive effect to starch liquefacation effect.Greatly The starch liquefying process of technical scale mainly uses instantaneous high-temperature injection liquefaction, and in the process, the temperature of starch wine with dregs is reachable 105 DEG C or more, this requires used amylase to have good thermal stability, it can be incited somebody to action when acting on starch slurry solution The viscosity of starch slurry solution reduces rapidly.Currently, the a- amylase that starch liquefacation uses in industrial large-scale production is main For the high temperature a- amylase of Novi's letter and the exploitation of Jie Neng section, the enzyme source is in bacillus licheniformis (B.licheniformis), warp Spending the half-life period of the transformation of the protein engineering enzyme at 95 DEG C has reached up to 250min, however the activity of the enzyme and stability according to Rely the presence in calcium ion, the presence of calcium ion can seriously inhibit to prepare key enzyme grape in high fructose syrup by raw material of starch sugar The activity of sugared isomerase.

On the other hand, as starch sugar manufacturing enterprise further decreases the demand of production cost, high concentration liquefaction process by It gradually releases, starch milk initial concentration is stepped up by original 25-30 weight % to 35 weight % or more, starch milk initial concentration When raising, the increase of viscosity is so that liquefaction control becomes difficult, and proposes to starch enzyme viability new after starch gelatinization Demand, a- amylase needs that high temperature resistant, most suitable catalytic temperature range is wide in range, performance is stable and catalysis under high concentration liquefaction process The characteristics such as high-efficient are to realize quick viscosity reduction, improve apparatus working efficiency, guarantee starch liquefacation quality.And it is domestic at present for realization High concentration liquefaction, is substantially improving on liquefaction process, as disclosed a kind of continuous liquefaction in CN104520333A The method of high concentration starch, i.e., in the complex process using two stages liquefaction and the enzyme of a variety of degradable starch, technological operation step Rapid more, enzyme preparation dosage is cumbersome；A kind of preprocess method for promoting high concentration starch liquefacation is disclosed in CN104293863A, is adopted The method for compounding Thermostable α-Amylase with mesophilicα-diastase, but this method viscosity reducing effect is limited, and also adds enzyme system The cost of agent；CN104911234A provides the method for the substep liquefaction high concentration cornstarch similar with CN104293863A, It is multiple using mesophilicα-diastase and Thermostable α-Amylase using the high concentration corn starch milk of 40-65 weight % as reaction substrate It liquefies with substep, however the dosage of enzyme preparation is 6-370 times of CN104293863A method, the liquefaction that just can be properly saccharified Liquid；In addition, disclosing a kind of method of high concentration starch consecutive spraying fluidification in CN201610726308.3, this method is using dense The starch milk of degree 40~50% is back to jet pump feed inlet according to 15-35 weight % to spray the liquefier after the completion of liquefaction, To expect that Dai Shui carries out viscosity reduction to the dextrin after injection instantaneous high-temperature.And in terms of diastase, it is concentrated mainly on to present The high temperature resistant type and catalytic activity of existing medium temperature amylase and alpha-amylase are transformed, or the high temperature resistant starch that exploitation is new Enzyme, if patent CN107312763A, CN107312764A, CN107326020A and CN107345223A are from lichens The a- amylase of bacillus (B.licheniformis) is that template carries out different loci and the mutation of a few amino acids obtains a- Amylase mutant, the mutant can at 80-110 DEG C, pH value 5.0-5.8, under the conditions of carry out good starch liquefacation, implement The butt quality score that example uses carries out application effect appraisal to a- amylase mutant for the starch milk of 32 weight %, forms sediment simultaneously The maximum concentration of powder cream has also only been an attempt to 37 weight %, does not carry out the survey of high concentration starch (> 40 weight %) liquefaction effect Examination.Therefore, it is seen that there is presently no the special enzyme preparations for the exploitation of this special process demand of high concentration starch liquefacation.

Summary of the invention

The present invention, which is directed to current existing commercialized a- catalyzed by amylase activity, has calcium ion dependence and highly concentrated Spend starch liquefying process to a- amylase in high temperature resistant, most suitable catalytic temperature range is wide, performance is stable, high catalytic efficiency and rapidly The demand functionally such as viscosity reduction provides a kind of a- amylase mutant, under high concentration starch liquefying process have high temperature resistant, Most suitable catalytic temperature range is wide, performance is stable, high catalytic efficiency and the characteristics of rapid viscosity reduction, and does not have calcium ion dependence, So as to meet the needs of modern starch sugar manufacturing enterprise further decreases production cost, to overcome the shortcomings of existing product.

To achieve the goals above, in a first aspect, the present invention provides a kind of a- amylase mutant, the a- amylase Mutant has amino acid sequence shown in SEQ ID NO:2；Or the base with the amino acid sequence shown in SEQ ID NO:2 By replacing, missing or adding one or several amino acid and the constant amino as derived from SEQ ID NO:2 of enzymatic activity on plinth Acid sequence.

Second aspect, the present invention provides the genes that one kind can encode a- amylase mutant described in first aspect.

The third aspect, the present invention provides a kind of recombinant expression carriers containing gene described in second aspect.

Fourth aspect, the present invention provides a kind of recombinant bacterial strains containing recombinant expression carrier described in the third aspect.

5th aspect, the present invention provides a kind of methods for preparing a- amylase mutant, method includes the following steps: (1) recombinant bacterial strain described in the third aspect is cultivated, the gene expression for encoding a- amylase mutant is made；(2) separating-purifying institute table The a- amylase mutant reached.

6th aspect, the present invention provides a kind of enzyme preparation for high concentration starch liquefacation, which contains first A- amylase mutant is as active constituent described in aspect.

7th aspect, the present invention provides described in a- amylase mutant, second aspect described in a kind of first aspect Enzyme system described in recombinant expression carrier described in gene, the third aspect, recombinant bacterial strain described in fourth aspect and the 6th aspect Application of the agent in high concentration starch liquefacation.

Artificial synthesized a- amylase mutant of the invention can be in pH4.5-6.0,60-110 DEG C of temperature and without calcium ion liquid Under change system, the hydrolysis of efficient stable is carried out to high-concentration starch milk initial concentration, it is rapid to reach high concentration starch liquefying process The requirement of viscosity reduction.Meanwhile the addition for reducing calcium ion is more advantageous to the Downstream processing application for realizing product.

Other features and advantages of the present invention will the following detailed description will be given in the detailed implementation section.

Detailed description of the invention

The drawings are intended to provide a further understanding of the invention, and constitutes part of specification, with following tool Body embodiment is used to explain the present invention together, but is not construed as limiting the invention.In the accompanying drawings:

Fig. 1 is protokaryon shuttle plasmid pNCMO2-amyMU carrier schematic diagram.

Specific embodiment

Detailed description of the preferred embodiments below.It should be understood that described herein specific Embodiment is merely to illustrate and explain the present invention, and is not intended to restrict the invention.

The endpoint of disclosed range and any value are not limited to the accurate range or value herein, these ranges or Value should be understood as comprising the value close to these ranges or value.For numberical range, between the endpoint value of each range, respectively It can be combined with each other between the endpoint value of a range and individual point value, and individually between point value and obtain one or more New numberical range, these numberical ranges should be considered as specific open herein.

In the present invention, in the absence of explanation to the contrary, the i.e. enzyme dosage of size of the term " enzyme activity " used How much, indicated with enzyme activity unit, i.e. enzyme unit (U), in the present invention definition of enzyme unit be: under the conditions of pH5.5,70 DEG C, 1 Enzyme amount required for minute liquefies 1g soluble starch completely is an enzyme activity unit.

Term " gene rearrangement " used herein refers to that the present invention passes through in the future to obtain a- amylase mutant The coded sequence of the different a- amylase in source carries out DNasel digestion PCR denaturation renaturation and realizes homology mispairing recombination between gene, Form a kind of enzyme molecule lactam enzyme by directional anagenesis in vitro technology of fusion.

And term " target gene " used herein refers to import, express by target gene and/or knock out and foundation The gene that methods known in the art design.

In the present invention, amyMU is the gene for encoding a- amylase mutant of the present invention, and nucleotide sequence can be such as SEQ Shown in ID NO.:1, amino acid sequence can be as shown in SEQ ID NO.:2；

Bsta is gene of the coding from the a- amylase of Bacillus stearothermophilus, multicore glycosides Acid sequence can be as shown in SEQ ID NO.:3, and amino acid sequence is as shown in SEQ ID NO.:4；

Bla is gene of the coding from the a- amylase of Bacillus licheniformis, polynucleotide sequence Can be as shown in SEQ ID NO.:5, amino acid sequence is as shown in SEQ ID NO.:6；

Tsa is gene of the coding from the a- amylase of Thermococcus siculi, and polynucleotide sequence can be such as Shown in SEQ ID NO.:7, amino acid sequence is as shown in SEQ ID NO.:8；

Baa is gene of the coding from the a- amylase of Bacillus amyloliquefaciens, polynucleotides Sequence can be as shown in SEQ ID NO.:9, and amino acid sequence is as shown in SEQ ID NO.:10.

In a first aspect, the present invention provides a kind of a- amylase mutant, the a- amylase mutant has SEQ ID Amino acid sequence shown in NO:2；Or have on the basis of the amino acid sequence shown in SEQ ID NO:2 by replacing, lacking Or add one or several amino acid and the constant amino acid sequence as derived from SEQ ID NO:2 of enzymatic activity.

SEQ ID NO:2

MALNNGTMMQYFEWYLPNDGQHWKRLQNDSAYLAEHGITAVWIPPAYKGTSQADVYDPYDFFDLGEYY QKGTVETRFGTKELQDAIGSLHSRNVQVYGDVVLNHKAGADEWNPFVGDYTWTDFSGVASGKYTANYLDFHPNEVK CCDEGTFGDYPDIAHEKSWDQHWLWASDESYAAYLRSIGVDAWRFDYVKGYGAWVIKDWLDWWGGWAVGEYWDTNV DALLNWAYNSNAKVFDFPLYYKMDEAFDNKNIPALVDALRYGQTVVSRDPFKAVTFVANHDTDIIWNKYPAYAFIL TRESGYPQVFYGDMYGTKGDSQREIPALKHKIEPILKARKEYAYGPQHDYIDHPDVIGWTREGDSSAAKSGLAALI TDGPGGAKRMYVGRQNAGETWHDITGNRSDTVTITSDGWGEFHVNDGSVSIYVQK*

Wherein, enzymatic activity is constant refers under identical determination condition, has the amino acid sequence as derived from SEQ ID NO:2 The enzyme activity of the enzyme activity of the a- amylase of column and the a- amylase mutant with amino acid sequence shown in SEQ ID NO:2 Between percentage (relative activity) be not less than 95% (or 96% or 97% or 98% or 99% or 100%).

20 kinds of amino acid residues of constitutive protein matter, are segmented into four classes according to pendant polar: 1, nonpolar amino acid: Alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met), phenylalanine (Phe), tryptophan (Trp) and proline (Pro)；2, the uncharged amino acid of polarity: glycine (Gly), serine (Ser), threonine (Thr), cysteine (Cys), aspartic acid (Asn), glutamine (Gln) and tyrosine (Tyr)；3, band The amino acid of positive charge: arginine (Arg), lysine (Lys) and histidine (His)；4, negatively charged amino acid: asparagus fern ammonia Sour (Asp) and glutamic acid (Glu) (referring to " biochemistry " (second edition) first volume, Shen Tong, Wang Jingyan, the 82-83 pages, high religion Educate publishing house, December nineteen ninety).If it happens the amino acid residue that a classification is belonged in protein replaces, such as is taken by Arg Replace Ile for Lys or by Leu, effect of the residue played in protein domain (for example provides positive charge or formed and dredged The effect of water bag structure) do not change, therefore the stereochemical structure of protein can't be had an impact, therefore still can be real The function of existing albumen.The amino acid residue for belonging to a classification, which replaces, can occur appointing in above-mentioned a- amylase mutant It anticipates on an amino acid residue position.

As previously mentioned, a- amylase mutant provided by the invention can also be modified or is mutated, derivative egg is obtained White matter." derivative protein " of the present invention, which refers to, has amino acid with the a- amylase mutant with above-mentioned amino acid sequence Difference in sequence can also have not the difference on the modified forms for influencing sequence, or have both at the same time.These albumen include day Right or induction genetic variant.The induction variant can be obtained by various technologies, such as radiation or mutagens generate Random mutation, the technology of such as fixed-point mutation method or other known molecular biology can also be passed through." the derivative albumen Matter " further includes the analog (such as D type amino acid) with the residue of natural L-form amino acid, and have it is non-naturally occurring or The analog of the amino acid (such as beta-amino acids, gamma-amino acid) of synthesis.

(not changing primary structure usually, i.e., the do not change amino acid sequence) form of modification includes: internal or external albumen Chemical derivative form such as acetylation or carboxylated.Modification further include glycosylation, such as those in the synthesis and processing of albumen or Albumen that is glycosylation modified and generating is carried out in further processing step.This modification can be by being exposed to progress sugar for albumen The enzyme (glycosylase or deglycosylation enzyme of such as mammal) of base and complete.Modified forms further include with phosphorylation amino The sequence of sour residue (such as phosphotyrosine, phosphoserine, phosphothreonine).It further include being modified to improve its anti-egg White hydrolysis property or the albumen for optimizing solubility property.

In order to facilitate purifying, can also using the common label in this field to the amino acid sequence of a- amylase mutant into Row addition modification, for example, amino terminal and/or the carboxyl end of the amino acid sequence in a- amylase mutant can be passed through Label (at least one in such as Poly-Arg, Poly-His, FLAG, Strep-tag II and c-myc shown in end connection the following table 1 Kind) and obtain.The label will not influence the activity of a- amylase mutant of the invention, in actual application, can be with Addition label is chosen whether according to demand.

Table 1

Label	Residue number	Amino acid sequence
			Poly-Arg	5-6 (usually 5)	RRRRR (SEQ ID NO:11)
Poly-His	2-10 (usually 6)	HHHHHH (SEQ ID NO:12)
			FLAG	8	DYKDDDDK (SEQ ID NO:13)
Strep-tagⅡ	8	WSHPQFEK (SEQ ID NO:14)
			c-myc	10	EQKLISEEDL (SEQ ID NO:15)

Above-mentioned a- amylase mutant can be obtained by artificial synthesized, can also first synthesize its encoding gene, then pass through Biological expression obtains.

Second aspect, the present invention also provides the genes that can encode above-mentioned a- amylase mutant.

It is known in the art that in 20 kinds of different amino acid of constitutive protein matter, except Met (ATG) or Trp (TGG) are respectively Single password coding is outer, other 18 kinds of amino acid encode (Sambrook etc., molecular cloning, cold spring by 2-6 codon respectively Publishing house, Cold Spring Harbor Laboratory, New York, the U.S., the second edition, 1989, see the Appendix D of page 950).I.e. due to the degeneracy of genetic codon, Determine the most more than one of the codon of an amino acid, the displacement of third nucleotide, tends not in triplet codon Change the composition of amino acid, therefore the nucleotide sequence for encoding the gene of same protein can be different.Those skilled in the art are according to public affairs The password sublist known, from amino acid sequence disclosed by the invention, and the a- starch enzyme mutant obtained by the amino acid sequence The constant amino acid sequence of body activity, can derive the nucleotide sequence that can encode their gene completely, pass through biology Method (such as PCR method, mutation method) or chemical synthesis process obtain the nucleotide sequence, therefore the partial nucleotide sequence Column should be construed as being included in the scope of the invention.On the contrary, using DNA sequence dna disclosed herein, it can also be by well known in the art The method (molecular cloning, CSH Press, New York, the U.S., the second edition, 1989) of method, such as Sambrook etc. It carries out, by modifying nucleic acid sequence provided by the invention, obtains and the consistent ammonia of a- amylase mutant activity of the present invention Base acid sequence.

Preferably, the gene has nucleotide sequence shown in SEQ ID NO:1.

SEQ ID NO:1

ATGGCTCTTAACAACGGCACAATGATGCAATACTTCGAATGGTACCTTCCTAACGATGGCCAACATTGG AAACGTCTTCAAAACGATTCTGCTTACCTTGCTGAACATGGCATCACAGCTGTTTGGATCCCTCCTGCTTACAAAGG CACATCTCAAGCTGATGTTTACGATCCTTACGATTTCTTCGATCTTGGCGAATACTACCAAAAAGGCACAGTTGAAA CACGTTTCGGCACAAAAGAACTTCAAGATGCTATCGGCTCTCTTCATTCTCGTAACGTTCAAGTTTACGGCGATGTT GTTCTTAACCATAAAGCTGGCGCTGATGAATGGAACCCTTTCGTTGGCGATTACACATGGACAGATTTCTCTGGCGT TGCTTCTGGCAAATACACAGCTAACTACCTTGATTTCCATCCTAACGAAGTTAAATGCTGCGATGAAGGCACATTCG GCGATTACCCTGATATCGCTCATGAAAAATCTTGGGATCAACATTGGCTTTGGGCTTCTGATGAATCTTACGCTGCT TACCTTCGTTCTATCGGCGTTGATGCTTGGCGTTTCGATTACGTTAAAGGCTACGGCGCTTGGGTTATCAAAGATTG GCTTGATTGGTGGGGCGGCTGGGCTGTTGGCGAATACTGGGATACAAACGTTGATGCTCTTCTTAACTGGGCTTACA ACTCTAACGCTAAAGTTTTCGATTTCCCTCTTTACTACAAAATGGATGAAGCTTTCGATAACAAAAACATCCCTGCT CTTGTTGATGCTCTTCGTTACGGCCAAACAGTTGTTTCTCGTGATCCTTTCAAAGCTGTTACATTCGTTGCTAACCA TGATACAGATATCATCTGGAACAAATACCCTGCTTACGCTTTCATCCTTACACGTGAATCTGGCTACCCTCAAGTTT TCTACGGCGATATGTACGGCACAAAAGGCGATTCTCAACGTGAAATCCCTGCTCTTAAACATAAAATCGAACCTATC CTTAAAGCTCGTAAAGAATACGCTTACGGCCCTCAACATGATTACATCGATCATCCTGATGTTATCGGCTGGACACG TGAAGGCGATTCTTCTGCTGCTAAATCTGGCCTTGCTGCTCTTATCACAGATGGCCCTGGCGGCGCTAAACGTATGT ACGTTGGCCGTCAAAACGCTGGCGAAACATGGCATGATATCACAGGCAACCGTTCTGATACAGTTACAATCACATCT GATGGCTGGGGCGAATTCCATGTTAACGATGGCTCTGTTTCTATCTACGTTCAAAAA

As described above, correspondingly, the end 5' and/or the end 3' of the nucleotide sequence can also be connected with shown in table 1 The coded sequence of label.

Nucleotide sequence provided by the invention can usually use polymerase chain reaction (PCR) amplification, recombination method or people Work synthetic method obtains.For example, those skilled in the art according to the present invention provided by nucleotide sequence, can be easy to To template and primer, amplification is carried out using PCR and obtains related sequence.

A kind of preferred embodiment according to the present invention, nucleotide sequence provided by the invention can pass through gene rearrangement Technology obtains, according to the present invention a kind of preferred embodiment, obtains bsta, bla, tsa and baa genetic fragment, and make with this For template, PCR amplification obtains bsta, bla, tsa and baa genetic fragment respectively, passes through agarose gel electrophoresis and glue recycling examination Agent box purifies bsta, bla, tsa and baa genetic fragment, carries out DNAase endonuclease reaction, passes through agarose gel electrophoresis after digestion The DNA small fragment of 50-100bp is subjected to glue recycling, then using genetic recombination reaction system and reaction condition carry out bsta, The external rearrangement of bla, tsa and baa gene, obtains nucleotide sequence of the invention.

Once obtaining related nucleotide sequence, so that it may obtain related amino acid sequence with recombination method is large batch of.It is logical Often gained nucleotide sequence is cloned into carrier, then transgene engineering bacteria, then through conventional method after proliferation The isolated related nucleotide sequence of host cell.

In addition, also related nucleotide sequence can be synthesized with well known artificial chemistry synthetic method.

The third aspect, recombinant expression carrier provided by the invention contain gene provided by the invention.

Various carriers known in the art can be selected in " carrier " used in recombinant expression carrier, such as commercially available each germplasm Grain, clay, bacteriophage and retrovirus etc., the preferred pNCMO2-amyMU plasmid of the present invention, schematic diagram are as shown in Figure 1.Weight Group expression vector establishment, which can be used, to have the various endonucleases of cleavage site to carry out digestion in vector multiple cloning site Linear plasmid is obtained, is connect with the genetic fragment using the cutting of identical nucleic acid restriction endonuclease, obtains recombinant plasmid.

Fourth aspect, recombinant bacterial strain provided by the invention contain recombinant expression carrier provided by the invention.

The recombinant expression carrier can be converted, transduceed or is transfected into host cell by the method for this field routine It is preferably electroporated such as Calcium Chloride Method chemical conversion, electroporation in (bacterial strain).The host cell can be protokaryon Cell or eukaryocyte, preferably prokaryotic cell, further preferably rod bacterium, such as Escherichia coli (Escherichia coli) Or bacillus subtilis (Bacillus subtilis).

5th aspect, the present invention provide a kind of method for preparing a- amylase mutant, method includes the following steps: (1) recombinant bacterial strain as described above is cultivated, the gene expression for encoding a- amylase mutant is made；(2) expressed by separating-purifying A- amylase mutant.

The condition of culture is conventional condition of culture, when the recombinant strain is recombinated bacillus, be can be used TM culture medium containing neomycin cultivates 82h under the conditions of 200rpm, 32 DEG C with fermentation expression recombinant protein, and then ultrasound is broken A- amylase mutant can be obtained using purifying in chopping fine born of the same parents, and specific method is the routine techniques hand of those skilled in the art Section, details are not described herein.

6th aspect, the present invention provides a kind of enzyme preparation for high concentration starch liquefacation, which contains this hair The a- amylase mutant of bright offer is as active constituent.

According to the present invention, on the basis of the total weight of the enzyme preparation, the content of the a- amylase mutant can be 10-90 weight %.It can also be containing well known to a person skilled in the art solvent (such as glycerol, carbohydrate and albumen in the enzyme preparation The protein protective agents such as enzyme inhibitor) etc..

7th aspect, the present invention also provides a- amylase mutant as described above, gene as described above, institutes as above The enzyme preparation of recombinant expression carrier, recombinant bacterial strain as described above and the high concentration starch liquefacation as described above stated is highly concentrated Spend the application in starch liquefacation.

In the present invention, the method using a- amylase mutant liquefaction high concentration starch may include: by high concentration Farinaceous size is contacted with the a- amylase mutant, and is liquefied according to the starch liquefaction methods of this field routine.

Wherein, the high concentration starch is preferably starch milk of the solid content more than 36 weight %；More preferably solid content The starch milk of 36-65 weight %, the even more preferably starch milk for solid content 36-48 weight %.

Herein it should be noted that a- amylase mutant provided by the invention not only be applicable in solid content 36 weight % with On starch milk liquefaction, but compared to existing a- amylase, a- amylase mutant provided by the invention is to 36 weights When measuring the starch milk liquefaction of % or more, there is more preferably effect, but liquefy for starch milk of the solid content more than 36 weight % When, effect is also due to existing a- amylase.

The present invention will be described in detail by way of examples below.

(1) method and apparatus used in embodiment

Experimental method described in following embodiments and equipment are unless otherwise specified conventional method (such as under Standardization program described in column works is implemented: Sambrook etc., Molecular Cloning:A Laboratory Manual (the 3rd edition), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2001)；Davis etc., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (1995)；And Current Protocols in Cell Biology (CPCB) (Juan S.Bonifacino etc. writes, John Wiley and Sons, Inc.)) and equipment.Institute in following embodiments Experimental material, unless otherwise instructed, whole reagents are purchased from OXOID company.

(2) culture medium used in embodiment, thallus and enzyme

LB solid medium (LB plate): tryptone 10g/L；Yeast extract 5g/L；Agar 15g/L；Sodium chloride 20- 100g/L；pH 7.0；121 DEG C of sterilizing 15min.

SOC media components concentration: 2% (W/V) Tryptone；0.5% (W/V) Yeast Extract；0.05% (W/V)NaCl；2.5mM KCl；10mM MgCl₂；20mM glucose；121 DEG C of sterilizing 15min.

Bacillus pumilus MT fluid nutrient medium: glucose 10g/L；Beef extract powder 5g/L；Yeast powder 2g/L； FeSO₄.7H₂O 0.01g/L；MnSO₄.4H₂O 0.01g/L；ZnSO₄.7H₂O 0.01g/L；MgCl₂4g/L；121 DEG C of sterilizings 15min.(MT solid medium then adds agar 15g/L on the basis of MT liquid)

Mutant library screening and culturing medium (plate): tryptone 10g/L；Yeast extract 5g/L；Agar 15g/L；2% can Soluble starch；5mM EDTA；7.0,121 DEG C of sterilizing 15min of pH.

Bacillus pumilus seed culture medium (SM liquid): glucose 10g/L；Yeast extract 15g/L；FeSO₄.7H₂O 0.01g/L；MnSO₄.4H₂O 0.01g/L；ZnSO₄.7H₂O 0.01g/L；121 DEG C of sterilizing 15min.

Bacillus pumilus fermentation medium (TM liquid): glucose 10g/L；Beef extract powder 5g/L；Yeast powder 2g/L； FeSO₄.7H₂O 0.01g/L；MnSO₄.4H₂O 0.01g/L；ZnSO₄.7H₂O 0.01g/L；121 DEG C of sterilizing 15min.

Escherichia coli cloning and expression host cell are purchased from Beijing Quanshijin Biotechnology Co., Ltd；

Electrotransformation e. coli Trans109 competence is purchased from Beijing Quanshijin Biotechnology Co., Ltd；

Bacillus pumilus (Brevibacillus choshinensis SP3) and expression plasmid pNCMO2 are with reagent Box-like formula is purchased from TaKaRa；

Double digestion reagent is purchased from NEB, conventional reagent；

It connects reaction reagent and is purchased from NEB, conventional reagent.

(3) sequence

Target gene sequence and primer used are synthesized by Jin Weizhi company；Sequencing is limited by the raw work bioengineering in Shanghai Company completes.

The primer sequence used is as shown in table 2:

2. primer sequence of table

Primer	Sequence (5 ' -3 ')	Sequence number
			M13-RV-F	GAGCGGATAACAATTTCACACAGG	SEQ ID NO.:16
M13-47-R	CGCCAGGGTTTTCCCAGTCACGAC	SEQ ID NO.:17
			pNC-F	CGCTTGCAGGATTCGG	SEQ ID NO.:18
pNC-D	CAATGTAATTGTTCCCTACCTGC	SEQ ID NO.:19

(4) reaction system and reaction condition

Double enzyme digestion reaction system and reaction condition are as shown in table 3 below:

Table 3.Nco I/Hind III double enzyme digestion reaction system and reaction condition

Coupled reaction system and reaction condition are as shown in table 4 below:

Table 4.T4 DNA ligase linked system and reaction condition

Endonuclease reaction system and reaction condition are as shown in table 5 below:

5 DNAase endonuclease reaction system of table and condition

PCR reaction system and reaction condition is as shown in the following table 6 and table 7:

6 PCR reaction condition of table

7 PCR system of table is 15 μ L, and each component ratio is as follows:

dNTP(10mM)	1.0μL
		10 × rTaq archaeal dna polymerase buffer	1.5μL
Upstream primer (10 μM)	0.5μL
		Downstream primer (10 μM)	0.5μL
Bacterium colony	1μL
		RTaq archaeal dna polymerase (5U/ μ L)	0.5μL
ddH2O	10.0μL

Genetic recombination (DNAShuffling) reaction system and reaction condition is as shown in the following table 8 and the following table 9:

8 primer free PCR amplification of table

The amplification of 9 linker-adapter-PCR of table

(5) measuring method

Starch milk viscosimetric analysis:

The starch milk for taking 30g to be determined pours into the sample jar of RVA viscosity analyser, and setting program is as follows: at 60 DEG C 1.5min is kept the temperature, 95 DEG C is then raised temperature to, is 6min the time required to heating, 5.5min is kept the temperature at 95 DEG C, is then cooled to 50 DEG C, it is 2.5min the time required to cooling, revolving speed is set as 250rpm, measures the viscosity of liquefaction process, takes liquefaction process viscosity bent The peak-peak of line is as sample viscosity evaluation index, unit cP.

Amylase activity detection and definition:

The definition of amylase enzyme activity: under the conditions of pH5.5,70 DEG C, enzyme required for 1g soluble starch liquefying completely in 1 minute Amount is defined as 1U.

Enzyme activity determination: 1 weight % gelatinization of starch solution of 10mL is prepared in test tube, and (25mL is added in 1.0g soluble starch 25mL 0.4M CH is added after cooling in 0.4M NaOH, 60 DEG C of 5min₃COOH is settled to 100mL), 70 DEG C of heat preservation 10min add Enter the enzyme solution after 1.0mL dilutes, 10min is heated in 70 DEG C of thermostat water baths, 1mL 1M hydrochloric acid is added after cooling and terminates reaction, takes 1mL reaction solution is added installs 10mL work iodine solution (0.5g I in advance₂Ground in 5.0g KI water, be settled to 1000mL) examination Pipe shakes up and measures light absorption value (OD under 660nm wavelength rapidly₆₆₀), the identical of enzyme solution (water for adding same enzyme liquid product) is not added Reaction solution records it for control and measures light absorption value (OD under 660nm wavelength⁰ ₆₆₀), amylase activity calculation formula is as follows:

Enzyme activity=[(OD₆₆₀-OD⁰ ₆₆₀)*100/OD₆₆₀* 10] extension rate of * enzyme

The measuring method of sample liquid DE value:

(1) solution is prepared

(a) methylene blue indicator solution (10g/L): weighing methylene blue 1.0g, is dissolved in water and is settled to 100mL.

(b) 100 ± 2 DEG C of benchmark DEXTROSE ANHYDROUS 0.5000g that drying to constant weight glucose standards solution (2g/L): are weighed (claims standard to 0.0001g), be dissolved in water and be settled to 250mL.

(c) Fehling's solution is prepared

Fehling's solution I: copper sulphate (CuSO is weighed₄·5H₂O) 69.3g is dissolved in water and is settled to 1000mL, stores in brown In bottle；

Fehling's solution II: sodium potassium tartrate tetrahydrate (KNaC is weighed₄H₄O₆·4H₂O) 346.0g and sodium hydroxide 100.0g, adds water 1000mL is dissolved and be settled to, is stored in rubber stopper vial.If having precipitating using preceding, supernatant use is drawn；

(2) it demarcates: when pre- titration, successively drawing Fehling's solution II, each 5.0mL of Fehling's solution I in conical flask, add water 20mL is added bead 3, and glucose standards solution 24mL is added, shakes up solution in conical flask, and conical flask is heated, and controls Solution processed boiling in 120 ± 15s, and keep slightly boiled, methylene blue indicator solution 2 is added and drips, continues with glucose standards solution Titration, until it is its terminal that blue, which just disappears, entire titration operation should be completed in 3min.When formal titration, ratio is added in advance The glucose standards solution of above-mentioned titration consumption lacks the glucose standards solution of 1mL, does parallel test, records consumption of glucose mark Quasi- total volume, takes its arithmetic mean of instantaneous value.

The quality of consumption of glucose when 5mL each according to formula [1] calculating Fehling's solution II, I:

In formula:

The quality of consumption of glucose, g when each 5mL of RP: Fehling's solution II, I；

m₁: weigh the amount of benchmark DEXTROSE ANHYDROUS, g；

V₁: the total volume of consumption of glucose standard solution, mL.

(3) the DE value in sample liquid is measured

(a) preparation of sample liquid

A certain amount of sample liquid is weighed, sampling amount contains reduction sugar amount 125- to meet titration for condition in every 100mL sample liquid 200mg (claims standard to be advisable to 0.0001g), adds hot water dissolving, and be settled to 250mL with water.

(b) pre- titration

Fehling's solution II, each 5.0mL of Fehling's solution I are successively drawn in conical flask, adds water 20mL, is added bead 3, It is pre- that part sample liquid is added, conical flask, the boiling in 120 ± 15s of control solution are heated, and keep slightly boiled, with the speed of every 2 seconds 1 drops Sample liquid is added dropwise in degree, until methylene blue indicator solution 2 is added and drips when solution blue will disappear, is further continued for that sample liquid is added dropwise until blue Just disappearing is its terminal, the total volume of record consumption sample liquid.

(c) formal titration

Fehling's solution II, each 5.0mL of Fehling's solution I are successively drawn in conical flask, is added 1mL fewer than titrating sample liquid in advance Sample liquid heats conical flask, the boiling in 120 ± 15s of control solution in conical flask, and keeps slight boiling condition, is dripped with every 2 seconds 1 Speed sample liquid is added dropwise, until when solution blue will disappear, methylene blue indicator solution 2 is added and drips, be further continued for that sample liquid is added dropwise until It is its terminal that blue, which just disappears, and entire titration operation must be completed in 3min, the total volume of record consumption sample liquid, according to formula [2] the DE value in sample liquid is calculated:

In formula:

DE value: dextrose equivalent value in sample liquid, %；

The quality of consumption of glucose, g when each 5mL of RP: Fehling's solution II, I；

m₂: sampling amount, g；

V₂: the total volume of sample liquid, mL are consumed when titration；

DMC: the content of syrup dry matter (solid content), % in sample liquid.

4, liquifying method is sprayed:

The temperature of jet port is 108 DEG C and maintains in pipe 3-10 minutes, steam pressure 0.6MPa, sprays product after liquefaction It is flashed, temperature is down to 98 DEG C, then keeps the temperature 2h at 95 DEG C, obtains liquefier.

Embodiment 1

The present embodiment is used to illustrate the foundation in a- amylase mutant library

Trust money Wei Zhi company carries out gene chemical synthesis gene bsta, bla, tsa and baa, and nucleotide sequence is respectively such as SEQ Shown in ID NO.:3,5,7,9, when synthesis each gene 5 ' end insertion Nco I restriction enzyme site sequence, 3 ' end insertion Hind III enzymes Enzyme site sequence, the sequence after synthesis are both connected on pUC57 carrier.

Using the above sequence as template, is expanded respectively using M13-RV-F and M13-47-R as primer and obtain bsta, bla, tsa With baa genetic fragment, bsta, bla, tsa and baa gene are purified by 1.2% agarose gel electrophoresis and plastic recovery kit Segment carries out DNAase endonuclease reaction, is carried out the DNA small fragment of 50-100bp by 2% agarose gel electrophoresis after digestion Glue recycling, then carries out the external rearrangement of bsta, bla, tsa and baa gene using genetic recombination reaction system and reaction condition, Gene after being reset with primer M13-RV-F and M13-47-R amplification, with 2% agarose gel electrophoresis recycling 1500bp's or so It is spare repeatedly to carry out above-mentioned steps acquisition 10-20 μ g genetic recombination segment for genetic recombination segment.

After the recombination segment of above-mentioned acquisition passes through Nco I and Hind III double digestion, with 2% agarose gel electrophoresis Genetic fragment (need to recycle nearly 5 μ g) after recycling digestion, is connected on pNCMO2 shuttle vector；It is connected referring to T4DNA ligase System and reaction condition, point 5 batches and every batch of carry out the connection reaction of 10 systems parallel, obtain the connection product of 750 μ L； Desalination recycling is carried out to connection product using gel reclaims kit, recovery product uses the ddH of 100 μ L₂O is eluted, and is obtained Obtain about 85 μ L desalination connection products.

5 μ L are taken gently to be mixed into the Electroporation-competent cells Trans109 of 100 μ L, carrying out electrotransformation, (electricity turns condition: 0.2cm Electrotransformation cup, 200 ohm, 25 μ Fd, 2.5 kilovolts), electrotransformation is rapidly added the preheated SOC culture medium of 37 DEG C of 900 μ L, presses 85 μ L desalination connection products have been totally converted by the method.

Conversion fluid is placed at 37 DEG C and cultivates 1 hour progress cell recovery, after recovery every 100 μ L or so be coated on containing On the LB solid plate of 100 μ g/mL ampicillins, according to said method conversion fluid all will be put down all on LB solid plate Plate is placed at 37 DEG C and cultivates 15 hours；Take out 10 transformants of the random picking of each plate of plate carry out bacterium colony PCR identification (with PNC-F and pNC-D is upstream and downstream primer), positive transformant ratio is 80% or so as the result is shown.

1mL LB liquid medium is added on each plate simultaneously and is eluted the bacterium colony on plate with spreading rod, it will Bacterium colony on all plates elutes, and the plasmid eluted in bacterium solution is extracted using plasmid extraction kit, extracts obtain altogether Obtain 100 μ L of plasmid (total 2 μ g).

Referring to the operating instruction of TaKaRa bacillus pumilus kit, bacillus pumilus competent cell is kit It provides, using the method for the NTP conversion that kit provides, each competent cell is transferred to 5 μ L plasmids (about 100ng), corotation Change 20 bacillus pumilus, the conversion fluid 3000rpm after recovering will be converted, be centrifuged under the conditions of 4 DEG C, thallus is collected, by thallus It is applied in the MT culture medium containing 10 μ g/mL neomycins, 37 DEG C are incubated overnight and calculate clump count, establish a- starch enzyme mutant Body library.

Above-mentioned work is repeated several times, to combine later period screening that there is biggish a- amylase mutant library could obtain mesh Marking mutant, (the mutant library storage capacity that the present invention establishes is 2 × 10⁶)。

Embodiment 2

The present embodiment is used to illustrate screening and the heterogenous expression of a- amylase mutant

By the a- amylase mutant library (being grown in the bacterium colony on MT culture medium flat plate) of above-mentioned foundation, chosen with QPix450 It clones by the way of machine uses similar panel photocopy, by the colony lift on MT culture medium flat plate to containing 10 μ g/mL neomycins Mutant library screening flat board on, 37 DEG C be incubated overnight after be put into 4 DEG C of refrigerators and place two days, selecting after taking-up has obvious hydrolysis The single colonie of transparent circle is first round screening without Ca²⁺The a- amylase mutant (total to obtain 456) of dependent form.

The mutant that the screening of all first round obtains is inoculated into (48 holes in the SM culture medium containing 10 μ g/mL neomycins Deep-well plates culture), using inoculated culture medium be placed in 200rpm, under the conditions of 32 DEG C culture 12h as seed liquor；Absorption is cultivated Seed liquor, (48 hole deep-well plates cultures) are seeded in the TM culture medium containing 10 μ g/mL neomycins with 5% inoculum concentration, in 200rpm, 82h is cultivated under the conditions of 32 DEG C with fermentation expression recombinant protein, 48 hole deep-well plates are in 4000rpm, 4 DEG C of conditions after culture Lower centrifugation, collecting fermented supernatant fluid is mutant crude enzyme liquid.

200 μ L, 1% gelatinization of starch solution is added in every hole in 48 orifice plates, and the mutation of 20 μ L is added in 70 DEG C of heat preservation 10min Body crude enzyme liquid (with culture orifice plate correspond), prepare parallel orifice plate 5, be respectively placed in 90 DEG C, 95 DEG C, 100 DEG C, 105 DEG C, 10min is heated in 110 DEG C of thermostat water baths, every hole is added 20 μ L 1M hydrochloric acid and terminates reaction after cooling, and 20 μ L reaction solutions is taken to be added In 48 orifice plates for installing 200 μ L work iodine solution in advance, shakes up and measure light absorption value (OD660) under 660nm wavelength rapidly, the above work Make the screening that the second wheel thermostableα-amylase mutant is carried out using the liquor removing workstation of Beckman, by the above repeatedly screening Obtaining altogether has greater activity at 8 100 DEG C of mutant, wherein 2 still have equal active at 105 DEG C, it is ordered respectively Entitled amyMU1 and amyMU2.

The recombination bacillus pumilus monoclonal of amyMU1 and amyMU2a- amylase mutant is seeded to 20mL to contain In the SM culture medium of 10 μ g/mL neomycins, by inoculated culture medium as culture 12h under the conditions of 200rpm, 32 DEG C as kind Sub- liquid；Cultured seed liquor is drawn, TM of the 300mL (1L shaking flask) containing 10 μ g/mL neomycins is seeded to 5% inoculum concentration In culture medium, 82h is cultivated under the conditions of 200rpm, 32 DEG C with fermentation expression recombinant protein, while with not antibiotic identical It is blank control that culture medium and cultural method culture, which do not have the bacillus pumilus of expression alien gene, after culture 4000rpm, it is centrifuged under the conditions of 4 DEG C, it is spare as crude enzyme liquid to collect fermented supernatant fluid.

Embodiment 3

The present embodiment is used to illustrate the enzymatic property and stability of a- amylase mutant

(1) enzyme activity is tested

Take above-mentioned crude enzyme liquid to carry out SDS-PAGE analysis, mutant amyMU1 and amyMU2 molecular weight be respectively 52KD and 47KD；Enzyme activity assay is carried out to above-mentioned crude enzyme liquid, mutant amyMU1 and amyMU2 enzyme activity is respectively 52U/mL and 60U/mL.

(2) mutant catalysis reaction optimal pH detection

Mutant amyMU1 and amyMU2 (each reaction enzyme amount is 0.05U) are prepared from different pH (3-7) respectively 1% gelatinized starch is reacted, and is detected according to enzyme activity determination method, different pH (3-7) buffer used are as follows: 0.1mol/L is sweet Propylhomoserin-hydrochloride buffer (pH3.0)；0.1mol/L acetate buffer solution (pH 4.0-5.0)；0.1mol/L kaliumphosphate buffer (pH6.0—7.0)；A reaction condition is set every 0.5pH value, being set as relative activity with the highest pH value point of enzyme activity is 100%, remaining enzyme activity is denoted as the pH value point multiplied by 100% divided by highest point enzyme activity according to the site enzyme activity lower than highest point Relative activity, the results are shown in Table 10.

Table 10: pH is reacted in the most suitable catalysis of different mutants

By table 10 as it can be seen that the effective pH value range of mutant amyMU1 catalysis reaction is 5.0-7.0, optimum pH range is 5.5-6.5；The effective pH value range of mutant amyMU2 catalysis reaction is 4.5-7.0, and optimum pH range is 5.0-6.0；It is prominent Variant amyMU2 ratio amyMU1 is more suitable for the reaction of the catalysis under acidic environment and effective pH value range is broad.

(3) Detection of Stability under mutant difference pH value

By mutant amyMU1 and amyMU2 (each reaction enzyme amount is 0.1U) pH different from 50mM's (4.5,5.0, 5.5,6.0) buffer 1:1 mix, and 25 DEG C of placements are sampled in different time intervals respectively, detected according to enzyme activity determination method, with The enzyme activity of zero moment calculates remaining enzyme activity under different pH standing times as 100%, detection mutant amyMU1 and The ph stability of amyMU2, the results are shown in Table 11.

Table 11: the stability of mutant at various ph values

By table 11 as it can be seen that stability of the mutant amyMU2 under acidic environment is significantly stronger than amyMU1, amyMU2 exists Placed under pH4.5 remain within 3 hours 50% or more catalytic activity, certain mutant amyMU1 and amyMU2 is in more partial neutral Its stability of environment is better, illustrates that a- amylase mutant of the invention all has good acid resistance.

(4) mutant catalysis reaction optimum temperature detection

Mutant amyMU1 and amyMU2 (each reaction enzyme amount is 0.05U) distinguish with pH 1% gelatinized starch for being 6.0 It is reacted under different temperatures (50-120 DEG C), relative activity is set as 100% with the highest temperature of enzyme activity, remaining enzyme activity is lower than Highest point is denoted as relative activity at this temperature multiplied by 100% divided by highest point enzyme activity according to the site enzyme activity, as a result sees Table 12.

Table 12: the most suitable catalytic reaction temperature of different mutants

Temperature DEG C	50	60	70	80	90	100	110	120
									amyMU1	52%	64%	80%	91%	100%	81%	45%	23%
amyMU2	63%	79%	86%	94%	100%	90%	87%	41%

By table 12 as it can be seen that the most suitable catalytic temperature of mutant amyMU1 and amyMU2 is 90 DEG C or so, mutant amyMU2 The effective temperature scope of catalysis reaction is 60-110 DEG C, and the effective temperature scope of mutant amyMU1 catalysis reaction is 70-100 ℃；Illustrate that a- amylase mutant of the invention belongs to high temperature enzyme, there is fine high-temperature catalytic performance；Wherein mutant amyMU2 Simultaneously also with very wide in range effective catalytic temperature, still there is 63% catalytic activity when temperature is 50 DEG C, and temperature At up to 110 DEG C with nearly 90% catalytic activity.

(5) Detection of Stability under mutant different temperatures

The crude enzyme liquid of mutant amyMU1 and amyMU2 20U/mL, amyMU1 and amyMU2 is adjusted to first respectively to take 1mL be put in respectively 90 DEG C, 100 DEG C, keep the temperature 180min in 110 DEG C of water-baths, sample 2.5 μ L in different time intervals respectively, according to Enzyme activity determination method detection, using the enzyme activity of zero moment as 100% calculate different temperatures standing time under remaining enzyme activity, inspection The temperature stability for surveying mutant amyMU1 and amyMU2, the results are shown in Table 13.

Table 13: the stability of mutant at different temperatures

By table 13 as it can be seen that mutant amyMU2 keeps the temperature the activity that 180min still has 50% or more at 90 DEG C, protected at 110 DEG C Warm 40min have 60% or more activity, show preferable high temperature resistance, compared to lower mutant amyMU1 temperature tolerance compared with Difference, 90 DEG C of heat preservation 60min just activity less than 50%.

According to the above-mentioned analysis to mutant amyMU1 and amyMU2 enzymatic property and stability, the present invention finally determines prominent Variant amyMU2 is the a- amylase mutant (SEQ ID NO:2) of the claims in the present invention, is named as amyMU, this hair The effective pH value range of bright a- amylase mutant amyMU catalysis reaction is 4.5-7.0, and optimum pH range is 5.0-6.0, Most suitable catalytic temperature is 90 DEG C or so, and effective temperature scope is 60-110 DEG C, and effective catalytic temperature range is very wide, can be simultaneously The catalytic function of medium temperature amylase and alpha-amylase is carried out, there is extraordinary acid resistance and thermal stability.

Embodiment 4

The present embodiment is used to illustrate the application of a- amylase mutant (amyMU) provided by the invention

(1) a- amylase mutant amyMU tests the liquefaction of various concentration corn starch milk and viscosity reducing effect

Test condition: to dry cornstarch as raw material, butt quality score difference is prepared in 60 DEG C of tank of sizing mixing It, will with 8M hydrochloric acid for the starch milk of 36wt%, 38wt%, 40wt%, 42wt%, 44wt%, 46wt%, 48wt%, 50wt% PH is adjusted to 5.5.The a- amylase mutant amyMU crude enzyme liquid of 20U/g starch is added into the tank of sizing mixing, 60 after mixing DEG C heat preservation 0.5h, then reaction system heating carries out injection liquefaction and obtains liquefier, and liquefier carries out DE value and viscosity detection, together When observation sample protein flocculate situation, the results are shown in Table 14.

Table 14: amyMU liquefaction effect under different cornstarch concentration

The results show that a- amylase mutant amyMU can be applied to the liquefaction process of various concentration starch milk, efficient liquefaction Concentration is 36-48wt%；It is up to 48wt% in starch concentration, a- amylase mutant amyMU of the invention remains to normal liquid Change, and reach the demand of liquefaction process, while protein coagulation is obvious, swims on liquefier, starch milk is viscous when high temperature Degree range of decrease effect is fairly obvious, and viscosity reducing effect reaches high concentration starch liquefying process condition, with No. 101 150mm diameter filter paper mistakes Filter, the rate of filtration are that every 4min filters out 42mL；Illustrate that a- amylase mutant amyMU of the invention is very suitable for high concentration shallow lake Powder liquefaction process uses.

(2) difference a- amylase is to high concentration starch liquefacation measure of merit

Test condition: a- amylase mutant amyMU of the present invention, Liquozyme Supre (being purchased from Novozymes), SuperLIQ 3.0T (this is outstanding purchased from Nanjing hundred), to dry cornstarch as raw material, prepares butt in 60 DEG C of tank of sizing mixing Mass fraction is respectively the starch milk of 38wt%, and pH is adjusted to 5.5 with 8M hydrochloric acid.20U/g is added into the tank of sizing mixing to form sediment The different a- amylase of powder, in 60 DEG C of heat preservation 0.5h after mixing, then reaction system heating carries out injection liquefaction and obtains liquefier, Liquefier carries out DE value and viscosity detection, while observing sample protein flocculation situation, the results are shown in Table 15.

Table 15: different a- amylase are to high concentration starch liquefacation effect

A- amylase	Mutant amyMU	Liquozyme Supre	SuperLIQ 3.0T
				DE value (%)	22.4	19.7	19.6
Viscosity (cP)	5342	12307	12048

As a result as it can be seen that under the conditions of identical liquefaction process, a- amylase mutant amyMU of the invention and current quotient Industry amylase liquefaction effect is suitable, and albumen coagulation is preferable, illustrates that a- amylase mutant amyMU of the invention can be answered Liquefaction process section for starch sugar industry；In addition, a- amylase mutant amyMU of the invention is in the liquefaction process condition Under, the viscosity reducing effect of starch milk is substantially better than commercial amylases act, illustrates that a- amylase mutant amyMU of the invention is more suitable for It is used in high concentration starch liquefying process.

(3) different liquefaction process are to high concentration starch liquefacation measure of merit

Test condition: (a) step liquefies: to dry cornstarch as raw material, preparing butt in 60 DEG C of tank of sizing mixing Mass fraction is respectively the starch milk of 44wt%, and pH is adjusted to 5.5 with 8M hydrochloric acid；20U/g is added into the tank of sizing mixing to form sediment The a- amylase mutant amyMU of powder, in 60 DEG C of heat preservation 0.5h after mixing, then reaction system heating spray liquefying and be obtained Liquefier, liquefier carry out DE value and viscosity detection, while observing sample protein flocculation situation.(b) substep liquefies: to dry jade Rice starch prepares the starch milk that butt quality score is respectively 32wt%, with 8M hydrochloric acid as raw material in 50 DEG C of tank of sizing mixing PH is adjusted to 6.0；The mesophilicα-diastase (purchased from this is outstanding purchased from Nanjing hundred) of 20U/g starch is added into the tank of sizing mixing, The first starch milk is obtained in 55 DEG C of heat preservation 1h after mixing；Into first starch milk, cornstarch is added in stirring, obtains butt Mass fraction is the second starch milk of 44wt%, and Xiang Suoshu sizes mixing, and the high-temperatureα-amylase of 20U/g starch is added in tank (SuperLIQ 3.0T be purchased from this is outstanding purchased from Nanjing hundred), in 55 DEG C of heat preservation 0.5h or more after mixing, then reaction system heat up into Row injection liquefaction obtains liquefier, and liquefier carries out DE value and viscosity detection, while observing sample protein flocculation situation.As a result see Table 16.

Table 16: different liquefaction process are to high concentration starch liquefacation effect

Liquefaction process	The liquefaction of one step	Substep liquefies
			Amylase	Mutant amyMU	Mesophilicα-diastase+SuperLIQ 3.0T
DE value (%)	19.4	20.3
			Viscosity (cP)	7120	7031

As a result as it can be seen that in the high concentration starch liquefacation of 44wt%, only with a- amylase mutant amyMU of the invention An a kind of step liquefaction process of enzyme can reach the substep liquefaction effect of mild alpha-amylase in use, and protein is wadded a quilt with cotton It is solidifying normal；Illustrate that a- amylase mutant amyMU of the invention is not only suitable for the use of high concentration starch liquefying process, while can In enforcement the effect of mild alpha-amylase, the liquefaction effect of mild alpha-amylase in being used in combination is reached in highly concentrated liquefaction Fruit, the working efficiency that the highly concentrated liquefaction process of a step can also be achieved simple process, reduce production cost, improve starch sugar industry.

The preferred embodiment of the present invention has been described above in detail, still, during present invention is not limited to the embodiments described above Detail within the scope of the technical concept of the present invention can be with various simple variants of the technical solution of the present invention are made, this A little simple variants all belong to the scope of protection of the present invention.

It is further to note that specific technical features described in the above specific embodiments, in not lance In the case where shield, can be combined in any appropriate way, in order to avoid unnecessary repetition, the present invention to it is various can No further explanation will be given for the combination of energy.

In addition, various embodiments of the present invention can be combined randomly, as long as it is without prejudice to originally The thought of invention, it should also be regarded as the disclosure of the present invention.

SEQUENCE LISTING

<110>Cofco Group Co., Ltd.

Jilin COFCO Biochemical Co., Ltd.

<120>application for the a- amylase mutant and its encoding gene and they of high concentration starch liquefacation

<130>fast I56030COF

<160> 19

<170> PatentIn version 3.3

<210> 1

<211> 1281

<212> DNA

<213>a- amylase mutant nucleotide sequence

<400> 1

atggctctta acaacggcac aatgatgcaa tacttcgaat ggtaccttcc taacgatggc 60

caacattgga aacgtcttca aaacgattct gcttaccttg ctgaacatgg catcacagct 120

gtttggatcc ctcctgctta caaaggcaca tctcaagctg atgtttacga tccttacgat 180

ttcttcgatc ttggcgaata ctaccaaaaa ggcacagttg aaacacgttt cggcacaaaa 240

gaacttcaag atgctatcgg ctctcttcat tctcgtaacg ttcaagttta cggcgatgtt 300

gttcttaacc ataaagctgg cgctgatgaa tggaaccctt tcgttggcga ttacacatgg 360

acagatttct ctggcgttgc ttctggcaaa tacacagcta actaccttga tttccatcct 420

aacgaagtta aatgctgcga tgaaggcaca ttcggcgatt accctgatat cgctcatgaa 480

aaatcttggg atcaacattg gctttgggct tctgatgaat cttacgctgc ttaccttcgt 540

tctatcggcg ttgatgcttg gcgtttcgat tacgttaaag gctacggcgc ttgggttatc 600

aaagattggc ttgattggtg gggcggctgg gctgttggcg aatactggga tacaaacgtt 660

gatgctcttc ttaactgggc ttacaactct aacgctaaag ttttcgattt ccctctttac 720

tacaaaatgg atgaagcttt cgataacaaa aacatccctg ctcttgttga tgctcttcgt 780

tacggccaaa cagttgtttc tcgtgatcct ttcaaagctg ttacattcgt tgctaaccat 840

gatacagata tcatctggaa caaataccct gcttacgctt tcatccttac acgtgaatct 900

ggctaccctc aagttttcta cggcgatatg tacggcacaa aaggcgattc tcaacgtgaa 960

atccctgctc ttaaacataa aatcgaacct atccttaaag ctcgtaaaga atacgcttac 1020

ggccctcaac atgattacat cgatcatcct gatgttatcg gctggacacg tgaaggcgat 1080

tcttctgctg ctaaatctgg ccttgctgct cttatcacag atggccctgg cggcgctaaa 1140

cgtatgtacg ttggccgtca aaacgctggc gaaacatggc atgatatcac aggcaaccgt 1200

tctgatacag ttacaatcac atctgatggc tggggcgaat tccatgttaa cgatggctct 1260

gtttctatct acgttcaaaa a 1281

<210> 2

<211> 427

<212> PRT

<213>a- amylase mutant amino acid sequence

<400> 2

Met Ala Leu Asn Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr Leu

1 5 10 15

Pro Asn Asp Gly Gln His Trp Lys Arg Leu Gln Asn Asp Ser Ala Tyr

20 25 30

Leu Ala Glu His Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys

35 40 45

Gly Thr Ser Gln Ala Asp Val Tyr Asp Pro Tyr Asp Phe Phe Asp Leu

50 55 60

Gly Glu Tyr Tyr Gln Lys Gly Thr Val Glu Thr Arg Phe Gly Thr Lys

65 70 75 80

Glu Leu Gln Asp Ala Ile Gly Ser Leu His Ser Arg Asn Val Gln Val

85 90 95

Tyr Gly Asp Val Val Leu Asn His Lys Ala Gly Ala Asp Glu Trp Asn

100 105 110

Pro Phe Val Gly Asp Tyr Thr Trp Thr Asp Phe Ser Gly Val Ala Ser

115 120 125

Gly Lys Tyr Thr Ala Asn Tyr Leu Asp Phe His Pro Asn Glu Val Lys

130 135 140

Cys Cys Asp Glu Gly Thr Phe Gly Asp Tyr Pro Asp Ile Ala His Glu

145 150 155 160

Lys Ser Trp Asp Gln His Trp Leu Trp Ala Ser Asp Glu Ser Tyr Ala

165 170 175

Ala Tyr Leu Arg Ser Ile Gly Val Asp Ala Trp Arg Phe Asp Tyr Val

180 185 190

Lys Gly Tyr Gly Ala Trp Val Ile Lys Asp Trp Leu Asp Trp Trp Gly

195 200 205

Gly Trp Ala Val Gly Glu Tyr Trp Asp Thr Asn Val Asp Ala Leu Leu

210 215 220

Asn Trp Ala Tyr Asn Ser Asn Ala Lys Val Phe Asp Phe Pro Leu Tyr

225 230 235 240

Tyr Lys Met Asp Glu Ala Phe Asp Asn Lys Asn Ile Pro Ala Leu Val

245 250 255

Asp Ala Leu Arg Tyr Gly Gln Thr Val Val Ser Arg Asp Pro Phe Lys

260 265 270

Ala Val Thr Phe Val Ala Asn His Asp Thr Asp Ile Ile Trp Asn Lys

275 280 285

Tyr Pro Ala Tyr Ala Phe Ile Leu Thr Arg Glu Ser Gly Tyr Pro Gln

290 295 300

Val Phe Tyr Gly Asp Met Tyr Gly Thr Lys Gly Asp Ser Gln Arg Glu

305 310 315 320

Ile Pro Ala Leu Lys His Lys Ile Glu Pro Ile Leu Lys Ala Arg Lys

325 330 335

Glu Tyr Ala Tyr Gly Pro Gln His Asp Tyr Ile Asp His Pro Asp Val

340 345 350

Ile Gly Trp Thr Arg Glu Gly Asp Ser Ser Ala Ala Lys Ser Gly Leu

355 360 365

Ala Ala Leu Ile Thr Asp Gly Pro Gly Gly Ala Lys Arg Met Tyr Val

370 375 380

Gly Arg Gln Asn Ala Gly Glu Thr Trp His Asp Ile Thr Gly Asn Arg

385 390 395 400

Ser Asp Thr Val Thr Ile Thr Ser Asp Gly Trp Gly Glu Phe His Val

405 410 415

Asn Asp Gly Ser Val Ser Ile Tyr Val Gln Lys

420 425

<210> 3

<211> 1581

<212> DNA

<213>B. stearothermophilus a- amylase nucleotide sequence

<400> 3

atgcttttct gccctacagg ccaacctgct aaagctgctg ctcctttcaa cggcacaatg 60

atgcaatact tcgaatggta ccttcctgat gatggcacac tttggacaaa agttgctaac 120

gaagctaaca acctttcttc tcttggcatc acagctcttt ggcttcctcc tgcttacaaa 180

ggcacatctc gttctgatgt tggctacggc gtttacgatc tttacgatct tggcgaattc 240

aaccaaaaag gcacagttcg tacaaaatac ggcacaaaag ctcaatacct tcaagctatc 300

caagctgctc atgctgctgg catgcaagtt tacgctgatg ttgttttcga tcataaaggc 360

ggcgctgatg gcacagaatg ggttgatgct gttgaagtta acccttctga tcgtaaccaa 420

gaaatctctg gcacatacca aatccaagct tggacaaaat tcgatttccc tggccgtggc 480

aacacatact cttctttcaa atggcgttgg taccatttcg atggcgttga ttgggatgaa 540

tctcgtaaac tttctcgtat ctacaaattc cgtggcatcg gcaaagcttg ggattgggaa 600

gttgatacag aaaacggcaa ctacgattac cttatgtacg ctgatcttga tatggatcat 660

cctgaagttg ttacagaact taaaaactgg ggcaaatggt acgttaacac aacaaacatc 720

gatggcttcc gtcttgatgc tgttaaacat atcaaattct ctttcttccc tgattggctt 780

tcttacgttc gttctcaaac aggcaaacct cttttcacag ttggcgaata ctggtcttac 840

gatatcaaca aacttcataa ctacatcaca aaaacaaacg gcacaatgtc tcttttcgat 900

gctcctcttc ataacaaatt ctacacagct tctaaatctg gcggcgcttt cgatatgcgt 960

acacttatga caaacacact tatgaaagat caacctacac ttgctgttac attcgttgat 1020

aaccatgata cagaacctgg ccaagctctt caatcttggg ttgatccttg gttcaaacct 1080

cttgcttacg ctttcatcct tacacgtcaa gaaggctacc ctggcgtttt ctacggcgat 1140

tactacggca tccctcaata caacatccct tctcttaaat ctaaaatcga tcctcttctt 1200

atcgctcgtc gtgattacgc ttacggcaca caacatgatt accttgatca ttctgatatc 1260

atcggctgga cacgtgaagg cgttacagaa aaacctggct ctggccttgc tgctcttatc 1320

acagatggcc ctggcggctc taaatggatg tacgttggca aacaacatgc tggcaaagtt 1380

ttctacgatc ttacaggcaa ccgttctgat acagttacaa tcacatctga tggctggggc 1440

gaattcaaag ttaacggcgg ctctgtttct gtttgggttc ctcgtaaaac aacagtttct 1500

acaatcacac gtcctatcac aacacgtcct tggacaggcg aattcgttcg ttggacagaa 1560

cctcgtcttg ttgcttggcc t 1581

<210> 4

<211> 526

<212> PRT

<213>B. stearothermophilus a- amylase amino acid sequence

<400> 4

Leu Phe Cys Pro Thr Gly Gln Pro Ala Lys Ala Ala Ala Pro Phe Asn

1 5 10 15

Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr Leu Pro Asp Asp Gly Thr

20 25 30

Leu Trp Thr Lys Val Ala Asn Glu Ala Asn Asn Leu Ser Ser Leu Gly

35 40 45

Ile Thr Ala Leu Trp Leu Pro Pro Ala Tyr Lys Gly Thr Ser Arg Ser

50 55 60

Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr Asp Leu Gly Glu Phe Asn

65 70 75 80

Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys Ala Gln Tyr Leu

85 90 95

Gln Ala Ile Gln Ala Ala His Ala Ala Gly Met Gln Val Tyr Ala Asp

100 105 110

Val Val Phe Asp His Lys Gly Gly Ala Asp Gly Thr Glu Trp Val Asp

115 120 125

Ala Val Glu Val Asn Pro Ser Asp Arg Asn Gln Glu Ile Ser Gly Thr

130 135 140

Tyr Gln Ile Gln Ala Trp Thr Lys Phe Asp Phe Pro Gly Arg Gly Asn

145 150 155 160

Thr Tyr Ser Ser Phe Lys Trp Arg Trp Tyr His Phe Asp Gly Val Asp

165 170 175

Trp Asp Glu Ser Arg Lys Leu Ser Arg Ile Tyr Lys Phe Arg Gly Ile

180 185 190

Gly Lys Ala Trp Asp Trp Glu Val Asp Thr Glu Asn Gly Asn Tyr Asp

195 200 205

Tyr Leu Met Tyr Ala Asp Leu Asp Met Asp His Pro Glu Val Val Thr

210 215 220

Glu Leu Lys Asn Trp Gly Lys Trp Tyr Val Asn Thr Thr Asn Ile Asp

225 230 235 240

Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Phe Ser Phe Phe Pro

245 250 255

Asp Trp Leu Ser Tyr Val Arg Ser Gln Thr Gly Lys Pro Leu Phe Thr

260 265 270

Val Gly Glu Tyr Trp Ser Tyr Asp Ile Asn Lys Leu His Asn Tyr Ile

275 280 285

Thr Lys Thr Asn Gly Thr Met Ser Leu Phe Asp Ala Pro Leu His Asn

290 295 300

Lys Phe Tyr Thr Ala Ser Lys Ser Gly Gly Ala Phe Asp Met Arg Thr

305 310 315 320

Leu Met Thr Asn Thr Leu Met Lys Asp Gln Pro Thr Leu Ala Val Thr

325 330 335

Phe Val Asp Asn His Asp Thr Glu Pro Gly Gln Ala Leu Gln Ser Trp

340 345 350

Val Asp Pro Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu Thr Arg

355 360 365

Gln Glu Gly Tyr Pro Gly Val Phe Tyr Gly Asp Tyr Tyr Gly Ile Pro

370 375 380

Gln Tyr Asn Ile Pro Ser Leu Lys Ser Lys Ile Asp Pro Leu Leu Ile

385 390 395 400

Ala Arg Arg Asp Tyr Ala Tyr Gly Thr Gln His Asp Tyr Leu Asp His

405 410 415

Ser Asp Ile Ile Gly Trp Thr Arg Glu Gly Val Thr Glu Lys Pro Gly

420 425 430

Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro Gly Gly Ser Lys Trp

435 440 445

Met Tyr Val Gly Lys Gln His Ala Gly Lys Val Phe Tyr Asp Leu Thr

450 455 460

Gly Asn Arg Ser Asp Thr Val Thr Ile Thr Ser Asp Gly Trp Gly Glu

465 470 475 480

Phe Lys Val Asn Gly Gly Ser Val Ser Val Trp Val Pro Arg Lys Thr

485 490 495

Thr Val Ser Thr Ile Thr Arg Pro Ile Thr Thr Arg Pro Trp Thr Gly

500 505 510

Glu Phe Val Arg Trp Thr Glu Pro Arg Leu Val Ala Trp Pro

515 520 525

<210> 5

<211> 1452

<212> DNA

<213>B. licheniformis a- amylase nucleotide sequence

<400> 5

gctgctaacc ttaacggcac acttatgcaa tacttcgaat ggtacatgcc taacgatggc 60

caacattgga aacgtcttca aaacgattct gcttaccttg ctgaacatgg catcacagct 120

gtttggatcc ctcctgctta caaaggcaca tctcaagctg atgttggcta cggcgcttac 180

gatctttacg atcttggcga attccatcaa aaaggcacag ttcgtacaaa atacggcaca 240

aaaggcgaac ttcaatctgc tatcaaatct cttcattctc gtgatatcaa cgtttacggc 300

gatgttgtta tcaaccataa aggcggcgct gatgctacag aagatgttac agctgttgaa 360

gttgatcctg ctgatcgtaa ccgtgttatc tctggcgaac atcttatcaa agcttggaca 420

catttccatt tccctggccg tggctctaca tactctgatt tcaaatggca ttggtaccat 480

ttcgatggca cagattggga tgaatctcgt aaacttaacc gtatctacaa attccaaggc 540

aaagcttggg attgggaagt ttctaacgaa aacggcaact acgattacct tatgtacgct 600

gatatcgatt acgatcatcc tgatgttgct gctgaaatca aacgttgggg cacatggtac 660

gctaacgaac ttcaacttga tggcttccgt cttgatgctg ttaaacatat caaattctct 720

ttccttcgtg attgggttaa ccatgttcgt gaaaaaacag gcaaagaaat gttcacagtt 780

gctgaatact ggcaaaacga tcttggcgct cttgaaaact accttaacaa aacaaacttc 840

aaccattctg ttttcgatgt tcctcttcat taccaattcc atgctgcttc tacacaaggc 900

ggcggctacg atatgcgtaa acttcttaac ggcacagttg tttctaaaca tcctcttaaa 960

tctgttacat tcgttgataa ccatgataca caacctggcc aatctcttga atctacagtt 1020

caaacatggt tcaaacctct tgcttacgct ttcatcctta cacgtgaatc tggctaccct 1080

caagttttct acggcgatat gtacggcaca aaaggcgatt ctcaacgtga aatccctgct 1140

cttaaacata aaatcgaacc tatccttaaa gctcgtaaac aatacgctta cggcgctcaa 1200

catgattact tcgatcatca tgatatcgtt ggctggacac gtgaaggcga ttcttctgtt 1260

gctaactctg gccttgctgc tcttatcaca gatggccctg gcggcgctaa acgtatgtac 1320

gttggccgtc aaaacgctgg cgaaacatgg catgatatca caggcaaccg ttctgaacct 1380

gttgttatca actctgaagg ctggggcgaa ttccatgtta acggcggctc tgtttctatc 1440

tacgttcaac gt 1452

<210> 6

<211> 484

<212> PRT

<213>B. licheniformis a- amylase amino acid sequence

<400> 6

Ala Ala Asn Leu Asn Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr Met

1 5 10 15

Pro Asn Asp Gly Gln His Trp Lys Arg Leu Gln Asn Asp Ser Ala Tyr

20 25 30

Leu Ala Glu His Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys

35 40 45

Gly Thr Ser Gln Ala Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp

50 55 60

Leu Gly Glu Phe His Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr

65 70 75 80

Lys Gly Glu Leu Gln Ser Ala Ile Lys Ser Leu His Ser Arg Asp Ile

85 90 95

Asn Val Tyr Gly Asp Val Val Ile Asn His Lys Gly Gly Ala Asp Ala

100 105 110

Thr Glu Asp Val Thr Ala Val Glu Val Asp Pro Ala Asp Arg Asn Arg

115 120 125

Val Ile Ser Gly Glu His Leu Ile Lys Ala Trp Thr His Phe His Phe

130 135 140

Pro Gly Arg Gly Ser Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His

145 150 155 160

Phe Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile Tyr

165 170 175

Lys Phe Gln Gly Lys Ala Trp Asp Trp Glu Val Ser Asn Glu Asn Gly

180 185 190

Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Tyr Asp His Pro Asp

195 200 205

Val Ala Ala Glu Ile Lys Arg Trp Gly Thr Trp Tyr Ala Asn Glu Leu

210 215 220

Gln Leu Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Phe Ser

225 230 235 240

Phe Leu Arg Asp Trp Val Asn His Val Arg Glu Lys Thr Gly Lys Glu

245 250 255

Met Phe Thr Val Ala Glu Tyr Trp Gln Asn Asp Leu Gly Ala Leu Glu

260 265 270

Asn Tyr Leu Asn Lys Thr Asn Phe Asn His Ser Val Phe Asp Val Pro

275 280 285

Leu His Tyr Gln Phe His Ala Ala Ser Thr Gln Gly Gly Gly Tyr Asp

290 295 300

Met Arg Lys Leu Leu Asn Gly Thr Val Val Ser Lys His Pro Leu Lys

305 310 315 320

Ser Val Thr Phe Val Asp Asn His Asp Thr Gln Pro Gly Gln Ser Leu

325 330 335

Glu Ser Thr Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile

340 345 350

Leu Thr Arg Glu Ser Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr

355 360 365

Gly Thr Lys Gly Asp Ser Gln Arg Glu Ile Pro Ala Leu Lys His Lys

370 375 380

Ile Glu Pro Ile Leu Lys Ala Arg Lys Gln Tyr Ala Tyr Gly Ala Gln

385 390 395 400

His Asp Tyr Phe Asp His His Asp Ile Val Gly Trp Thr Arg Glu Gly

405 410 415

Asp Ser Ser Val Ala Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly

420 425 430

Pro Gly Gly Ala Lys Arg Met Tyr Val Gly Arg Gln Asn Ala Gly Glu

435 440 445

Thr Trp His Asp Ile Thr Gly Asn Arg Ser Glu Pro Val Val Ile Asn

450 455 460

Ser Glu Gly Trp Gly Glu Phe His Val Asn Gly Gly Ser Val Ser Ile

465 470 475 480

Tyr Val Gln Arg

<210> 7

<211> 1308

<212> DNA

<213>Thermococcus siculi a- amylase nucleotide sequence

<400> 7

atggctaaat accttgaact tgaagaaggc ggcgttatca tgcaagcttt ctactgggat 60

gttcctggcg gcggcatctg gtgggataca atccgttcta aaatccctga ttggtacgat 120

gctggcatct ctgctatctg gatccctcct gcttctaaag gcatgtctgg cggctactct 180

atgggctacg atccttacga tttcttcgat cttggcgaat actaccaaaa aggcacagtt 240

gaaacacgtt tcggctctaa agaagaactt gttaacatga tcaacacagc tcatgcttac 300

ggcatcaaag ttatcgctga tatcgttatc aaccatcgtg ctggcggcga tcgtgaatgg 360

aaccctttcg ttggcgatta cacatggaca gatttctctg gcgttgcttc tggcaaatac 420

acagctaact accttgattt ccatcctaac gaagttaaat gctgcgatga aggcacattc 480

ggcgattacc ctgatatcgc tcatgaaaaa tcttgggatc aacattggct ttgggcttct 540

gatgaatctt acgctgctta ccttcgttct atcggcgttg atgcttggcg tttcgattac 600

gttaaaggct acggcgcttg ggttatcaaa gattggcttg attggtgggg cggctgggct 660

gttggcgaat actgggatac aaacgttgat gctcttctta actgggctta caactctaac 720

gctaaagttt tcgatttccc tctttactac aaaatggatg aagctttcga taacaaaaac 780

atccctgctc ttgttgatgc tcttcgttac ggccaaacag ttgtttctcg tgatcctttc 840

aaagctgtta cattcgttgc taaccatgat acagatatca tctggaacaa ataccctgct 900

tacgctttca tccttacata cgaaggccaa cctgttatct tctaccgtga ttacgaagaa 960

tggcttaaca aagatcgtct taaaaacctt atctggatcc atgatcatct tgctggcggc 1020

tctacatcta tcgtttacta cgattctgat gaacttatct tcgttcgtaa cggctacggc 1080

gatcgtcctg gccttatcac atacatcaac cttggctctt ctaaagctgg ccgttgggtt 1140

tacgttccta aattcgctgg ctcttgcatc catgaataca caggcaacct tggcggctgg 1200

gttgataaat gggttgattc tggcggctgg gtttaccttg aagctcctgc tcatgatcct 1260

gctaacggcc aatacggcta ctctgtttgg tcttactgcg gcgttggc 1308

<210> 8

<211> 435

<212> PRT

<213>Thermococcus siculi a- amylase amino acid sequence

<400> 8

Ala Lys Tyr Leu Glu Leu Glu Glu Gly Gly Val Ile Met Gln Ala Phe

1 5 10 15

Tyr Trp Asp Val Pro Gly Gly Gly Ile Trp Trp Asp Thr Ile Arg Ser

20 25 30

Lys Ile Pro Asp Trp Tyr Asp Ala Gly Ile Ser Ala Ile Trp Ile Pro

35 40 45

Pro Ala Ser Lys Gly Met Ser Gly Gly Tyr Ser Met Gly Tyr Asp Pro

50 55 60

Tyr Asp Phe Phe Asp Leu Gly Glu Tyr Tyr Gln Lys Gly Thr Val Glu

65 70 75 80

Thr Arg Phe Gly Ser Lys Glu Glu Leu Val Asn Met Ile Asn Thr Ala

85 90 95

His Ala Tyr Gly Ile Lys Val Ile Ala Asp Ile Val Ile Asn His Arg

100 105 110

Ala Gly Gly Asp Arg Glu Trp Asn Pro Phe Val Gly Asp Tyr Thr Trp

115 120 125

Thr Asp Phe Ser Gly Val Ala Ser Gly Lys Tyr Thr Ala Asn Tyr Leu

130 135 140

Asp Phe His Pro Asn Glu Val Lys Cys Cys Asp Glu Gly Thr Phe Gly

145 150 155 160

Asp Tyr Pro Asp Ile Ala His Glu Lys Ser Trp Asp Gln His Trp Leu

165 170 175

Trp Ala Ser Asp Glu Ser Tyr Ala Ala Tyr Leu Arg Ser Ile Gly Val

180 185 190

Asp Ala Trp Arg Phe Asp Tyr Val Lys Gly Tyr Gly Ala Trp Val Ile

195 200 205

Lys Asp Trp Leu Asp Trp Trp Gly Gly Trp Ala Val Gly Glu Tyr Trp

210 215 220

Asp Thr Asn Val Asp Ala Leu Leu Asn Trp Ala Tyr Asn Ser Asn Ala

225 230 235 240

Lys Val Phe Asp Phe Pro Leu Tyr Tyr Lys Met Asp Glu Ala Phe Asp

245 250 255

Asn Lys Asn Ile Pro Ala Leu Val Asp Ala Leu Arg Tyr Gly Gln Thr

260 265 270

Val Val Ser Arg Asp Pro Phe Lys Ala Val Thr Phe Val Ala Asn His

275 280 285

Asp Thr Asp Ile Ile Trp Asn Lys Tyr Pro Ala Tyr Ala Phe Ile Leu

290 295 300

Thr Tyr Glu Gly Gln Pro Val Ile Phe Tyr Arg Asp Tyr Glu Glu Trp

305 310 315 320

Leu Asn Lys Asp Arg Leu Lys Asn Leu Ile Trp Ile His Asp His Leu

325 330 335

Ala Gly Gly Ser Thr Ser Ile Val Tyr Tyr Asp Ser Asp Glu Leu Ile

340 345 350

Phe Val Arg Asn Gly Tyr Gly Asp Arg Pro Gly Leu Ile Thr Tyr Ile

355 360 365

Asn Leu Gly Ser Ser Lys Ala Gly Arg Trp Val Tyr Val Pro Lys Phe

370 375 380

Ala Gly Ser Cys Ile His Glu Tyr Thr Gly Asn Leu Gly Gly Trp Val

385 390 395 400

Asp Lys Trp Val Asp Ser Gly Gly Trp Val Tyr Leu Glu Ala Pro Ala

405 410 415

His Asp Pro Ala Asn Gly Gln Tyr Gly Tyr Ser Val Trp Ser Tyr Cys

420 425 430

Gly Val Gly

435

<210> 9

<211> 1452

<212> DNA

<213>B. amyloliquefaciens a- amylase nucleotide sequence

<400> 9

atggttaacg gcacacttat gcaatacttc gaatggtaca cacctaacga tggccaacat 60

tggaaacgtc ttcaaaacga tgctgaacat ctttctgata tcggcatcac agctgtttgg 120

atccctcctg cttacaaagg cctttctcaa tctgataacg gctacggccc ttacgatctt 180

tacgatcttg gcgaattcca acaaaaaggc acagttcgta caaaatacgg cacaaaatct 240

gaacttcaag atgctatcgg ctctcttcat tctcgtaacg ttcaagttta cggcgatgtt 300

gttcttaacc ataaagctgg cgctgatgct acagaagatg ttacagctgt tgaagttaac 360

cctgctaacc gtaaccaagt tacatctgaa gaataccaaa tcaaagcttg gacagatttc 420

cgtttccctg gccgtggcaa cacatactct gatttcaaat ggcattggta ccatttcgat 480

ggcgctgatt gggatgaatc tcgtaaaatc tctcgtatct tcaaattccg tggcgaaggc 540

aaagcttggg attgggaagt ttcttctgaa aacggcaact acgattacct tatgtacgct 600

gatgttgatt acgatcatcc tgatgttgtt gctgaaacaa aaaaatgggg catctggtac 660

gctaacgaac tttctcttga tggcttccgt atcgatgctg ctaaacatat caaattctct 720

ttccttcgtg attgggttca agctgttcgt caagctacag gcaaagaaat gttcacagtt 780

gctgaatact ggcaaaacaa cgctggcaaa cttgaaaact accttaacaa aacatctttc 840

aaccaatctg ttttcgatgt tcctcttcat ttcaaccttc aagctgcttc ttctcaaggc 900

ggcggctacg atatgcgtcg tcttcttgat ggcacagttg tttctcgtca tcctgaaaaa 960

gctgttacat tcgttgaaaa ccatgataca caacctggcc aatctcttga atctacagtt 1020

caaacatggt tcaaacctct tgcttacgct ttcatcctta cacgtgaatc tggctaccct 1080

caagttttct acggcgatat gtacggcaca aaaggcacat ctcctaaaga aatcccttct 1140

cttaaagata acatcgaacc tatccttaaa gctcgtaaag aatacgctta cggccctcaa 1200

catgattaca tcgatcatcc tgatgttatc ggctggacac gtgaaggcga ttcttctgct 1260

gctaaatctg gccttgctgc tcttatcaca gatggccctg gcggctctaa acgtatgtac 1320

gctggcctta aaaacgctgg cgaaacatgg tacgatatca caggcaaccg ttctgataca 1380

gttaaaatcg gctctgatgg ctggggcgaa ttccatgtta acgatggctc tgtttctatc 1440

tacgttcaaa aa 1452

<210> 10

<211> 483

<212> PRT

<213>B. amyloliquefaciens a- amylase amino acid sequence

<400> 10

Val Asn Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr Thr Pro Asn Asp

1 5 10 15

Gly Gln His Trp Lys Arg Leu Gln Asn Asp Ala Glu His Leu Ser Asp

20 25 30

Ile Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly Leu Ser

35 40 45

Gln Ser Asp Asn Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu Gly Glu

50 55 60

Phe Gln Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys Ser Glu

65 70 75 80

Leu Gln Asp Ala Ile Gly Ser Leu His Ser Arg Asn Val Gln Val Tyr

85 90 95

Gly Asp Val Val Leu Asn His Lys Ala Gly Ala Asp Ala Thr Glu Asp

100 105 110

Val Thr Ala Val Glu Val Asn Pro Ala Asn Arg Asn Gln Val Thr Ser

115 120 125

Glu Glu Tyr Gln Ile Lys Ala Trp Thr Asp Phe Arg Phe Pro Gly Arg

130 135 140

Gly Asn Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe Asp Gly

145 150 155 160

Ala Asp Trp Asp Glu Ser Arg Lys Ile Ser Arg Ile Phe Lys Phe Arg

165 170 175

Gly Glu Gly Lys Ala Trp Asp Trp Glu Val Ser Ser Glu Asn Gly Asn

180 185 190

Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Tyr Asp His Pro Asp Val

195 200 205

Val Ala Glu Thr Lys Lys Trp Gly Ile Trp Tyr Ala Asn Glu Leu Ser

210 215 220

Leu Asp Gly Phe Arg Ile Asp Ala Ala Lys His Ile Lys Phe Ser Phe

225 230 235 240

Leu Arg Asp Trp Val Gln Ala Val Arg Gln Ala Thr Gly Lys Glu Met

245 250 255

Phe Thr Val Ala Glu Tyr Trp Gln Asn Asn Ala Gly Lys Leu Glu Asn

260 265 270

Tyr Leu Asn Lys Thr Ser Phe Asn Gln Ser Val Phe Asp Val Pro Leu

275 280 285

His Phe Asn Leu Gln Ala Ala Ser Ser Gln Gly Gly Gly Tyr Asp Met

290 295 300

Arg Arg Leu Leu Asp Gly Thr Val Val Ser Arg His Pro Glu Lys Ala

305 310 315 320

Val Thr Phe Val Glu Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu

325 330 335

Ser Thr Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu

340 345 350

Thr Arg Glu Ser Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly

355 360 365

Thr Lys Gly Thr Ser Pro Lys Glu Ile Pro Ser Leu Lys Asp Asn Ile

370 375 380

Glu Pro Ile Leu Lys Ala Arg Lys Glu Tyr Ala Tyr Gly Pro Gln His

385 390 395 400

Asp Tyr Ile Asp His Pro Asp Val Ile Gly Trp Thr Arg Glu Gly Asp

405 410 415

Ser Ser Ala Ala Lys Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro

420 425 430

Gly Gly Ser Lys Arg Met Tyr Ala Gly Leu Lys Asn Ala Gly Glu Thr

435 440 445

Trp Tyr Asp Ile Thr Gly Asn Arg Ser Asp Thr Val Lys Ile Gly Ser

450 455 460

Asp Gly Trp Gly Glu Phe His Val Asn Asp Gly Ser Val Ser Ile Tyr

465 470 475 480

Val Gln Lys

<210> 11

<211> 5

<212> PRT

<213>Poly-Arg label

<400> 11

Arg Arg Arg Arg Arg

1 5

<210> 12

<211> 6

<212> PRT

<213>Poly-His label

<400> 12

His His His His His His

1 5

<210> 13

<211> 8

<212> PRT

<213>FLAG label

<400> 13

Asp Tyr Lys Asp Asp Asp Asp Lys

1 5

<210> 14

<211> 8

<212> PRT

<213>II label of Strep-tag

<400> 14

Trp Ser His Pro Gln Phe Glu Lys

1 5

<210> 15

<211> 10

<212> PRT

<213>c-myc label

<400> 15

Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu

1 5 10

<210> 16

<211> 24

<212> DNA

<213> M13-RV-F

<400> 16

gagcggataa caatttcaca cagg 24

<210> 17

<211> 24

<212> DNA

<213> M13-47-R

<400> 17

cgccagggtt ttcccagtca cgac 24

<210> 18

<211> 16

<212> DNA

<213> pNC-F

<400> 18

cgcttgcagg attcgg 16

<210> 19

<211> 23

<212> DNA

<213> pNC-D

<400> 19

caatgtaatt gttccctacc tgc 23

Claims (12)

1. a kind of a- amylase mutant, which is characterized in that the amino acid sequence of the a- amylase mutant such as SEQ ID Shown in NO:2.

2. the gene that one kind can encode a- amylase mutant described in claim 1.

3. gene according to claim 2, wherein the nucleotide sequence of the gene is as shown in SEQ ID NO:1.

4. a kind of recombinant expression carrier, which is characterized in that the recombinant expression carrier contains gene described in claim 2 or 3.

5. a kind of recombinant bacterial strain, which is characterized in that the recombinant bacterial strain contains recombinant expression carrier as claimed in claim 4.

6. recombinant bacterial strain according to claim 5, wherein the bacterial strain is prokaryotic cell.

7. recombinant bacterial strain according to claim 6, wherein the bacterial strain be Escherichia coli (Escherichia coli) or Bacillus subtilis (Bacillus subtilis).

8. a kind of method for preparing a- amylase mutant, which is characterized in that method includes the following steps:

(1) recombinant bacterial strain described in any one of claim 5-7 is cultivated, the gene table for encoding a- amylase mutant is made It reaches；

(2) a- amylase mutant expressed by separating-purifying.

9. a kind of enzyme preparation for high concentration starch liquefacation, which is characterized in that the enzyme preparation contains a- described in claim 1 Amylase mutant is as active constituent.

10. a- amylase mutant described in claim 1 perhaps gene described in claim 2 or 3 or claim 4 The recombinant expression carrier is perhaps described in recombinant bacterial strain or claim 9 described in any one of claim 5-7 High concentration starch liquefacation application of the enzyme preparation in high concentration starch liquefacation；

Wherein, the high concentration starch is starch milk of the solid content more than 36 weight %.

11. application according to claim 10, wherein the high concentration starch is the starch of solid content 36-65 weight % Cream.

12. application according to claim 10, wherein the high concentration starch is the starch of solid content 36-48 weight % Cream.