CN107164346B - A kind of alkalinity salt tolerant Pullulanase PulA and its gene and application - Google Patents

Abstract

The present invention relates to genetic engineering fields, in particular it relates to a kind of alkalinity salt tolerant Pullulanase PulA and its gene and application.Alkaline salt tolerant Pullulanase PulA, amino acid sequence is as shown in SEQ ID NO.1, and the genome encoding gene of the above-mentioned Pullulanase of codingPulA, nucleotide sequence is as shown in SEQ ID NO.2, and recombinant vector and recombinant bacterial strain comprising the gene.Pullulanase PulA optimal pH 9.0, the enzyme activity of residue 77% when 10.0.50 DEG C of the enzyme optimal reactive temperature, the enzyme activity of 50% or more residue in the presence of 0.25-3.0 M NaCl, and it is highly stable under the concentration of 3 M and 4 M NaCl.Pullulanase of the invention has the characteristics that alkaline salt tolerant, can be applied to the industrial circles such as detergent, food.

Description

A kind of alkalinity salt tolerant Pullulanase PulA and its gene and application

Technical field

The present invention relates to genetic engineering field, in particular it relates to a kind of alkalinity salt tolerant Pullulanase PulA and its Gene and application.

Background technique

Starch is the producing high-molecular being polymerized by α-D glucose monomer by α-Isosorbide-5-Nitrae-glycosidic bond or α -1,6- glycosidic bond Close object.There is a large amount of starch in animals and plants.Containing the starch of higher proportion in all kinds of plants, starch is plant storage battalion A kind of mode of substance is supported, the starch in plant is stored in the form of spherical particle, and seed, root, stem tuber etc. are distributed in.

Amylase (amylase) refers to the general name that can hydrolyze the class of enzymes of starch and glycogen.Amylase is as a kind of catalysis Agent has been widely used in each industrial circle.Most plant, animal all contain amylase in microorganism.According to Amylorrhexis substrate generate sugared isomers type difference and be classified as α and beta amylase.According to the most suitable anti-of amylase The difference of temperature and thermal stability is answered, it can be divided into: alpha-amylase, medium temperature amylase and low-temperature amylase.According to shallow lake The optimal pH of powder enzyme and the difference of pH stability can be classified as: acid, neutral and alkali starch enzyme.According to amylorrhexis sugar The difference of the mode of glycosidic bond can be broadly divided into: alpha-amylase, beta amylase, glucoamylase, isoamylase, branch Amylase etc..

Pullulanase is a kind of starch debranching enzymes, can specificity hydrolysis glycogen and amylopectin branch point in α -1,6- Glycosidic bond, the entire side chain for cutting amylopectin are become amylose.Pullulanase is different with isoamylase, Pu Lu Blue enzyme can be hydrolyzed by side chain that two to three glucose residues are constituted and isoamylase cannot.By the difference of hydrolytic sites, Pullulanase can be divided into two types, and one is I type Pullulanase, specificity hydrolyzing alphas -1,6- glycosidic bond；Another kind is II type Pullulanase, also referred to as starch Pullulanase have hydrolyzing alpha -1,6- glycosidic bond and inscribe oligosaccharide α-Isosorbide-5-Nitrae-sugar simultaneously The ability of glycosidic bond.Pullulanase is as a kind of important glycoside hydrolase, in starch processing, glucose syrup production, Beer Brewage There is huge application value with medication chemistry etc..

There are many microorganism for the production Pullulanase reported at present, wherein big portion is bacterium, such as bacillus, Exiguobacterium sp With thermophilic cold genus Shewanella etc..In addition, being found from hyperthermophilic archaeon strain and fungi there are also part Pullulanase.Because of the sugar of starch Change process needs (pH 4.5~5.5) under higher temperature (55~60 DEG C), slightly acidic condition to carry out, acidic pullulanase Utilization rate can be improved in syrup industry, reduce grain consumption and improve product quality, to report that more Pullulanase is big at present It is all acidicenzym.Alkaline pullulanase is with important application prospects in detergent industry.However at present for alkaline general Shandong The research report of blue enzyme is seldom.In addition, the patent about alkaline pullulanase also rarely has report.

The present invention has separated one plant of thermophilic salt basophilic that can produce alkaline salt tolerant Pullulanase from big cloth Soviet Union salt alkali lake bed mud BacteriumAlkalibacteriumSp., the alkalinity salt tolerant Pullulanase is obtained using the method for molecular biology and genetic engineering Encoding gene, and the gene is recombinantly expressed, recombinase has the spy of high activity and high stability under alkaline condition Point.In addition, the enzyme has good salt tolerance and stability, 45% or more there remains in the presence of 4 M NaCl Enzyme activity.In addition, the enzyme is trisaccharide to the hydrolysate of pulullan, show that the enzyme is I type Pullulanase.Therefore, the Propiram Possessed by enzyme the characteristics of alkalinity salt tolerant, there is very big application potential in detergent and field of food industry.

Summary of the invention

The object of the present invention is to provide a kind of alkalinity salt tolerant Pullulanase PulA and its gene and applications.

It is a further object of the present invention to provide the recombinant vectors comprising said gene.

It is a further object of the present invention to provide the recombinant bacterial strains comprising said gene.

It is a further object of the present invention to provide a kind of gene engineering methods for preparing above-mentioned alkaline salt tolerant Pullulanase.

Another object of the present invention provides the application of above-mentioned alkaline salt tolerant Pullulanase.

The present invention obtains a kind of alkaline salt tolerant Pullulanase PulA first, has very high enzyme under alkalinity and high salt conditions It is living.These properties meet the requirement in detergent industry to the alkalinity of Pullulanase, enhance dirt-removing power.

Alkalinity salt tolerant Pullulanase PulA of the present invention, amino acid sequence is as shown in SEQ ID NO. 1.

Pullulanase PulA of the invention contains 1184 amino acid in total, through software prediction no signal peptide, theoretical molecular weight For 132.09 kDa, theoretical isoelectric point is 4.07.

The optimal pH of Pullulanase PulA of the invention is 9.0, the enzyme activity of residue 77.4% in pH 10.0.The Propiram Enzyme is very stable between pH 6.0-10.0, remaining enzymatic activity is handled after 60min within the scope of this pH 75% or more, this explanation This enzyme has preferable pH stability.

The optimum temperature of Pullulanase PulA of the invention is 50 DEG C, the enzyme activity of 50% or more residue at 40-55 DEG C.? There is no in the case where substrate, the enzyme is more stable at 45 DEG C, the enzyme activity of residue 50% or so after processing one hour.The enzyme is at 50 DEG C When thermal stability it is not so good, processing one hour only residue 20% or so enzyme activity.

Pullulanase PulA of the invention also has the tolerance and stability of good salt.In 0.25-3.0 M NaCl In the presence of residue 55% or more enzyme activity.And 0.25M NaCl there are when the enzyme activity be promoted, it is living Power is the 105% of control.In addition, the enzyme handles 60 min under the concentration of 3 M and 4 M NaCl, remaining 95% or more enzyme It is living, show that the enzyme has good salt tolerance.

The present invention also provides the genes for encoding above-mentioned alkaline salt tolerant Pullulanase PulA, and the base sequence of the gene is as such as Shown in SEQ ID NO. 2.

The present invention has cloned this Pullulanase gene by the method separation of PCRPulA, DNA complete sequence analysis result Show Pullulanase PulA structural genePulAOverall length 3555bp, initiation codon ATG, terminator codon TAA, GC 50 % of content, encodes the polypeptide (PulA) of 1184 amino acid composition, no signal peptide sequence, and N-terminal is the 41st family Carbohydrate binding domain (CBM), C-terminal be a Pullulanase catalyst structure domain.Comparison in GenBank The result shows that it withAlkalibacterium pelagium (SEK70797) the Pullulanase highest consistency of prediction is 93%, and the Pullulanase gene in database with its similitude greater than 50% or more did not did property research, showed PulA It is a new Pullulanase.

The present invention also provides include above-mentioned alkaline salt tolerant Pullulanase genePulARecombinant vector, preferablypET22b-PulA.By Pullulanase gene of the inventionPulAIt is inserted between suitable restriction enzyme cleavage sites of the expression vector, Make its nucleotide sequence is operable to be linked to the expression control sequence.As the most preferred embodiment of the invention, Preferably by Pullulanase genePulAIt is inserted on plasmid pET22b (+)NcoI andNotBetween I restriction enzyme site, Obtain recombinant expression plasmidpET22b- PulA。

The present invention also provides include above-mentioned alkaline salt tolerant Pullulanase genePulARecombinant bacterial strain, the preferably described bacterial strain For Escherichia coli, saccharomycete, bacillus and filamentous fungi, preferably recombinant bacterial strain BL21 (DE3)/PulA.

The present invention also provides a kind of methods for preparing alkaline salt tolerant Pullulanase PulA, comprising the following steps:

1) host cell is converted with above-mentioned recombinant vector, obtains recombinant bacterial strain；

2) recombinant bacterial strain, the expression of induction recombination Pullulanase are cultivated；

3) it recycles and purifies expressed Pullulanase PulA.

Wherein, the preferably described host cell is Bacillus coli cells, is preferably converted expression of recombinant e. coli plasmid big Coli cell BL21 (DE3), obtains recombinant bacterial strainBL21/ PulA。

The present invention also provides the applications of above-mentioned alkaline salt tolerant Pullulanase PulA.

The present invention provides a kind of good properties, be suitable for new Pullulanase in washing and Applications in Food Industry PulA.Pullulanase PulA optimal reactive temperature provided by the present invention is 50 DEG C, and the optimal pH of the enzyme is 9.0, in pH10.0 When residue 77.4% enzyme activity.The Pullulanase also has good pH tolerance, highly stable in pH6.0~11.0.This Outside, the enzyme activity of the enzyme 50% or more residue in the presence of 0.25-3.0 M NaCl, and in the concentration of 3 M and 4M NaCl Under it is highly stable.Pullulanase PulA of the invention has the characteristics that alkaline salt tolerant, therefore alkaline salt tolerant Propiram of the invention Enzyme PulA can be used in detergent industry, enhance dirt-removing power.In addition, the enzyme can also be applied to food industry.

Detailed description of the invention

Fig. 1: in the SDS-PAGE analysis of the recombination Pullulanase PulA of expression in escherichia coli.Wherein, 1: low molecule Measure protein Marker；2: what is do not induced contains Pullulanase genophore pET-PulAE. coli culture supernatant liquid；3: Induction contains Pullulanase genophore pET-PulAE. coli culture supernatant concentrated liquor；4: passing through ni-sepharose purification Reach electrophoretically pure PulA albumen.

Fig. 2: the optimal pH of recombination Pullulanase PulA.

Fig. 3: the pH stability of recombination Pullulanase PulA.

Fig. 4: the optimum temperature of recombination Pullulanase PulA.

Fig. 5: the thermal stability of recombination Pullulanase PulA.

Fig. 6: calcium ion is on the recombination active influence of Pullulanase PulA.

Fig. 7: the salt tolerance of recombination Pullulanase PulA.

Fig. 8: the salt-stable of recombination Pullulanase PulA.

Fig. 9: the catabolite to pulullan of recombination Pullulanase PulA is analyzed.

Specific embodiment

Test material and reagent

1, bacterial strain and carrier: coli expression carrier pET22b (+) and bacterial strainEscherichia coli BL21 (DE3) it is purchased from Novagen company.

2, enzyme and other biochemical reagents: restriction enzyme, T4 DNA ligase, archaeal dna polymerase, dNTPs and PMD 18-T carrier is purchased from Japanese TaKaRa company.Genome extraction kit is purchased from Beijing Tiangen company, and purifying and plasmid extract Kit is purchased from OMEGA company, the U.S..Pulullan (Pullulan) is purchased from Sigma Co., USA；Peptone (Tryptone), Yeast extract (Yeast Extract) is Britain OXOID Products, remaining reagent is that domestic analysis is pure.

3, culture medium:

(1) LB culture medium (g/l): yeast powder 5.0, peptone 10.0, NaCl 10.0, pH7.0.

(2) plate screening culture medium (g/l): yeast powder 5.0, peptone 10.0, NaCl 10.0, agar 15.0, pH7.0。

Illustrate: not making the experimental methods of molecular biology illustrated in following embodiment, referring to " Molecular Cloning: A Laboratory Guide " specific method listed in book of (third edition) J. Pehanorm Brooker one carries out, or according to kit and product description It carries out.

Embodiment 1: Pullulanase genePulAClone

The extraction of thermophilic salt basophilic bacterium genomic DNA: big from Jilin Qianan using the screening and culturing medium that starch is sole carbon source The microorganism of isolated one plant of production alkaline pullulanase, is accredited as thermophilic salt basophilic Pseudomonas through 16S in the bed mud of cloth Soviet Union salt alkali lake. The genomic DNA of the thermophilic salt basophilic bacterium, concrete operations are extracted using the bacterial genomes extracts kit (DP302) of Tiangeng company It is carried out fully according to the specification of the kit.

Degenerate primer Pul- has been synthesized according to conservative (HVRDFTSDP and FDMMGDHD) sequence design of Pullulanase gene F and Pul-R:

Pul-F:5'-GTNCGNGAYTTYACNTCNGAYC-3';

Pul-R:5'- TCRTGRTCNCCCATCATRTC-3'

PCR amplification is carried out by template of above-mentioned thermophilic salt basophilic bacterium genome DNA.Base is carried out using touchdown PCR method Because of the amplification of segment.Response parameter are as follows: 94 DEG C of 5 min of initial denaturation；94 DEG C of denaturation 30 sec, 50-45 DEG C of annealing 30 sec, 72 DEG C extend 30 sec, 10 circulations (each circulation landing 0.5 DEG C).Then 94 DEG C of denaturation 30 sec, 45 DEG C of 30 sec of annealing, 72 DEG C of 30 sec of extension, 30 circulations, 72 DEG C of 5 min of heat preservation.Obtain an about 570bp segment, by the segment recycling after with PMD 18-T carrier, which is connected, is sent to handsome company's sequencing.

According to the nucleotide sequence for the conserved region that obtained Pullulanase gene is sequenced, upstream and downstream each three are designed TAIL-PCR specific primer: design direction is to need the zone of ignorance direction that expands, the Position Design of sp2 in the inside of sp1, Sp3 is located at the inside of sp2.The distance between every two primer without strict regulations (for convenience of electrophoresis recognition result, sp2 and General 100 bp of spacing of sp3), general 22~30 nt of primer length, annealing temperature is at 62 DEG C.And they are respectively designated as SL4-uSP1, SL4-uSP2, SL4-uSP3 (upstream specific primer); SL4-dSP1, SL4-dSP2, SL4-dSP3 (downstream specific primer) is shown in Table 1.Both ends flanking sequence is expanded according to the program in TAIL-PCR.

Primer needed for 1 Pullulanase PulA TAIL-PCR of table amplification and overall length amplification

The flanking sequence of known sequence is obtained by TAIL-PCR, amplification send handsome company to survey after obtaining product recycling Sequence.The upstream and downstream flanking sequence of the segment is obtained by sequence assembly, complete sequence is total to long 4.2kb, finds one and completely opens Reading frame (ORF).Pullulanase genePulABy 3555 base compositions, 1184 amino acid and a termination codon are encoded Son, the comparison result in GenBank show it and derive fromAlkalibacterium pelagium(SEK70797) The Pullulanase highest consistency of prediction is 93%.And the Pullulanase gene in database with its similitude greater than 50% or more Property research was not done.PulACoding albumen estimated molecular weight is 132 kDa, isoelectric point 4.07.Predicted, PulA is without letter Number peptide sequence, centre are the carbohydrate domains of the 41st family, and C-terminal is a Pullulanase catalyst structure domain.

The preparation of the recombination Pullulanase of embodiment 2.

To be introduced respectively in gene 5 ' and 3 ' endsNcoI andNotI restriction enzyme sitePulA-m-F andPulA-m-R is primer To (see Table 1), thermophilic salt basophilic bacterium genomic DNA is template, carries out PCR amplification.PCR response parameter are as follows: 94 DEG C of initial denaturations 5 min；94 DEG C of 30 sec of denaturation, 54 DEG C of 30 sec of annealing, 72 DEG C of 4 min of extension, 30 recycle, 72 DEG C of 5 min of heat preservation.By table Up to carrier pET22b (+) carry out double digestion (NcoI+ NotI), while the gene of Pullulanase will be encodedPulADouble digestion (EcoRI+ NotI), the genetic fragment of the coding Pullulanase cut is connect with expression vector pET22b (+), is obtained containing general Shandong orchid enzyme genePulARecombinant plasmid pET22b-PulAAnd e. coli bl21 (DE3) is converted, obtain recombination bacillus coli Bacterial strain BL21/ PulA.

It takes containing recombinant plasmid pET22b-PulABL21 bacterial strain, being inoculated in 5 mL LB, (the ammonia benzyl of 100 μ g/mL is green Mycin) in culture solution, 37 DEG C of overnight incubations.Cultured bacterium solution is inoculated in 20 mL LB (added with the ammonia of 100 μ g/mL by 1% Parasiticin), the inducer of 1 mmol/L of final concentration is added in 37 DEG C of about 2 ~ 3 h (OD600 reaches 0.5) of shaken cultivation afterwards IPTG, 30 DEG C of 180 12 h of rpm shake culture.12000 rpm are centrifuged 5 min, collect culture medium supernatant.DNS method measures general Shandong The vigor of blue enzyme.SDS-PAGE result (Fig. 1) shows that recombinate Pullulanase is expressed in Escherichia coli.Expressed For Pullulanase after purifying, the content of protein reaches 95% of total protein or more.

Embodiment 3 recombinates the property measurement of Pullulanase PulA

1, the activity analysis of Pullulanase is recombinated

3,5- dinitrosalicylic acid (DNS) method is taken in the determination of activity of recombination Pullulanase: the specific method is as follows: in pH Under the conditions of 9.0,50 DEG C, the reaction system of 1 mL includes 100 μ L dilution enzyme solution appropriate, 900 μ L substrate (the general Shandongs of 1% beech Blue sugar), 10 min are reacted, 1.5 mL DNS are added and terminate reaction, 5 min of boiling water boiling.540 nm measure OD value after cooling.1 Enzyme-activity unit (U) is defined as releasing the required enzyme amount of 1 μm of ol reduced sugar per minute under given conditions.

2, the optimal pH of Pullulanase PulA and the measurement of pH stability are recombinated

Optimal pH measurement: purified recombination Pullulanase PulA is slow with 0.1M pH4.0-11.0 at 37 DEG C Enzymatic reaction is carried out in fliud flushing.The pH Stability Determination of enzyme: enzyme solution being placed in the buffer of 0.1M pH4.0-11.0, 1h is handled at 37 DEG C, enzymatic reaction is then carried out at pH9.0 and 50 DEG C, using untreated enzyme solution as control.Buffer are as follows: 0.1 M McIlvaine buffer(pH4.0-7.0), 0.1 M Tris-HCl buffer(pH7.0-9.0) and 0.1M Glycine-NaOH(pH9.0-11.0).Using 1% pulullan as substrate, 10min is reacted, the vigor of PulA is measured.As a result table Bright: the optimal pH of PulA is 9.0, in the enzyme activity (Fig. 2) of pH10.0 residue 77.4%；This Pullulanase is in pH 6.0-10.0 Between it is very stable, remaining enzymatic activity is handled after 60min within the scope of this pH 75% or more, this illustrate this enzyme have compared with Good pH stability (Fig. 3).

3, the optimum temperature and thermal stability determination of Pullulanase PulA are recombinated

The measurement of the optimum temperature of enzyme: in the buffer of pH 9.0, enzymatic reaction is carried out at 10-60 DEG C.The heat of enzyme Stability Determination: the enzyme solution of same enzyme amount is placed in 45 DEG C, 50 DEG C and 55 DEG C, after handling 0-60min, in pH9.0 and 50 Enzymatic reaction is carried out at DEG C, using untreated enzyme solution as control.Using 1% pulullan as substrate, 10min is reacted.Enzyme reaction is most Thermophilic degree measurement result (Fig. 4) shows that optimum temperature is 50 DEG C.The thermal stability of enzyme is experiments have shown that (Fig. 5), recombinase At 45 DEG C, stability is preferable.60 min are kept the temperature at 55 DEG C, remaining enzymatic activity is 13%,.

4, recombinate Pullulanase PulA'sV _maxAnd K_mMeasurement

With the pulullan of various concentration (1,0.8,0.4,0.2,0.15 and 0.1%) for substrate, measured under optimum condition Enzymatic activity calculates corresponding reaction speed, acquires Km value and Vmax using the double counting backward techniques of Michaelis-Menten equation.The result shows that: the enzymeV _maxFor 38.02 ± 2.55 μm of ol min^–1mg^–1, K_mFor 0.09 ± 0.01 mg mL^–1。

5, influence measurement of the different metal ions chemical reagent to PulA enzyme activity

The metal ion and chemical reagent of 5 mM are added in enzymatic reaction system, studies its influence to enzymatic activity.? Under the conditions of 50 DEG C and pH 9.0, enzymatic activity is measured by substrate of pulullan.As a result (table 2) shows: Ca²⁺, Zn²⁺, Mn²⁺, Fe³⁺ And Co²⁺It can be respectively increased 40% or more enzyme activity； Cu²⁺And Hg²⁺Almost inhibitory enzyme activity；Ni²⁺, EDTA and SDS pairs Enzyme activity has faint inhibiting effect.Other reagents have faint facilitation to enzyme activity.

Two metal ion of table and chemical reagent are on the recombination active influence of pulullan PulA

6, various concentration Ca²⁺Active influence on recombination Pullulanase PulA

Contain Ca in Pullulanase PulA²⁺Binding site, therefore have studied various concentration Ca²⁺Influence to the enzymatic activity: The Ca of various concentration is added in enzymatic reaction system²⁺, study its influence to enzymatic activity.Under the conditions of 50 DEG C and 9.0 pH, Enzymatic activity is measured by substrate of pulullan.The result shows that concentration is 30mM Ca below²⁺All there is promotion to make the enzymatic activity With activity is maximum when wherein concentration is 5nM, for 1.42 times (Fig. 6) that calcium ion is not added.

7, activity and stable measurement of the various concentration NaCl to recombination Pullulanase PulA

Activity influence of the NaCl to recombination Pullulanase PulA: being added the NaCl of various concentration in enzymatic reaction system, Study its influence to enzymatic activity.Under the conditions of 50 DEG C and 9.0 pH, enzymatic activity is measured by substrate of pulullan.NaCl counterweight The influence of the stability of group Pullulanase PulA: enzyme solution is respectively placed in the buffer of the pH7.0 containing 3M and 4 M, 37 1h is handled at DEG C, enzymatic reaction is then carried out at pH9.0 and 50 DEG C, using untreated enzyme solution as control.The result shows that: In the presence of 0.25-4.0 M NaCl, which is capable of the enzyme activity (Fig. 7) of 45% or more residue.In the dense of 3 M and 4M NaCl Degree is lower to handle 60 min, the enzyme activity (Fig. 8) of 95% or more residue.

8, recombination Pullulanase PulA analyzes the catabolite of pulullan

Sample handling processes are as follows: the 1% pulullan substrate using excessive pure enzyme solution and pH9.0 buffer configuration is mixed It is even, it is placed on water-bath in the most stable of temperature of recombinase, is sampled respectively under 10 min, 30 min, 1h, 2h, 4h and 8h, Boiling water bath 5min inactivates enzyme solution, and 13000 r/min are centrifuged 5 min after being cooled to room temperature, and takes the sample point sample of same volume in silicon On offset plate.Thin-layer chromatography experiment shows that the enzyme only has three pools to the hydrolysate of pulullan, shows that the enzyme is I type Propiram Enzyme (Fig. 9).

It should be understood that for those of ordinary skills, it can be modified or changed according to the above description, And all these modifications and variations should all belong to the protection domain of appended claims of the present invention.

SEQUENCE LISTING

<110>University of Fuzhou

<120>a kind of alkalinity salt tolerant Pullulanase PulA and its gene and application

<130> 10

<160> 10

<170> PatentIn version 3.3

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<211> 1184

<212> PRT

<213>amino acid sequence

<400> 1

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

1 5 10 15

Ile Ala Glu Glu Ile Glu Thr Pro Asp Asp Ser Asp Glu Gln Leu Glu

20 25 30

Glu Thr Glu Val Glu Glu Gly Phe Phe Arg Ile His Phe Ala Ser Leu

35 40 45

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

50 55 60

Lys Glu Pro Ser Glu Asn Arg Gly Ala Trp Pro Asn Gly Ala Thr Ser

65 70 75 80

Phe Thr Glu Ala Val Gln Thr Asp Tyr Gly Trp Tyr Met Asp Ile Glu

85 90 95

Leu Glu Glu Asn Pro Gln Ser Ile Gly Phe Leu Ile Asn Ser Val Ser

100 105 110

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

115 120 125

Met Asn Gln Val Trp Leu Asp His Glu Tyr Ala Met Thr Pro Tyr Glu

130 135 140

Pro Leu Leu Glu Lys Asp Met Ile Arg Ile Asn Tyr Lys Arg Asp Asn

145 150 155 160

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

165 170 175

Pro Thr Glu Asn Trp Pro Ala Gly Ala Gln Asp Ser Asp Gly Val Gly

180 185 190

Pro Asn Gly Thr Tyr Phe Asn Leu Thr Leu Ala Glu Asp Ser Asp Gln

195 200 205

Ile Gly Phe Leu Phe Leu Asn Lys Ala Asp Gly Ser Gln Thr Arg Asp

210 215 220

Tyr Thr Phe Ser Asn Leu Ser Ala His Ser Gln Leu Phe Met Arg Glu

225 230 235 240

Gly Asp Asp Thr Ile Tyr Thr Asn Pro Tyr Tyr Val Ser Glu Ala Gly

245 250 255

Met Ile Arg Ala Glu Leu Ile Ser Glu Thr Glu Ile Glu Val Phe Phe

260 265 270

His Ser Thr Glu Gly Leu Glu Glu Ala Asp Leu Leu Glu Leu Ile Gln

275 280 285

Leu Thr Asp Ala Glu Gly Arg Asp Val Leu Phe Asp Ala Ala Val Asp

290 295 300

His Asp Arg Arg Val Val Ser Leu Thr Gly Asp Phe Ser Val Glu His

305 310 315 320

Ala Pro Tyr Thr Val Thr Phe Asp Glu Thr Glu Ala Glu Ala Arg Met

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

Val Val Val Arg Asp Asp Ile Glu Met Thr Ala Glu Glu Ser Gly Val

385 390 395 400

Trp Arg Val Val Leu Asp Glu Asp Thr Thr Gly Leu Asp Asp Val Thr

405 410 415

Gly Tyr Phe Tyr His Phe Ala Ile Glu Arg Asn Gly Glu Thr Val Leu

420 425 430

Ala Leu Asp Pro Tyr Ala Arg Ser Met Ala Ala Trp Asn Ser Ser Asp

435 440 445

Pro Asp Asn Tyr Ile Gly Lys Ala Ala Ile Val Asn Pro Ser Glu Ile

450 455 460

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

465 470 475 480

Asp Ala Ile Ile Tyr Glu Ile His Val Arg Asp Phe Thr Ser Asp Pro

485 490 495

Ser Ile Glu Asp Glu Leu Thr Ser Gln Phe Gly Thr Phe Ser Ala Phe

500 505 510

Ile Glu Lys Leu Asp Tyr Ile Glu Ser Leu Gly Val Thr His Val Gln

515 520 525

Leu Leu Pro Val Met Ser Tyr Phe Phe Ala Asn Glu Phe Glu Asn Ala

530 535 540

Glu Arg Met Leu Asp Tyr Gly Ser Thr Gln Thr Asn Tyr Asn Trp Gly

545 550 555 560

Tyr Asp Pro Gln Ser Tyr Phe Ser Leu Thr Gly Met Tyr Ser Glu Asn

565 570 575

Pro Lys Asp Pro Ala Arg Arg Ile Glu Glu Phe Lys Asn Leu Ile Asp

580 585 590

Ala Ile His Ser His Gly Met Gly Val Ile Leu Asp Val Val Tyr Asn

595 600 605

His Thr Ala Arg Glu His Ile Phe Glu Asp Leu Glu Pro Asn Tyr Tyr

610 615 620

His Phe Met Asp Ala Asp Gly Thr Ser Arg Thr Ser Phe Gly Gly Gly

625 630 635 640

Arg Leu Gly Thr Thr His Glu Met Ala Arg Arg Ile Leu Val Asp Ser

645 650 655

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

660 665 670

Met Met Gly Asp His Asp Ala Glu Ser Ile Gln Met Ala Phe Asp Glu

675 680 685

Ala Gln Lys Leu Asn Pro Asn Ile Leu Met Ile Gly Glu Gly Trp Arg

690 695 700

Thr Phe Val Gly Asp Glu Gly Tyr Glu Asp Val Met Pro Ala Asp Gln

705 710 715 720

Asp Trp Met Gln His Thr Gln Ala Val Gly Ser Phe Ser Asp Asp Phe

725 730 735

Arg Asn Glu Leu Lys Ser Gly Phe Gly Ser Glu Gly Glu Pro Arg Phe

740 745 750

Ile Thr Gly Gly Thr Arg Ser Ile Gln Arg Ile Phe Asp Asn Leu Thr

755 760 765

Ala Asn Pro His Asn Phe Met Ala Thr Asp Pro Gly Asp Val Val Pro

770 775 780

Tyr Ile Ala Ala His Asp Asn Leu Thr Leu His Asp Val Ile Ala Gln

785 790 795 800

Ser Ile Gln Lys Asp Pro Glu Tyr His Gln Glu Glu Ile His Gln Arg

805 810 815

Ile Arg Leu Gly Asn Leu Met Val Leu Thr Ser Gln Gly Thr Pro Phe

820 825 830

Val His Ala Gly Gln Glu Tyr Gly Arg Thr Lys Gln Phe Arg Asp Pro

835 840 845

Asp Phe Ile Glu Pro Val Ala Asn Asp Gln Val Pro Tyr Lys Ser Thr

850 855 860

Phe Met Thr Asp Glu Asp Gly Asn Pro Phe Leu Tyr Pro Tyr Phe Ile

865 870 875 880

His Asp Ser Tyr Asp Ser Thr Asp Ala Val Asn Arg Phe Glu Trp Asp

885 890 895

Lys Val Thr Asp Ala Glu Ala Tyr Pro Ile Asn Thr Gln Thr Gln Ser

900 905 910

Tyr Thr Ser Gly Leu Ile Ala Leu Arg Arg Ser Thr Asp Ala Phe Ser

915 920 925

Lys Gly Thr Met Glu Glu Ile Ala Asp Met Val Ser Leu Val Asp Ala

930 935 940

Pro Glu Ile Glu Asp Glu Asp Leu Val Ile Val Tyr Arg Ala Glu Asp

945 950 955 960

Ser Asn Gly Asp Arg Tyr Tyr Val Phe Val Asn Ala Asp Asp Ser Glu

965 970 975

Arg Thr Leu Thr Leu Asp Ser Asp Leu Thr Glu Gly His Val Leu Val

980 985 990

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

995 1000 1005

Thr Val Glu Gln Ala Gly Val Thr Leu Ala Pro Leu Thr Ala Ser

1010 1015 1020

Val Val Leu Leu Thr Asp Arg Glu Ile Glu Pro Val Glu Glu Ser

1025 1030 1035

Asp Glu Asp Gly Asp Glu Gly Thr Asp Pro Gly Asn Gly Glu Gln

1040 1045 1050

Pro Gly Gly Glu Ser Gly Pro Gly Thr Asp Gln Gly Ser Asp Gly

1055 1060 1065

Asp Asp Pro Val Ser Gly Gly Glu Glu Thr Ala Asp Pro Glu Arg

1070 1075 1080

Asp Ala Glu Gly Asp Asp Tyr Pro Glu Asp Asp Thr Asp Leu Ser

1085 1090 1095

Glu Asp Pro Gly Ala Gly Gln Asp Ser Gly Asp Ser Ile Ala Asp

1100 1105 1110

Gly Asp Gln Gly His Ser Asp Gly Pro Leu Asp Gly Pro Asp Gly

1115 1120 1125

Asp Glu Thr Gly Lys Ala Glu Gly Asp Ser Ser Glu Ser Gln Thr

1130 1135 1140

Gly Glu Gln Asn Gly Glu Arg Leu Pro Ser Thr Ala Thr Leu Leu

1145 1150 1155

Trp Thr Val Gly Ala Val Gly Leu Met Ser Leu Leu Thr Gly Val

1160 1165 1170

Val Val Arg Gln Ile Lys Lys Lys Asn Lys Thr

1175 1180

<210> 2

<211> 3555

<212> DNA

<213>gene order

<400> 2

atggcagacg gcggaacagc actcctgaca gtggataacg ggtcactgat tgcggaagaa 60

attgagacac cggatgattc agatgaacag ctagaagaaa ccgaggtgga agaaggattt 120

tttcgcatcc attttgcgtc gcttccgtca gatgatcgcg agactcttgg gctctggatc 180

tggaacgatg taaaagagcc ttctgaaaac agaggggcat ggccgaatgg ggcgacctca 240

tttacagagg ctgttcagac cgactacggc tggtatatgg atattgaatt ggaagagaat 300

ccacagtcga ttggcttcct tatcaattcc gtctcgggga acaatctgtc aggagatatc 360

gtcctgcggc ttctgacttc tgagatgaat caggtatggc ttgatcatga gtatgccatg 420

acaccttatg agcctttact tgaaaaggac atgatccgga tcaactacaa acgagacaac 480

aatgactacg atgactgggg cctctggacc tgggacgatg tggcagaacc aacagaaaac 540

tggcctgcag gcgctcagga cagcgatggc gtcgggccta acggaaccta ttttaatctt 600

acgctggcag aagattccga tcagatcggt tttctgttcc tgaataaagc tgatggttcc 660

cagacccggg attacacatt ctcgaacttg tccgctcaca gccagctatt tatgcgtgaa 720

ggagacgata ctatttatac caatccgtac tatgtcagtg aagccggcat gatccgagcc 780

gagctgattt ctgaaactga aatcgaagtg tttttccatt cgacagaagg actggaggag 840

gctgatctgc ttgaattaat tcagctgacc gatgcagaag gacgagacgt gctatttgat 900

gcggctgtcg atcatgaccg acgagtcgtt agtctgaccg gtgacttctc agtggagcat 960

gctccttata ccgtgacttt tgacgaaacg gaagcagaag cgcgaatggg ctggcgcctg 1020

aaggatgcgc tctatgccta tgacggagaa cttggtctga cttttaacga agacgggacc 1080

gctgatctga aagtctggtc tccaagtgcc gacgcagtga cggtcgtttt atatgataaa 1140

gatgatcaga ctgtcgttgt cagagatgat atcgaaatga ctgccgagga gtcaggcgtg 1200

tggcgtgttg ttcttgatga agacacgacc ggactggacg atgtgacggg gtacttctac 1260

cattttgcga ttgaaagaaa tggcgaaacc gtcctggcac tggatcctta tgcccgctca 1320

atggccgcat ggaacagcag tgatccggat aattacatcg gcaaagctgc catcgtgaat 1380

ccgagtgaga tcggtcggga actggattat gctcagatcg aaggctatga caagcgcgaa 1440

gatgcgatca tttatgaaat tcatgtcaga gacttcacat ccgatccgtc aattgaagat 1500

gaactgacca gtcagttcgg gacgttcagt gctttcattg aaaagctcga ttatatcgaa 1560

agtctgggtg tgacccatgt tcagctgctt ccggtcatga gctatttctt tgcaaatgaa 1620

ttcgaaaacg ccgaacggat gttggactac ggatcgactc agaccaacta taactggggt 1680

tacgatccgc agagctactt ctctctgacg ggaatgtatt cagaaaatcc aaaagacccg 1740

gcgagacgca ttgaagaatt caaaaatctg atcgatgcga ttcattccca cggcatgggt 1800

gtcattcttg atgtggtcta taatcacacg gcacgggagc acatttttga agatctggaa 1860

ccgaactatt accatttcat ggatgcggac ggcacgtcac gaacaagttt cggaggcggc 1920

cgactaggca ccactcatga gatggcccgc cgtattctgg tcgattcgat cacatactgg 1980

gtggaagaat ataaagttga cggcttccgc ttcgatatga tgggcgatca cgatgccgaa 2040

agcattcaga tggcatttga cgaagcacag aaactgaatc cgaatatcct gatgatcggt 2100

gagggctggc ggacctttgt cggtgacgaa ggctatgaag acgtcatgcc ggctgaccag 2160

gactggatgc agcacacaca agccgttgga tcgttctcgg atgacttcag aaatgaactg 2220

aaatccggat tcggcagtga gggagaaccg cgctttatta ccggaggcac acggtccatc 2280

caacgaattt ttgacaatct gacagccaat ccacataact ttatggcgac agatccgggc 2340

gatgtggtcc cttatatcgc ggcgcacgac aacctgacgc ttcatgatgt catcgctcaa 2400

agcattcaga aagatccgga ataccaccag gaagagatcc atcagcgtat ccgtctgggc 2460

aatctgatgg tgctgacttc tcagggcacg ccattcgtcc atgccggtca ggaatacgga 2520

cggaccaagc aattccgtga tcctgacttt atcgaacctg tagcaaacga tcaggttcct 2580

tacaagtcga cattcatgac agatgaagac gggaatccgt tcctttaccc gtatttcatt 2640

catgactcat acgattcaac ggatgcggtc aaccgttttg aatgggataa agtgacggat 2700

gctgaagctt atccgattaa tacgcagacc cagtcttaca catctggtct gattgcattg 2760

cggcgcagta cggatgcctt cagtaaagga acgatggaag agatcgcgga catggtgtcg 2820

ctagtggatg cgccggaaat cgaggacgaa gacctggtta tcgtctatcg tgcagaagat 2880

tccaatggcg atcgttacta cgtctttgtg aatgcagatg attccgaacg aacgctgaca 2940

cttgattctg atttgactga agggcacgtc ctggtcgata gtcagcaggc cggcacacga 3000

gcgatcgcca gaccagaagg catcacggtc gaacaggctg gtgtcacgct ggctcctttg 3060

acggcatccg tggttttact tacggataga gaaattgagc cggttgaaga gagtgacgaa 3120

gatggcgatg aaggaactga cccaggcaac ggggaacagc ctggtggaga atctggacca 3180

ggaacagatc agggatccga tggcgacgat cctgtctcag gtggcgaaga aacggctgat 3240

ccagaaagag atgccgaggg tgatgattat ccggaagacg acactgattt gtctgaggat 3300

cctggtgctg gtcaggatag tggagattcg atagctgatg gcgaccaagg tcattcagat 3360

ggtccactag atgggccgga tggtgatgaa acaggcaaag cggaaggcga ttcttctgaa 3420

tcccagactg gtgagcagaa cggagaaagg ctcccctcta cagcaaccct actctggact 3480

gtaggagcag tcggactgat gagcctcttg acaggggtag tggtcagaca gatcaaaaag 3540

aaaaataaga cataa 3555

<210> 3

<211> 26

<212> DNA

<213>artificial sequence

<400> 3

gtcagttcgg gacgttcagt gctttc 26

<210> 4

<211> 28

<212> DNA

<213>artificial sequence

<400> 4

cgaaagtctg ggtgtgaccc atgttcag 28

<210> 5

<211> 30

<212> DNA

<213>artificial sequence

<400> 5

gtcattcttg atgtggtcta taatcacacg 30

<210> 6

<211> 30

<212> DNA

<213>artificial sequence

<400> 6

ccagtatgtg atcgaatcga ccagaatacg 30

<210> 7

<211> 31

<212> DNA

<213>artificial sequence

<400> 7

catccatgaa atggtaatag ttcggttcca g 31

<210> 8

<211> 28

<212> DNA

<213>artificial sequence

<400> 8

ggtctgagtc gatccgtagt ccaacatc 28

<210> 9

<211> 29

<212> DNA

<213>artificial sequence

<400> 9

gaaccatgga tggcagacgg cggaacagc 29

<210> 10

<211> 41

<212> DNA

<213>artificial sequence

<400> 10

ggagcggccg ctgtcttatt tttctttttg atctgtctga c 41

Claims (7)

1. a kind of alkalinity salt tolerant Pullulanase PulA, which is characterized in that its amino acid sequence is as shown in SEQ ID NO. 1.

2. a kind of alkaline salt tolerant Pullulanase gene for encoding alkaline salt tolerant Pullulanase PulA described in claim 1PulA, It is characterized in that, its nucleotide sequence is as shown in SEQ ID NO. 2.

3. including alkalinity salt tolerant Pullulanase gene described in claim 2PulARecombinant vector.

4. including alkalinity salt tolerant Pullulanase gene described in claim 2PulARecombinant bacterial strain.

5. recombinant bacterial strain according to claim 4, which is characterized in that the recombinant bacterial strain is Escherichia coli, saccharomycete, bud Spore bacillus or filamentous fungi.

6. a kind of method for preparing alkaline salt tolerant Pullulanase PulA, which comprises the following steps:

1) host cell is converted with the recombinant vector of claim 3, obtains recombinant bacterial strain；

2) recombinant bacterial strain, induction recombination Pullulanase expression are cultivated；And

3) it recycles and purifies expressed Pullulanase PulA.

7. alkalinity salt tolerant Pullulanase PulA described in claim 1 is preparing the application in laundry detergent compositions and detergent.