CN109022396A - The alpha-amylase mutant and its application that a kind of enzyme activity improves - Google Patents

The alpha-amylase mutant and its application that a kind of enzyme activity improves Download PDF

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CN109022396A
CN109022396A CN201811002140.7A CN201811002140A CN109022396A CN 109022396 A CN109022396 A CN 109022396A CN 201811002140 A CN201811002140 A CN 201811002140A CN 109022396 A CN109022396 A CN 109022396A
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amylase
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asp
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CN109022396B (en
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吴敬
宿玲恰
姚动邦
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Jiangnan University
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    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01001—Alpha-amylase (3.2.1.1)

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Abstract

The alpha-amylase mutant improved the invention discloses a kind of enzyme activity and its application, belong to genetic engineering and microbial engineering field.Mutant of the invention is by being glutamic acid by lysine mutation for the 82nd amino acids of the alpha-amylase that the amino acid sequence that sets out is SEQ ID NO.1 or amino acid sequence is that the 405th amino acids of the alpha-amylase of SEQ ID NO.1 are arginine by mutant serine or be simultaneously glutamic acid and the 405th amino acids by lysine mutation by the 82nd amino acids of the alpha-amylase that amino acid sequence is SEQ ID NO.1 by by mutant serine are what arginine obtained by；It will be purpose gene, using pHY300PLK as expression vector, the recombined bacillus subtilis engineering bacterium fermentation 48h constructed using bacillus subtilis (Bacillus subtilis) WS5 as expressive host using the gene for encoding mutant of the invention, the enzyme activity of alpha-amylase in fermentation liquid may make to improve to 474.7U/mL.

Description

The alpha-amylase mutant and its application that a kind of enzyme activity improves
Technical field
The alpha-amylase mutant improved the present invention relates to a kind of enzyme activity and its application, belong to genetic engineering and microorganism Field of engineering technology.
Background technique
Alpha-amylase (α-amylase, EC.3.2.1.1) is a kind of important glycoside hydrolase, with extensive substrate Preference and product specificities can cut off α-Isosorbide-5-Nitrae-glucoside bond in starch and related alpha-glucans molecule, and will Starch Hydrolysis be soluble dextrins, oligosaccharide and maltose and glucose, meanwhile, the alpha-isomer structure of product can be retained As.
Therefore, alpha-amylase is widely used in the industries such as food, washing, papermaking, weaving, alcohol and medicine.
According to the difference of operative temperature, alpha-amylase can also be divided into high temperature, in mild low temperature alpha-amylase.Wherein, high temperature Alpha-amylase has good thermal stability, and from a wealth of sources, can extract and obtain from plant, animal and microorganism.Plant, In animal and microbe-derived alpha-amylase, microbe-derived high-temperatureα-amylase production cost, fermenting stability and There is more obvious advantage in production time etc. than other sources；And in microbe-derived high-temperatureα-amylase, carefully The high-temperatureα-amylase thermal stability advantage in bacterium source becomes apparent.
Therefore, there is huge application prospect from the high-temperatureα-amylase of bacterium.
Currently, having there is high-temperatureα-amylase of many literature research from different bacterium in Escherichia coli or withered grass In the bacteriums such as bacillus the case where heterogenous expression.
Expression quantity of the high-temperatureα-amylase in Escherichia coli is than high in bacillus subtilis, but due to Escherichia coli The harmful substances such as endotoxin can be generated during carrying out enzymatic production, it is unfavorable to human body, greatly limit its application；And it is withered The cell wall of careless bacillus is free of endotoxin, is a kind of nonpathogenic edaphon, by Food and Drug Administration It is regarded as aliment security level bacterial strain GRAS (Generally recognized as safe) with Chinese relevant departments, but high temperature α- The amylase expression quantity of heterogenous expression and activity in bacillus subtilis is very low.
Drawbacks described above makes the high-temperatureα-amylase from bacterium industrially be difficult to be widely used.
Summary of the invention
To solve the above problems, the present invention provides a kind of alpha-amylase mutant of enzyme activity raising and its applications.This is prominent Variant is by being by lysine mutation by the 82nd amino acids of the alpha-amylase that the amino acid sequence that sets out is SEQ ID NO.1 405th amino acids of the alpha-amylase that the amino acid sequence that sets out is SEQ ID NO.1 are by glutamic acid by mutant serine Arginine or 82nd amino acids of the amino acid sequence for the alpha-amylase of SEQ ID NO.1 that will will set out simultaneously are dashed forward by lysine Becoming glutamic acid and the 405th amino acids by mutant serine is what arginine obtained；By the gene to encode this mutant It is expression place as expression vector, with bacillus subtilis (Bacillus subtilis) WS5 for purpose gene, using pHY300PLK It is main to construct obtained recombined bacillus subtilis engineering bacterium fermentation 48h, may make the enzyme activity of alpha-amylase in fermentation liquid improve to 474.7U/mL。
Technical scheme is as follows:
The present invention provides the alpha-amylase mutants that a kind of enzyme activity improves, and the enzyme mutant is by the amino that will set out Acid sequence is the 82nd amino acids of the alpha-amylase of SEQ ID NO.1 and/or the 405th amino acids are mutated.
In one embodiment of the invention, it is SEQ ID that the enzyme mutant, which is by the amino acid sequence that will set out, 82nd amino acids of the alpha-amylase of NO.1 are what glutamic acid obtained by lysine mutation；
Or the enzyme mutant is by will set out that amino acid sequence is SEQ ID NO.1 the 405th of alpha-amylase Amino acid is what arginine obtained by mutant serine；
Or the enzyme mutant be by simultaneously by set out amino acid sequence be SEQ ID NO.1 alpha-amylase the 82nd It by mutant serine is what arginine obtained that amino acids, which are glutamic acid and the 405th amino acids by lysine mutation,.
In one embodiment of the invention, the alpha-amylase is high-temperatureα-amylase.
In one embodiment of the invention, the source of the alpha-amylase is bacillus stearothermophilus (Bacillus stearothermophilus)。
In one embodiment of the invention, the amino acid sequence of the alpha-amylase mutant be SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4.
The present invention provides the genes for encoding the alpha-amylase mutant that a kind of above-mentioned enzyme activity improves.
The present invention provides the recombinant plasmids for carrying said gene.
In one embodiment of the invention, the construction method of the recombinant plasmid is first according to determining mutation position Point designs the mutant primer of rite-directed mutagenesis, further according to the mutant primer of design, to carry the carrier of alpha-amylase gene as template Carry out rite-directed mutagenesis.
In one embodiment of the invention, in the mutant primer, the nucleotides sequence of forward primer is classified as SEQ ID NO.5 or SEQ ID NO.7, the nucleotides sequence of reverse primer are classified as SEQ ID NO.6 or SEQ ID NO.8.
In one embodiment of the invention, the recombinant plasmid vector is pHY300PLK.
The present invention provides the host cells for carrying said gene or above-mentioned recombinant plasmid.
In one embodiment of the invention, the host cell is bacillus subtilis (Bacillus subtilis)WS5。
Bacillus subtilis (Bacillus subtilis) WS5 is recorded in the special of Publication No. CN106754466A In sharp text.
The alpha-amylase mutant or said gene that are improved the present invention provides a kind of above-mentioned enzyme activity or above-mentioned recombinant plasmid Or above-mentioned host cell answering in terms of hydrolysis starch, preparing dextrin, preparing oligosaccharide, prepare maltose and prepare glucose With.
The utility model has the advantages that
(1) present invention by will set out amino acid sequence be SEQ ID NO.1 alpha-amylase the 82nd amino acids by Lysine mutation be glutamic acid or by the 405th amino acids of the alpha-amylase that the amino acid sequence that sets out is SEQ ID NO.1 by Mutant serine is arginine or simultaneously by the 82nd bit amino of the alpha-amylase that the amino acid sequence that will set out is SEQ ID NO.1 Acid is glutamic acid and the 405th amino acids by the α-that mutant serine is that arginine has obtained enzyme activity raising by lysine mutation Amylase mutant；
(2) by using the gene for encoding mutant of the invention be purpose gene, using pHY300PLK as expression vector, with withered Careless bacillus (Bacillus subtilis) WS5 is the recombined bacillus subtilis engineering bacteria hair that expressive host constructs Ferment 48h may make the enzyme activity of alpha-amylase in fermentation liquid to improve to 474.7U/mL.
Detailed description of the invention
Fig. 1 is recombinant plasmid pHY-SPamyE- amyS (K82E/S405R) constructs flow chart；
Fig. 2 is recombinant plasmid pHY-SPamyEThe QuickCut of-amyS (K82E/S405R)TMHind III digestion verifying knot Fruit；
Wherein, M is DL1000DNA Marker, and swimming lane 1 is pHY-SPamyE-amyS QuickCutTMHind III digestion Band；
Fig. 3 is to contain recombinant plasmid pHY-SPamyEThe recombined bacillus subtilis shake flask fermentation of-amyS (K82E/S405R) SDS-PAGE electrophoresis；
Wherein, M is middle-molecular-weihydroxyethyl standard protein, and swimming lane 1 is Bacillus subtilis WS5/pHY-SPamyE-amyS (K82E/S405R) shake flask fermentation supernatant band.
Specific embodiment
The embodiment of the present invention is only used as the further explanation of the content of present invention, it is not as a limitation of the present invention in perhaps Range.
Detection method involved in following embodiments is as follows:
Enzyme activity assay method: by the phosphoric acid buffer of the 1% of 1mL soluble starch solution and 20mM, pH6.0 of 0.9mL Liquid mixes well, and in 70 DEG C of preheating 10min, 0.1mL crude enzyme liquid is added, and oscillation mixes, and 3mL DNS, vibration is added after reacting 5min Swing, boil 7min and cool down rapidly, distilled water is added to be settled to 15ml, surveyed under 540nm absorbance (using the enzyme solution of inactivation as catalyst into The same operation of row is used as blank).
Enzyme-activity unit definition: under the above conditions, enzyme amount needed for catalysis generation per minute is equivalent to 1 μm of ol glucose is defined For a Starch Hydrolysis unit of activity.
Culture medium involved in following embodiments is as follows:
Seed culture medium: the sodium chloride of the peptone of 8~12g/L, the yeast powder of 4~6g/L and 8~12g/L.
Fermentation medium: the glycerol of the yeast extract of 20~25g/L, the soy peptone of 5~10g/L and 4~6g/L, Initial pH is 6~7.
LB solid medium: 10g/L peptone, the yeast extract of 5g/L, 10g/L NaCl, 0.2g/L agar powder.
LB liquid medium: 10g/L peptone, the yeast extract of 5g/L, 10g/L NaCl.
Growth medium: 10g/L peptone, the yeast extract of 5g/L, 10g/L NaCl, 0.5M sorbierite.
Electricity turns buffer: the sorbierite of 0.5M, the mannitol of 0.5M, 10% glucose.
The sorbierite of recovery medium RM:0.5M, the mannitol of 0.38M, 10g/L peptone, the yeast extract of 5g/L, 10g/ The NaCl of L.
Alpha-amylase involved in following embodiments is as follows:
The source of the alpha-amylase is bacillus stearothermophilus (Bacillus stearothermophilus), ammonia Base acid sequence is as shown in SEQ ID NO.1.
In the following, the 82nd lysine (K) of amino acid sequence alpha-amylase as shown in SEQ ID NO.1 is sported Glutamic acid (E) resulting mutant is named as K82E；By the of amino acid sequence alpha-amylase as shown in SEQ ID NO.1 405 serines (S) sport arginine (R) resulting mutant and are named as S405R；By amino acid sequence such as SEQ ID 82nd lysine (K) of alpha-amylase shown in NO.1 sports glutamic acid (E), while by the 82nd lysine (K) It sports glutamic acid (E) resulting mutant and is named as K82E/S405R.
Embodiment 1: the preparation of alpha-amylase mutant
(1) preparation of alpha-amylase single mutant
According to amino acid sequence be SEQ ID NO.1 shown in alpha-amylase gene order, separately design and synthesize and draw The primer for entering K82E, S405R mutation is carried using fast PCR technology with carrying the recombination of gene of encoding wild type alpha-amylase Body pET20b-amyS template carries out rite-directed mutagenesis to alpha-amylase gene, measures DNA encoding sequence, identify the 82nd Lys codon becomes Glu codon, and the 405th Ser codon becomes Arg codon, obtain single mutation alpha-amylase；
Wherein, recombinant vector pET20b-amyS is that laboratory saves (Ref:L Z, D X, W J.Improving the thermostability and enhancing the Ca2+binding of the maltohexaose-formingα- amylase from Bacillus stearothermophilus.Journal of Biotechnology.2016；222: 65–72.)。
Introduce the rite-directed mutagenesis primer of K82E mutation are as follows:
Nucleotides sequence is classified as the forward primer of SEQ ID NO.5: 5 '-CAAGTACGGCACCGAGGCCCAGTAC-3 ' (under It is marked as mutating alkali yl)
Nucleotides sequence is classified as the reverse primer of SEQ ID NO.6: 5 '-GTACTGGGCCTCGGTGCCGTACTTG-3 ' (under It is marked as mutating alkali yl)
Introduce the rite-directed mutagenesis primer of S405R mutation are as follows:
Nucleotides sequence is classified as the forward primer of SEQ ID NO.7: 5 '-CTATCTGGACCACAGAGACATCATTGGCTGG- 3 ' (underscore is mutating alkali yl)
Nucleotides sequence is classified as the reverse primer of SEQ ID NO.8: 5 '-GGTCCAGCCAATGATGTCTCTGTGGTCCAGA- 3 ' (underscore is mutating alkali yl)
PCR system is shown in Table 1:
1 PCR reaction system of table
5xPhusion HF Reaction Buffer 10.0μL
dNTP 4.0μL
pET20b-amyS 0.5μL
Forward primer 0.5μL
Reverse primer 0.5μL
Primerstar DNA 0.5μL
ddH2O Up to 50μL
PCR condition: 94 DEG C of initial denaturation 4min；98 DEG C of denaturation 10s, 55 DEG C of annealing 5s, 72 DEG C of extension 1kb/min, 30 are followed Ring, 72 DEG C of extension 10min, 4 DEG C of heat preservations.
PCR product is detected with 1% agarose gel electrophoresis.
Correct PCR product will be verified to digest through Dpn I, convert e. coli jm109 competence, competent cell is in LB After solid medium (containing 100 μ g/mL ampicillins) overnight incubation, chooses and be cloned in LB liquid medium (containing 100 μ g/mL ammonia Parasiticin) in culture after extract plasmid, all mutant plasmids are sequenced correctly, and obtained recombinant bacterium is respectively designated as E.coli JM109/pET20b-amyS(K82E)、E.coli JM109/pET20b-amyS(S405)。
Correct mutant is sequenced, is seeded to LB culture medium from glycerol tube, is incubated overnight, extracts plasmid, plasmid is converted Expressive host e. coli bl21 (DE3) competent cell, obtained recombinant bacterium be respectively designated as E.coli BL21 (DE3)/ pET20b-amyS(K82E)、E.coli BL21(DE3)/pET20b-amyS(S405)。
(2) preparation of alpha-amylase double-mutant
Using single mutant K82E encoding gene obtained in (1) as template, designs and synthesizes and introduce drawing for S405R mutation Object, using fast PCR technology, the recombinant vector pET20b-amyS (K82E) with the gene of carrying coding single mutant K82E is Template carries out rite-directed mutagenesis to alpha-amylase gene, measures DNA encoding sequence, identify the 405th Ser codon and become Arg codon obtains the double-mutant of alpha-amylase.
Introduce rite-directed mutagenesis primer, the PCR reaction system, the measuring method of reaction condition and mutated gene of S405R mutation With (1).
Correct PCR product will be verified to digest through Dpn I, convert e. coli jm109 competence, competent cell is in LB After solid medium (containing 100 μ g/mL ampicillins) overnight incubation, chooses and be cloned in LB liquid medium (containing 100 μ g/mL ammonia Parasiticin) in culture after extract plasmid, all mutant plasmids are sequenced correctly, and obtained recombinant bacterium is named as E.coli JM109/pET20b-amyS(K82E/S405R)。
Correct mutant is sequenced, is seeded to LB culture medium from glycerol tube, is incubated overnight, extracts plasmid, plasmid is converted Expressive host e. coli bl21 (DE3) competent cell, obtained recombinant bacterium be named as E.coli BL21 (DE3)/ pET20b-amyS(K82E/S405R)。
Embodiment 2: alpha-amylase mutant shake flask fermentation producing enzyme in large intestine bacillus is verified
(1) alpha-amylase mutant shake flask fermentation producing enzyme in large intestine bacillus
Pick them separately recombinant bacterium E.coli BL21 (DE3)/pET20b-amyS (K82E), E.coli BL21 (DE3)/ PET20b-amyS (S405), E.coli BL21 (DE3)/pET20b-amyS (K82E/S405R) are trained at 37 DEG C in LB liquid It supports in base (containing 100 μ g/mL ampicillins) and grows 8~10h, seed fermentation liquid is connected to fermentation medium by 5% inoculum concentration In (containing 100 μ g/mL ampicillins), after cultivating 48h in 25 DEG C of shaking tables, fermentation liquid is centrifuged 10min in 4 DEG C, 8000rpm Except thallus, collects centrifuged supernatant and obtain crude enzyme liquid.
(2) alpha-amylase mutant is measured in large intestine bacillus shake-flask fermentation enzyme activity
Alpha-amylase mutant K82E, S405R, K82E/S405R is measured to send out in large intestine bacillus BL21 (DE3) shaking flask The shaking flask culture 48h enzyme activity of ferment enzyme activity, alpha-amylase single mutation and double-mutant enzyme is listed in Table 2 below, wherein double-mutant The enzyme activity of K82E/S405R is 2.1 times of wild type enzyme activity, and the enzyme activity of single mutant S405R is 1.9 times of wild type enzyme activity, and The enzyme activity of single mutant K82E is only 1.2 times of wild type enzyme activity.
The enzyme activity of the wild enzyme of 2 alpha-amylase of table and mutant
Enzyme Wild mushroom K82E S405R K82E/S405R
Enzyme activity/UmL-1 1208.3 1450.0 2295.8 2537.4
Embodiment 3: building recombinant vector pHY-SPamyE-amyS、pHY-SPamyE-amyS(K82E/S405R)
Specific step is as follows (building process is shown in Fig. 1):
(1) primer amyS-F, amyS-R containing homology arm are designed, with recombinant vector pET20b-amyS, pET20b- AmyS (K82E/S405R) is template, and PCR amplification goes out to have amyS, amyS (K82E/S405R) of homology arm
(2) design primer pHY-F, pHY-R, using recombinant vector pHY300PLK- β-CGTase as template, PCR amplification goes out to carry Body pHY-SPamyE, wherein recombinant vector pHY300PLK- β-CGTase be laboratory save (Ref:Zhang, K., Duan, X., Wu,J.2016.Multigene disruption in undomesticated Bacillus subtilis ATCC 6051a using the CRISPR/Cas9system.Scientific Reports,6.Bacillus subtilis)。
Primer sequence is shown in Table 3:
3 primer sequence of table
Note: dashed part is homology arm sequence.
PCR system is shown in Table 4:
4 PCR reaction system of table
PCR condition: 94 DEG C of initial denaturation 4min；98 DEG C of denaturation 10s, 55 DEG C of annealing 5s, 72 DEG C of extension 1kb/min, 30 are followed Ring, PCR product carry out glue recycling.
(3) by the two sections of segments for amplifying and having recycled in (1), (2) according toH Cloning Kit reagent Box requires to be that 2:1 is mixed according to the molar ratio of Insert Fragment and carrier, will after being attached using following linked system Connection product is transferred to E.coli JM109 competent cell, is incubated overnight after coated plate in 37 DEG C；
Linked system is shown in Table 5:
Table 5HD Cloning Kit kit linked system
System component Amounts of components
5X In-Fusion HD Enzyme Premix 2.0μL
pHY-SPamyE 1.2μL
amyS/amyS(K82E/S405R) 3.5μL
ddH2O 3.3μL
Condition of contact: 50 DEG C, 25min.
(4) single colonie is chosen from the plate being incubated overnight, connects LB liquid medium in 37 DEG C, 8~10h is cultivated under 200rpm Upgrading grain carries out QuickCut afterwardsTMHind III digestion verifies (verification result is shown in Fig. 2), and digestion verification success carries up to recombination Body pHY-SPamyE-amyS、pHY-SPamyE-amyS(K82E/S405R)。
Digestion system is shown in Table 6:
6 QuickCut of tableTMHind III digestion system
Digestion condition: 37 DEG C of endonuclease reaction 20min.
Embodiment 4: the bacillus subtilis recombinant bacterium containing alpha-amylase wild type and mutant respectively is constructed
Bacillus subtilis (Bacillus subtilis) WS5 is chosen as host cell, constructs recombinant bacterium, it is specific to walk It is rapid as follows:
(1) bacillus subtilis (Bacillus subtilis) WS5 frozen is picked with oese, then in LB plate Upper scribing line, 37 DEG C of overnight incubation activation；
(2) it is inoculated in 5ml LB liquid medium from the single colonie for choosing step (1) on LB plate, 37 DEG C, 200rpm mistake Night culture；
(3) 2.5mL is taken to transfer into 40mL growth medium, 37 DEG C, 4~5h of 200rpm shake culture；
(4) by bacterium solution ice bath 10min, then 5000g, is centrifuged 5min and collects thallus by 4 DEG C；
(5) turn buffer washing thalline with the electricity that 50ml is pre-chilled, 5000g, 4 DEG C, centrifugation 5min removes supernatant, so rinses 4 It is secondary；
(6) thallus after washing is resuspended in 1mL electricity to turn in buffer, is dispensed into 1.5mL EP pipe, every pipe fills 200 μ l Competent cell；
(7) the resulting recombinant plasmid of 10 μ L embodiment 3 will be separately added into 200 μ L competent cells, ice bath 18min adds Enter in the electric revolving cup (2mm) of pre-cooling, electric shock is primary, electroporation setting: 2.4kv, 25 μ F, 200 Ω；
(8) the slow pressure-vaccum of 1mL recovery medium RM is added after shocking by electricity immediately to mix, 37 DEG C, 200rpm, recovery 3h Afterwards, the plate for containing tetracyclin resistance (50ug/mL) is applied, positive transformant is selected and obtains containing recombinant vector pHY- respectively SPamyE-amyS、pHY-SPamyEBacillus subtilis (Bacillus subtilis) WS5 of-amyS (K82E/S405R).
Embodiment 5: the measurement of bacillus subtilis WS5 recombinant bacterium shake flask fermentation producing enzyme and alpha-amylase enzyme activity
By recombined bacillus subtilis strain inoculated obtained in embodiment 4 in 35~38 DEG C, 180 in seed culture medium 8~10h is cultivated under the conditions of~220rpm, obtains seed liquor, then seed liquor is forwarded in fermentation medium, in fermented and cultured 45~50h is cultivated in base under 30~37 DEG C, 180~220rpm, after fermentation, it is crude enzyme liquid that supernatant, which is collected by centrifugation,； (contain recombinant plasmid pHY-SPamyEThe SDS- of the recombined bacillus subtilis shake flask fermentation supernatant of-amyS (K82E/S405R) PAGE electrophoresis result is shown in Fig. 3)
Obtained crude enzyme liquid is subjected to Enzyme activity assay, testing result are as follows: contain recombinant vector pHY-SPamyE-amyS、pHY- SPamyEThe enzyme activity of bacillus subtilis (Bacillus subtilis) WS5 of-amyS (K82E/S405R) is respectively 206.4U/ mL、474.7U/mL。
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention Enclosing subject to the definition of the claims.
Sequence table
<110>Southern Yangtze University
<120>alpha-amylase mutant and its application that a kind of enzyme activity improves
<160> 12
<170> PatentIn version 3.3
<210> 1
<211> 482
<212> PRT
<213>artificial sequence
<400> 1
Met Ala Ala Pro Phe Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr
1 5 10 15
Leu Pro Asp Asp Gly Thr Leu Trp Thr Lys Val Ala Asn Glu Ala Asn
20 25 30
Asn Leu Ser Ser Leu Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala Tyr
35 40 45
Lys Gly Thr Ser Arg Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr
50 55 60
Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly
65 70 75 80
Thr Lys Ala Gln Tyr Leu Gln Ala Ile Gln Ala Ala His Ala Ala Gly
85 90 95
Met Gln Val Tyr Ala Asp Val Val Phe Asp His Lys Gly Gly Ala Asp
100 105 110
Gly Thr Glu Trp Val Asp Ala Val Glu Val Asn Pro Ser Asp Arg Asn
115 120 125
Gln Glu Ile Ser Gly Thr Tyr Gln Ile Gln Ala Trp Thr Lys Phe Asp
130 135 140
Phe Pro Gly Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp Tyr
145 150 155 160
His Phe Asp Gly Val Asp Trp Asp Glu Ser Arg Lys Leu Ser Arg Ile
165 170 175
Tyr Lys Phe Arg Gly Lys Ala Trp Asp Trp Glu Val Asp Thr Glu Phe
180 185 190
Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Leu Asp Met Asp His Pro
195 200 205
Glu Val Val Thr Glu Leu Lys Asn Trp Gly Lys Trp Tyr Val Asn Thr
210 215 220
Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Phe
225 230 235 240
Ser Phe Phe Pro Asp Trp Leu Ser Tyr Val Arg Ser Gln Thr Gly Lys
245 250 255
Pro Leu Phe Thr Val Gly Glu Tyr Trp Ser Tyr Asp Ile Asn Lys Leu
260 265 270
His Asn Tyr Ile Thr Lys Thr Asp Gly Thr Met Ser Leu Phe Asp Ala
275 280 285
Pro Leu His Asn Lys Phe Tyr Thr Ala Ser Lys Ser Gly Gly Ala Phe
290 295 300
Asp Met Arg Thr Leu Met Thr Asn Thr Leu Met Lys Asp Gln Pro Thr
305 310 315 320
Leu Ala Val Thr Phe Val Asp Asn His Asp Thr Glu Pro Val Gln Ala
325 330 335
Leu Gln Ser Trp Val Asp Pro Trp Phe Lys Pro Leu Ala Tyr Ala Phe
340 345 350
Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr Gly Asp Tyr
355 360 365
Tyr Gly Ile Pro Gln Tyr Asn Ile Pro Ser Leu Lys Ser Lys Ile Asp
370 375 380
Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly Thr Gln His Asp
385 390 395 400
Tyr Leu Asp His Ser Asp Ile Ile Gly Trp Thr Arg Glu Gly Gly Thr
405 410 415
Glu Lys Pro Gly Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro Gly
420 425 430
Gly Ser Lys Trp Met Tyr Val Gly Lys Gln His Ala Gly Lys Val Phe
435 440 445
Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile Asn Ser Asp
450 455 460
Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser Val Trp Val
465 470 475 480
Pro Arg
<210> 2
<211> 482
<212> PRT
<213>artificial sequence
<400> 2
Met Ala Ala Pro Phe Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr
1 5 10 15
Leu Pro Asp Asp Gly Thr Leu Trp Thr Lys Val Ala Asn Glu Ala Asn
20 25 30
Asn Leu Ser Ser Leu Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala Tyr
35 40 45
Lys Gly Thr Ser Arg Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr
50 55 60
Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly
65 70 75 80
Thr Glu Ala Gln Tyr Leu Gln Ala Ile Gln Ala Ala His Ala Ala Gly
85 90 95
Met Gln Val Tyr Ala Asp Val Val Phe Asp His Lys Gly Gly Ala Asp
100 105 110
Gly Thr Glu Trp Val Asp Ala Val Glu Val Asn Pro Ser Asp Arg Asn
115 120 125
Gln Glu Ile Ser Gly Thr Tyr Gln Ile Gln Ala Trp Thr Lys Phe Asp
130 135 140
Phe Pro Gly Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp Tyr
145 150 155 160
His Phe Asp Gly Val Asp Trp Asp Glu Ser Arg Lys Leu Ser Arg Ile
165 170 175
Tyr Lys Phe Arg Gly Lys Ala Trp Asp Trp Glu Val Asp Thr Glu Phe
180 185 190
Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Leu Asp Met Asp His Pro
195 200 205
Glu Val Val Thr Glu Leu Lys Asn Trp Gly Lys Trp Tyr Val Asn Thr
210 215 220
Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Phe
225 230 235 240
Ser Phe Phe Pro Asp Trp Leu Ser Tyr Val Arg Ser Gln Thr Gly Lys
245 250 255
Pro Leu Phe Thr Val Gly Glu Tyr Trp Ser Tyr Asp Ile Asn Lys Leu
260 265 270
His Asn Tyr Ile Thr Lys Thr Asp Gly Thr Met Ser Leu Phe Asp Ala
275 280 285
Pro Leu His Asn Lys Phe Tyr Thr Ala Ser Lys Ser Gly Gly Ala Phe
290 295 300
Asp Met Arg Thr Leu Met Thr Asn Thr Leu Met Lys Asp Gln Pro Thr
305 310 315 320
Leu Ala Val Thr Phe Val Asp Asn His Asp Thr Glu Pro Val Gln Ala
325 330 335
Leu Gln Ser Trp Val Asp Pro Trp Phe Lys Pro Leu Ala Tyr Ala Phe
340 345 350
Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr Gly Asp Tyr
355 360 365
Tyr Gly Ile Pro Gln Tyr Asn Ile Pro Ser Leu Lys Ser Lys Ile Asp
370 375 380
Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly Thr Gln His Asp
385 390 395 400
Tyr Leu Asp His Ser Asp Ile Ile Gly Trp Thr Arg Glu Gly Gly Thr
405 410 415
Glu Lys Pro Gly Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro Gly
420 425 430
Gly Ser Lys Trp Met Tyr Val Gly Lys Gln His Ala Gly Lys Val Phe
435 440 445
Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile Asn Ser Asp
450 455 460
Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser Val Trp Val
465 470 475 480
Pro Arg
<210> 3
<211> 482
<212> PRT
<213>artificial sequence
<400> 3
Met Ala Ala Pro Phe Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr
1 5 10 15
Leu Pro Asp Asp Gly Thr Leu Trp Thr Lys Val Ala Asn Glu Ala Asn
20 25 30
Asn Leu Ser Ser Leu Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala Tyr
35 40 45
Lys Gly Thr Ser Arg Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr
50 55 60
Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly
65 70 75 80
Thr Lys Ala Gln Tyr Leu Gln Ala Ile Gln Ala Ala His Ala Ala Gly
85 90 95
Met Gln Val Tyr Ala Asp Val Val Phe Asp His Lys Gly Gly Ala Asp
100 105 110
Gly Thr Glu Trp Val Asp Ala Val Glu Val Asn Pro Ser Asp Arg Asn
115 120 125
Gln Glu Ile Ser Gly Thr Tyr Gln Ile Gln Ala Trp Thr Lys Phe Asp
130 135 140
Phe Pro Gly Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp Tyr
145 150 155 160
His Phe Asp Gly Val Asp Trp Asp Glu Ser Arg Lys Leu Ser Arg Ile
165 170 175
Tyr Lys Phe Arg Gly Lys Ala Trp Asp Trp Glu Val Asp Thr Glu Phe
180 185 190
Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Leu Asp Met Asp His Pro
195 200 205
Glu Val Val Thr Glu Leu Lys Asn Trp Gly Lys Trp Tyr Val Asn Thr
210 215 220
Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Phe
225 230 235 240
Ser Phe Phe Pro Asp Trp Leu Ser Tyr Val Arg Ser Gln Thr Gly Lys
245 250 255
Pro Leu Phe Thr Val Gly Glu Tyr Trp Ser Tyr Asp Ile Asn Lys Leu
260 265 270
His Asn Tyr Ile Thr Lys Thr Asp Gly Thr Met Ser Leu Phe Asp Ala
275 280 285
Pro Leu His Asn Lys Phe Tyr Thr Ala Ser Lys Ser Gly Gly Ala Phe
290 295 300
Asp Met Arg Thr Leu Met Thr Asn Thr Leu Met Lys Asp Gln Pro Thr
305 310 315 320
Leu Ala Val Thr Phe Val Asp Asn His Asp Thr Glu Pro Val Gln Ala
325 330 335
Leu Gln Ser Trp Val Asp Pro Trp Phe Lys Pro Leu Ala Tyr Ala Phe
340 345 350
Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr Gly Asp Tyr
355 360 365
Tyr Gly Ile Pro Gln Tyr Asn Ile Pro Ser Leu Lys Ser Lys Ile Asp
370 375 380
Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly Thr Gln His Asp
385 390 395 400
Tyr Leu Asp His Arg Asp Ile Ile Gly Trp Thr Arg Glu Gly Gly Thr
405 410 415
Glu Lys Pro Gly Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro Gly
420 425 430
Gly Ser Lys Trp Met Tyr Val Gly Lys Gln His Ala Gly Lys Val Phe
435 440 445
Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile Asn Ser Asp
450 455 460
Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser Val Trp Val
465 470 475 480
Pro Arg
<210> 4
<211> 482
<212> PRT
<213>artificial sequence
<400> 4
Met Ala Ala Pro Phe Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr
1 5 10 15
Leu Pro Asp Asp Gly Thr Leu Trp Thr Lys Val Ala Asn Glu Ala Asn
20 25 30
Asn Leu Ser Ser Leu Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala Tyr
35 40 45
Lys Gly Thr Ser Arg Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr
50 55 60
Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly
65 70 75 80
Thr Glu Ala Gln Tyr Leu Gln Ala Ile Gln Ala Ala His Ala Ala Gly
85 90 95
Met Gln Val Tyr Ala Asp Val Val Phe Asp His Lys Gly Gly Ala Asp
100 105 110
Gly Thr Glu Trp Val Asp Ala Val Glu Val Asn Pro Ser Asp Arg Asn
115 120 125
Gln Glu Ile Ser Gly Thr Tyr Gln Ile Gln Ala Trp Thr Lys Phe Asp
130 135 140
Phe Pro Gly Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp Tyr
145 150 155 160
His Phe Asp Gly Val Asp Trp Asp Glu Ser Arg Lys Leu Ser Arg Ile
165 170 175
Tyr Lys Phe Arg Gly Lys Ala Trp Asp Trp Glu Val Asp Thr Glu Phe
180 185 190
Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Leu Asp Met Asp His Pro
195 200 205
Glu Val Val Thr Glu Leu Lys Asn Trp Gly Lys Trp Tyr Val Asn Thr
210 215 220
Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Phe
225 230 235 240
Ser Phe Phe Pro Asp Trp Leu Ser Tyr Val Arg Ser Gln Thr Gly Lys
245 250 255
Pro Leu Phe Thr Val Gly Glu Tyr Trp Ser Tyr Asp Ile Asn Lys Leu
260 265 270
His Asn Tyr Ile Thr Lys Thr Asp Gly Thr Met Ser Leu Phe Asp Ala
275 280 285
Pro Leu His Asn Lys Phe Tyr Thr Ala Ser Lys Ser Gly Gly Ala Phe
290 295 300
Asp Met Arg Thr Leu Met Thr Asn Thr Leu Met Lys Asp Gln Pro Thr
305 310 315 320
Leu Ala Val Thr Phe Val Asp Asn His Asp Thr Glu Pro Val Gln Ala
325 330 335
Leu Gln Ser Trp Val Asp Pro Trp Phe Lys Pro Leu Ala Tyr Ala Phe
340 345 350
Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr Gly Asp Tyr
355 360 365
Tyr Gly Ile Pro Gln Tyr Asn Ile Pro Ser Leu Lys Ser Lys Ile Asp
370 375 380
Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly Thr Gln His Asp
385 390 395 400
Tyr Leu Asp His Arg Asp Ile Ile Gly Trp Thr Arg Glu Gly Gly Thr
405 410 415
Glu Lys Pro Gly Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro Gly
420 425 430
Gly Ser Lys Trp Met Tyr Val Gly Lys Gln His Ala Gly Lys Val Phe
435 440 445
Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile Asn Ser Asp
450 455 460
Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser Val Trp Val
465 470 475 480
Pro Arg
<210> 5
<211> 25
<212> DNA
<213>artificial sequence
<400> 5
caagtacggc accgaggccc agtac 25
<210> 6
<211> 25
<212> DNA
<213>artificial sequence
<400> 6
gtactgggcc tcggtgccgt acttg 25
<210> 7
<211> 31
<212> DNA
<213>artificial sequence
<400> 7
ctatctggac cacagagaca tcattggctg g 31
<210> 8
<211> 31
<212> DNA
<213>artificial sequence
<400> 8
ggtccagcca atgatgtctc tgtggtccag a 31
<210> 9
<211> 40
<212> DNA
<213>artificial sequence
<400> 9
tcaaataagg agtgtcaaga atggcagccc cgttcaatgg 40
<210> 10
<211> 41
<212> DNA
<213>artificial sequence
<400> 10
gtttttttat taccaagctt ttagcgcgga acccacacac t 41
<210> 11
<211> 27
<212> DNA
<213>artificial sequence
<400> 11
tcttgacact ccttatttga ttttttg 27
<210> 12
<211> 25
<212> DNA
<213>artificial sequence
<400> 12
aagcttggta ataaaaaaac acctc 25

Claims (10)

1. the alpha-amylase mutant that a kind of enzyme activity improves, which is characterized in that the enzyme mutant is by the amino acid that will set out Sequence is the 82nd amino acids of the alpha-amylase of SEQ ID NO.1 and/or the 405th amino acids are mutated.
2. the alpha-amylase mutant that a kind of enzyme activity as described in claim 1 improves, which is characterized in that the enzyme mutant is By by lysine mutation being glutamic acid by the 82nd amino acids of the alpha-amylase that the amino acid sequence that sets out is SEQ ID NO.1 It obtains；
Or the enzyme mutant be by will set out amino acid sequence be SEQ ID NO.1 alpha-amylase the 405th bit amino Acid is what arginine obtained by mutant serine；
Or the enzyme mutant is by 82nd ammonia of the amino acid sequence for the alpha-amylase of SEQ ID NO.1 that will set out simultaneously It by mutant serine is what arginine obtained that base acid, which is glutamic acid and the 405th amino acids by lysine mutation,.
3. the alpha-amylase mutant that a kind of enzyme activity as claimed in claim 1 or 2 improves, which is characterized in that the alphalise starch The source of enzyme is bacillus stearothermophilus (Bacillus stearothermophilus).
4. the alpha-amylase mutant that a kind of enzyme activity a method according to any one of claims 1-3 improves, which is characterized in that the α-shallow lake The amino acid sequence of powder enzyme mutant is SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4.
5. encoding the gene for the alpha-amylase mutant that a kind of any enzyme activity of claim 1-4 improves.
6. carrying the recombinant plasmid of gene described in claim 5.
7. recombinant plasmid as claimed in claim 6, which is characterized in that the construction method of the recombinant plasmid is first according to determination Mutational site, the mutant primer of rite-directed mutagenesis is designed, further according to the mutant primer of design, to carry the load of alpha-amylase gene Body is that template carries out rite-directed mutagenesis.
8. carrying the host cell of gene described in claim 5 or the recombinant plasmid of claim 6 or 7.
9. host cell as claimed in claim 8, which is characterized in that the host cell is bacillus subtilis (Bacillus subtilis)WS5。
10. gene described in alpha-amylase mutant or claim 5 that a kind of any enzyme activity of claim 1-4 improves Or host cell described in recombinant plasmid described in claim 6 or 7 or claim 8 or 9 hydrolysis starch, prepare dextrin, Application in terms of preparing oligosaccharide, prepare maltose and preparing glucose.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112980755A (en) * 2019-12-13 2021-06-18 中国科学院天津工业生物技术研究所 Gene engineering bacterium for efficiently secreting isoamylase
CN115125226A (en) * 2022-07-26 2022-09-30 青岛蔚蓝生物集团有限公司 High specific activity amylase mutant
WO2023225459A2 (en) 2022-05-14 2023-11-23 Novozymes A/S Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections

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CN101457230A (en) * 2008-12-08 2009-06-17 江南大学 High efficiency preparation method of high temperature alpha-amylase and mutant thereof

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CN101457230A (en) * 2008-12-08 2009-06-17 江南大学 High efficiency preparation method of high temperature alpha-amylase and mutant thereof

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CORNELIUS等: "D irected evolution of a bac teria l A- amylase: Toward enhanced pH - performance and higher spec ific activity", 《PROTE IN SCI》 *
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陈磊等: "基于体外分子进化技术提高弯曲芽孢杆菌CCTCC 2015368 β-淀粉酶的热稳定性", 《生物工程学报》 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112980755A (en) * 2019-12-13 2021-06-18 中国科学院天津工业生物技术研究所 Gene engineering bacterium for efficiently secreting isoamylase
CN112980755B (en) * 2019-12-13 2023-05-12 中国科学院天津工业生物技术研究所 Genetically engineered bacterium capable of efficiently secreting isoamylase
WO2023225459A2 (en) 2022-05-14 2023-11-23 Novozymes A/S Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections
CN115125226A (en) * 2022-07-26 2022-09-30 青岛蔚蓝生物集团有限公司 High specific activity amylase mutant
CN115125226B (en) * 2022-07-26 2024-04-02 青岛蔚蓝生物集团有限公司 High specific activity amylase mutant

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