CN110172468A

CN110172468A - One kind is from the novel Pullulanase of series bacillus and its gene and application

Info

Publication number: CN110172468A
Application number: CN201910390539.5A
Authority: CN
Inventors: 崔堂兵; 苏红玉
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2019-05-10
Filing date: 2019-05-10
Publication date: 2019-08-27
Also published as: CN110592119A; CN110592119B

Abstract

The invention discloses a kind of from the novel Pullulanase and its encoding gene of series bacillus and application.The novel Pullulanase from series bacillus, amino acid sequence is as shown in SEQ ID NO.2.The encoding gene of the novel PullulanasePulA,Nucleotide sequence is as shown in SEQ ID NO.1.The Pullulanase is type Pullulanase, and heterogenous expression can be carried out in Escherichia coli BL21 (DE3).The molecular weight of the recombination Pullulanase is about 76.95 ku, and specific enzyme activity is up to 508.8 U/mg.Novel Pullulanase provided by the invention from series bacillus can be applied to the relevant industries such as starch processing, medicine, chemical industry, food.

Description

One kind is from the novel Pullulanase of series bacillus and its gene and application

Technical field

The invention belongs to genetic engineerings and technical field of enzyme engineering, and in particular to one kind is novel general from series bacillus Shandong orchid enzyme and its gene and application.

Background technique

Pullulanase (pullulanase, EC 3.2.1.41) is a kind of starch debranching enzyme, can be acted in specific manner Pulullan, amylopectin, the α-l in limit dextrin, 6 glycosidic bonds cut entire side shoot, form amylose.Pullulanase With carbohydrase and used time, the yield and purity of glucose can be improved, its DE value is made to reach 97%-98%；When itself and beta amylase When collective effect, 100% ground of starch can be converted to maltose, to obtain superhigh maltose syrup；In the sugar of brewing Pullulanase is added during changing, the content of fermentable sugar can be significantly increased, guarantee the mouthfeel of beer.Pullulanase These properties determine its extensive use in starch processing industry.

Produced in the nature reported out at present Pullulanase bacterial strain be concentrated mainly on bacillus, bacteroides thetaiotaomicron, gram The primary Salmonella of thunder, hot sulphur clostridium and streptococcus etc..The industrialized production bacterium of Pullulanase is seldom, only Denmark Novo company Bacillus acidopullulyticus and the Bacillus deramificans of Genencor company, the U.S. etc. are applied to reality In the production of border (Jensen B F, Norman B E.Bacillus acidopullulyticus Pullulanase: application and regulatory aspects for use in the food industry[J].Process Biochemistry,1984,19:129-134；Malviya S N,Malakar R,Tiwari A.Pullulanase:a potential enzyme for industrial application[J].International Journal of Materials Research,2010,1:10-20).It is domestic constantly to have the relevant report for producing Pullulanase bacterial strain, but wild mushroom The producing enzyme vigor of strain is generally relatively low, and commodity production demand is also not achieved in the fermentation level of most of genetic engineering bacteriums.China's starch Pullulanase used in processing industry relies primarily on import, and price is more high.Therefore, it is realized by technique for gene engineering different The high efficient expression of the Pullulanase of property provides more potential resources for the industrialized production of Pullulanase, to adapt to more extensively Industrial requirement, have stronger realistic meaning.

Summary of the invention

In order to overcome the shortcomings of the prior art, the object of the present invention is to provide a kind of new from series bacillus Type Pullulanase and its gene and application.

The present invention provides a kind of producing enzyme level is higher, the Pullulanase encoding gene with larger application potential (is denoted as pulA).The present invention provides the recombinant plasmids comprising above-mentioned Pullulanase encoding gene.The present invention provides include above-mentioned base Cause, the recombinant bacterial strain for capableing of high efficient expression Pullulanase.

The purpose of the present invention is realized at least through one of following technical solution.

It is provided by the invention a kind of from the novel general of series bacillus (Paenibacillus puldeungensis) The encoding gene pulA of Shandong orchid enzyme, nucleotide sequence is as shown in SEQ ID NO.1.

A kind of novel Pullulanase from series bacillus provided by the invention, amino acid sequence such as SEQ ID Shown in NO.2.

Further, the novel Pullulanase is I type Pullulanase.

A kind of recombinant plasmid provided by the invention inserts nucleotide sequence described in SEQ ID NO.1.

Further, the recombinant plasmid is by being cloned into more grams of expression vector pET-28a (+) for encoding gene pulA Grand site is obtained, and pET-28a (+)-pulA is denoted as.

Further, the recombinant plasmid contains above-mentioned Pullulanase gene；The plasmid is logical for pET-28a (+)-pulA Cross BamHI the and HindIII digestion that above-mentioned Pullulanase gene pulA is cloned into coli expression carrier pET-28a (+) Between site, it is linked to the expression control sequence objective gene sequence, obtains expression plasmid pET-28a (+)-pulA.

A kind of engineering strain (recombinant bacterial strain) provided by the invention, contains above-mentioned recombinant plasmid.The recombinant bacterium Strain being capable of high efficient expression Pullulanase.

Further, the engineering strain, host cell are e. coli bl21 (DE3).

The encoding gene pulA of novel Pullulanase provided by the invention from series bacillus, the recombination matter Grain and the engineering strain can be applied in the preparation novel Pullulanase for deriving from series bacillus.

Further, the application, comprises the following steps that

(1) clone of Pullulanase gene pulA；

(2) construction recombination plasmid；

(3) by recombinant plasmid transformed host cell described in step (2), recombinant bacterial strain is obtained, i.e., the described genetic engineering Bacterial strain；

(4) expression and purifying of the novel Pullulanase from series bacillus.

Novel Pullulanase provided by the invention from series bacillus can apply to starch processing, medicine, chemical industry, In the relevant industries such as food.

The present invention separates the bacterial strain LK18 of one plant of production I type Pullulanase from starch factory sewage, is accredited as Paenibacillus puldeungensis is named as Paenibacillus puldeungensis LK 18.It is domestic at present The report of the outer Pullulanase for deriving from Paenibacillus puldeungensis not yet and its related zymologic property.This Invention provides the clonal expression and zymologic property of the Pullulanase in the source Paenibacillus puldeungensis, success The recombinant bacterial strain of one plant of high efficient expression Pullulanase is constructed, has established base for the enzyme library building of Pullulanase and industrialized production Plinth.

The present invention obtains the Pullulanase gene in Paenibacillus puldeungensis LK18 using round pcr PulA, sequence 1968bp encode 655 amino acid, initiation codon TTG, terminator codon TAG, amino acid Sequence is as shown in SEQ ID NO.2.On-line analysis software (http://web.expasy.org/protparam/) shows: this is general The isoelectric point pI of Shandong orchid enzyme is 5.15, and total atom number is 10195, molecular formula C₃₂₈₆H₅₀₁₃N₈₈₉O₉₈₇S₂₀, unstability index (Instability index) is 38.88 (numerical value less than 40, protein stabilization), and liposoluble index (Aliphatic index) is 81.71；Overall average hydrophilicity (Grand average of hydropathicity:GRAVY) is -0.339.Pass through Clustal Omega online software (http://www.ebi.ac.uk/Tools/msa/clustalo/) compares the amino acid sequence, discovery The I type Pullulanase of itself and Paenibacillus barengoltzii CAU904 (GenBank accession number: KP714732.1) Sequence homology highest, similarity 73% illustrate that the Pullulanase is a kind of new Pullulanase.

The present invention provides the cloning process of the Pullulanase gene, compare NCBI data by ClustalX software first The I type Pullulanase gene order for the bacillus genus that library is announced, determines its conservative region, design degenerate primer (PF1 and PR1；PF2 and PR2；PF3 and PR3), using Paenibacillus puldeungensis LK18 full-length genome as template, amplification Its portion gene out, then by the sequencing result of portion gene with DNAMAN be compared with sequence assembly (as shown in Figure 1), and The ORF Finder that the sequence of splicing is committed on NCBI is analyzed, is redesigned primer (Pul-F and Pul-R), into one Step obtains pulA full length gene (see Fig. 2 a, Fig. 2 b, Fig. 2 c and Fig. 2 d).

The present invention provides the active Pullulanases for deriving from Paenibacillus puldeungensis, pass through Ni column affinity protein purification goes out the higher recombinant protein of purity, and measuring its specific enzyme activity is 508.8U/mg, SDS-PAGE detection display Its molecular weight is about 76.95ku (see Fig. 3).The optimal reactive temperature of the recombinase is 45 DEG C (as shown in Figure 4), at 35 DEG C -40 Heat preservation 120min still maintains 60% or more enzyme activity (as shown in Figure 5) under DEG C range；Most suitable action pH is 6.0 (as shown in Figure 6), After keeping the temperature 1h under the conditions of pH 6.0-8.0, still remaining 60% or more (as shown in Figure 7) of opposite enzyme activity；The K of 10mmol/L⁺And Mg² ⁺There is activation, and Zn to the recombinase²⁺、Mn²⁺、Ni²⁺、Fe²⁺、Cu²⁺、Co²⁺、Ca²⁺Deng there is different degrees of inhibition to it It acts on (Fig. 8).The present invention successfully obtains the recombinant bacterial strain of one plant of high efficient expression Pullulanase, and expressed Pullulanase can be applied In the relevant industries such as starch processing, medicine, chemical industry, food.

Compared with prior art, the invention has the advantages that and the utility model has the advantages that

(1) the present invention provides clone's tables of the novel Pullulanase in the source Paenibacillus puldeungensis Up to method and zymologic property, the present invention successfully constructs the recombinant bacterial strain of one plant of high efficient expression Pullulanase, can be Pullulanase Enzyme library building and industrialized production lay the foundation；

(2) novel Pullulanase molecular weight provided by the invention is smaller, only 655 amino acid, more conducively recombinant protein Expression；

(3) novel Pullulanase provided by the invention is suitable for the reaction environment of meta-acid, has in industrial application certain Advantage.

Detailed description of the invention

Fig. 1 is the gene order spliced map for deriving from the novel Pullulanase of series bacillus.

Fig. 2 a is the amplified production electrophoretogram of primer PF1 and PR1；

Fig. 2 b is the amplified production electrophoretogram of primer PF2 and PR2；

Fig. 2 c is the amplified production electrophoretogram of primer PF3 and PR3；

Fig. 2 d is the novel Pullulanase full length gene sequence electrophoretogram of primer Pul-F and Pul-R amplification；Wherein, M DL 5000DNA Marker。

Fig. 3 be after purification it is described derive from the novel Pullulanase SDS-PAGE analysis chart of series bacillus, wherein M be egg White Marker；Swimming lane 1 is unloaded bacterial strain broken wall supernatant；Swimming lane 2 is unpurified recombination Pullulanase (novel Pullulanase)；Swimming Road 3 is recombination Pullulanase (novel Pullulanase) after purification.

Fig. 4 is temperature to the enzyme activity influence diagram from the novel Pullulanase of series bacillus.

Fig. 5 is the temperature stability figure for deriving from the novel Pullulanase of series bacillus.

Fig. 6 is pH to the enzyme activity influence diagram from the novel Pullulanase of series bacillus.

Fig. 7 is the pH stability diagram for deriving from the novel Pullulanase of series bacillus.

Fig. 8 is metal ion to the enzyme activity influence diagram from the novel Pullulanase of series bacillus.

Specific embodiment

Specific implementation of the invention is described further below in conjunction with attached drawing and example, but implementation and protection of the invention It is without being limited thereto.If it is existing to be that those skilled in the art can refer to it is noted that there is the not special process of detailed description below Technology realize or understand.Reagents or instruments used without specified manufacturer, be considered as can by it is commercially available be commercially available it is normal Advise product.

Experimental material and reagent

1, bacterial strain and plasmid: Paenibacillus puldeungensis LK18 is that wild mushroom is sieved in laboratory certainly；JM110 Competent cell is purchased from Shanghai Wei Di Bioisystech Co., Ltd；Escherichia coil BL21 (DE3) competent cell, PEASY-Blunt Cloning vector is purchased from Beijing Quanshijin Biotechnology Co., Ltd；PET-28a (+) expression vector, Purchased from Invitrogen biotech firm.

2, enzyme and main agents: TransStart KD Plus DNA Polymerase, 2 × EasyTaq PCR SuperMix enzyme is purchased from Beijing Quanshijin Biotechnology Co., Ltd；Restriction enzyme (BamHI and HindIII), T4DNA Ligase, purchased from precious bioengineering (Dalian) Co., Ltd；Pulullan is purchased from Sigma Co., USA；Primer synthesis and sequencing Work is completed by Guangzhou Ai Ji Bioisystech Co., Ltd.

3, culture medium

LB culture medium includes: peptone 1.0% (w/v), yeast powder 0.5% (w/v), NaCl 0.5% (w/v), pH 7.0；1.5% (w/v) agar is added in solid medium on this basis, and the unit of w/v is g/mL.

Primary operational method of the invention is as follows, and the place of not being described further does not refer to " Molecular Cloning:A Laboratory guide " (J. Pehanorm cloth Shandong Gram, the Beijing the D.W. Russell Molecular Cloning:A Laboratory guide third edition [M]: Science Press, 2002.) or Ago-Gel DNA Purification kit specification (Tiangeng biochemical technology Co., Ltd).

The clone of 1 Pullulanase gene pulA of embodiment

The I type Pullulanase gene that a plurality of bacillus genus source is chosen in ncbi database, uses ClustalX Software carries out sequence alignment, determines its conserved region, in combination with codon preference, designs three pairs of degenerate primers: PF1 and PR1； PF2 and PR2；PF3 and PR3 passes through using the full-length genome of Paenibacillus puldeungensis LK18 as template Its portion gene of Touchdown PCR amplification, reaction condition are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s；65 DEG C of annealing 30s, 68 DEG C of extensions (extension of time 1min/kb), each cycle annealing temperature reduce by 0.5 DEG C, totally 30 circulations；Then 94 DEG C of changes Property 30s；50 DEG C of annealing 30s；68 DEG C of extensions (extension of time 1min/kb)；10 circulations；Last 68 DEG C of heat preservations 10min.It will amplification Product carries out the detection of 1% (w/v, unit g/mL) agarose gel electrophoresis, and glue recycles the sequencing of purpose band Hou Song company.

Upstream primer PF1:5 '-GCTTTCATYACKGCSTATTGCAAAGT-3 ', i.e., shown in SEQ ID NO.3.

Downstream primer PR1:5 '-TAATTCTTCGGATCDTASCCCCAGTT-3 ', i.e., shown in SEQ ID NO.4.

Upstream primer PF2:5 '-TGGAACGAGGCCGCCGAYCCNTAYGC-3 ', i.e., shown in SEQ ID NO.5.

Downstream primer PR2:5 '-AGGCCCATCAGGTCGAACCKRAANCCRTC-3 ', i.e., shown in SEQ ID NO.6.

Upstream primer PF3:5 '-GACGTGTAYAACCAYGTNTATGACGG-3 ', i.e., shown in SEQ ID NO.7.

Downstream primer PR3:5 '-TACCCYAGWATGAAKAGACCCGC-3 ', i.e., shown in SEQ ID NO.8.

The overlapping region of sequencing result is compared by DNAMAN, is carried out sequence assembly (as shown in Figure 1), is committed on NCBI ORF Finder analyzed, further obtain pulA gene order.According to pulA gene order and expression vector pET-28a The multiple cloning sites of (+) (this carrier is purchased from Invitrogen biotech firm) redesign primer (Pul-F, Pul-R), amplify PulA full length gene (as shown in Fig. 2 a, Fig. 2 b, Fig. 2 c and Fig. 2 d).PCR reaction condition are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of changes Property 30s；59 DEG C of annealing 30s；68 DEG C of extension 2min, totally 30 recycle；Last 68 DEG C of heat preservations 10min.Amplified production passes through 1% (w/v, unit g/mL) agarose gel electrophoresis is detected, and will connect the flat end pEASY-Blunt after the recycling of purpose band glue Carrier (this carrier is purchased from Beijing Quanshijin Biotechnology Co., Ltd), constructs cloned plasmids pEASY-Blunt-pulA, and turn Enter JM110 competent cell.Bacterium colony PCR verifying is carried out through the multiple single colonies of blue hickie screening picking, correct bacterial strain will be verified It send after expanding culture to company's sequencing；

Expand upstream primer Pul-F:5 '-CGCGGATCC(dashed part is TTGTCAGTACAGAAGGAAAGCAATG-3 ' BamHI restriction enzyme site), i.e. SEQ ID NO.9.

Expand downstream primer Pul-R:5 '-CCCAAGCTT(dashed part is CTACTCCGCGATGCTCAGCAC-3 ' HindIII restriction enzyme site), i.e. SEQ ID NO.10.

The building of 2 recombinant plasmid of embodiment

To verify after correct positive clone molecule expands culture and extract plasmid pEASY-Blunt-pulA, with Bam HI and HindIII carries out double digestion, by the glue recovery product and pET-28a (+) double digestion glue recovery product T4DNA after its double digestion Ligase connects under the conditions of 16 DEG C, Connection Time 12h.Connection product is converted thin to E.coli BL21 (DE3) competence It in born of the same parents, is coated on the LB plate containing 50 μ g/mL kanamycins, 37 DEG C of culture 12h.Picking monoclonal carries out bacterium colony PCR and tests Card, verifying correct bacterial strain is recombinant bacterial strain BL21 (DE3)/pET-28a (+)-pulA.

The preparation of the recombination Pullulanase of embodiment 3

Picking recombinant bacterial strain BL21 (DE3)/pET-28a (+)-pulA is inoculated into the LB liquid medium of 20mL (containing 50 μ g/ ML kanamycins) in, 37 DEG C, cultivate 12h under the conditions of 200r/min.Bacterium solution is inoculated into 50mL with the inoculum concentration of 1% (v/v) In LB liquid medium (containing 50 μ g/mL kanamycins), continue shaken cultivation to its OD₆₀₀When reaching 0.6, it is added final concentration of The IPTG of 0.5mmol/L, after cultivating 12h under the conditions of 22 DEG C, 200r/min, thalline were collected by centrifugation, and with 0.1mol/L phosphoric acid Buffer (pH 8.0) washing thalline.Then buffer is added and is made into cell concentration in the section 50mg/mL-100mg/mL Re-suspension liquid, on ice after ultrasonication 20min, 4 DEG C, 12000r/min centrifugation 30min collect supernatant, as crude enzyme liquid.Make Purified with the nickel column HisTrap HP of GE company to crude enzyme liquid, collects eluting peak, its albumen is measured using Bradford method Content, and be 5% concentration glue with mass percent concentration, mass percent concentration is that 10% separation gel carries out SDS-PAGE inspection It surveys.As the result is shown (as shown in Figure 3): after Ni column affinitive layer purification, recombinase has obvious single albumen one at 76.95ku Band, measuring its specific enzyme activity is 508.8U/mg.

4 Pullulanase vigour-testing method of embodiment

For the present invention using DNS method measurement recombination Pullulanase vigor, concrete operations are as follows: 100 μ L being taken to dilute suitable multiple It is slow with 100mmol/L PBS that the recombination Propiram enzyme solution of (extension rate is 60-120 times, is herein 100 times) is added to 100uL In 1% (w/v, unit g/mL) pulullan solution made of fliud flushing (pH 6.0) configuration, after reacting 30min at 45 DEG C, it is added 300 μ L DNS reagents terminate reaction, boiling water bath 10min.It is settled to 2.5mL with distilled water after taking-up is cooling, is measured at 540nm The light absorption value of reaction solution.To boil the enzyme solution after 10min inactivation treatment as control.Pullulanase vigor is defined as: in phase Under the conditions of answering, 1 enzyme activity unit (U) is defined as with enzyme amount needed for generating 1 μm of ol glucose per minute.

The characterization analysis of the recombination Pullulanase of embodiment 5

1, influence and temperature stability of the temperature to recombination Pullulanase vigor

Under the conditions of 6.0 pH, the recombination Propiram of (extension rate is 60-120 times, is herein 100 times) will be suitably diluted Enzyme solution and 1% (w/v, unit g/mL) pulullan are respectively placed under different temperatures (25,30,35,40,45,50,55,60 DEG C) 30min is reacted, measures its Pullulanase vigor respectively, is 100% control with enzyme activity soprano, explores the optimal reaction of the enzyme Temperature.The recombination Propiram enzyme solution that (appropriate extension rate is 60-120 times, is herein 100 times) will suitably be diluted is respectively placed in 35,15,30,45,60,75,90,105,120min are kept the temperature under the conditions of 40,45,50 DEG C, are then added to 1% (w/v, unit g/ ML 30min) is reacted in pulullan under optimum temperature, measures its remaining enzyme activity, respectively with the enzyme solution of non-isothermal holding Enzyme activity probes into the temperature stability of the Pullulanase as control.As the result is shown: the optimal reactive temperature of the recombinase is 45 DEG C (such as Fig. 4), relatively stable at 35 DEG C -40 DEG C, remaining enzyme activity is up to 60% or more after keeping the temperature 120min, when holding temperature is higher than 45 DEG C when, recombination enzyme activity stability is poor, and opposite enzyme activity is 43.65% (such as Fig. 5) after 30min is kept the temperature at 50 DEG C.

2, influence and pH stability of the pH to recombination Pullulanase vigor

At 45 DEG C, will appropriate diluted enzyme solution (extension rate is 60-120 times, is herein 100 times) respectively with pH 1% (w/v, unit g/mL) pulullan made of the buffer configuration of 3.0-10.0 reacts 30min, measures its Propiram enzyme activity Power is 100% control with enzyme activity soprano, studies the optimal reaction pH of the enzyme.It will appropriate diluted enzyme solution (appropriate dilution times Number is 60-120 times, is herein 100 times) it is respectively placed in pH 3.0-10.0 buffer after room temperature preservation 1h, in optimal pH and most Its remaining enzyme activity is measured at suitable temperature, using the enzyme activity of untreated enzyme solution as control, the pH for probing into the Pullulanase is steady It is qualitative.The results show that the optimal reaction pH of the recombinase is 6.0 (such as Fig. 6), high stability under the conditions of pH 6.0-8.0, Enzyme activity still 60% or more residue after heat preservation 1h, after keeping the temperature 1h under the conditions of 9.0 pH, remaining enzyme activity is for 45.32% (such as Fig. 7 institute Show).

3, influence of the metal ion to enzyme activity

Different metal ions is added in 1% (w/v, unit g/mL) pulullan, makes its final concentration of 10mmol/L, Under the conditions of 45 DEG C, 6.0 pH, with appropriate diluted enzyme solution (appropriate extension rate is 60-120 times, is herein 100 times) reaction 30min measures corresponding Pullulanase vigor, using be not added metal ion enzyme solution enzyme activity as control.As the result is shown (as shown in Figure 8): the K of 10mmol/L⁺And Mg²⁺Have activation to the recombinase, make its enzyme activity activate respectively to 119.36% and 123.68%；The Zn of 10mmol/L²⁺、Mn²⁺、Ni²⁺、Fe²⁺、Cu²⁺、Co²⁺、Ca²⁺Have in various degree to recombinase Inhibiting effect, wherein Cu²⁺It is most strong to the inhibiting effect of the recombinase, so that its enzyme activity is only remained 16.73%.

Above embodiments are only preferrred embodiment of the present invention, for explaining only the invention, are not intended to limit the present invention, this Field technical staff should belong to guarantor of the invention without departing from change made under spirit of the invention, replacement, modification etc. Protect range.

Sequence table

<110>South China Science & Engineering University

<120>a kind of from the novel Pullulanase of series bacillus and its gene and application

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<213>the bacterial strain LK18 (Paenibacillus puldeungensis LK18) of production type Pullulanase

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ggagcgacct acacgccggg gcagacgaag tttcgacttt gggcgccgac ggcttctgag 180

gcgaaggtga tattttacaa aacgtgggac ggggaaccgg agcgggaact gtccatgaag 240

cgggatgtgc agggaacttg gatactcacc gtcatggagg attgcgccaa tattttctac 300

acgtaccgtg ttaaggttgg cgatcagtgg aatgaagccg ctgacccgta tgccaaagcg 360

gtaggggtaa atggggataa agcggtagtc ctctatctgc gaagcacaga cccggaaggc 420

tggaacaagg agaagccact ttttgactct tccgtggacg cagtgattta tgagcttcat 480

gttcgcgatt tatcgattca tcctagcagc ggcattgatc ctcaaaacca aggaaagttt 540

ctgggccttg ctgaaagcgg tacaaaaggg cctggtggaa tcgccacagg tctcgatcat 600

attgccgggc ttggggtgac gcatgttcag cttctgccga tcttcgatta cgcaacggaa 660

agcgtggatg agcaaaagct cgaccagccg cactacaact gggggtacga tccgaaaaat 720

tacaacgtac ctgaaggctc ttacgcaacc gatccgtatt cccctgcgct gcgcattacg 780

gagctgaaac gaatgattca ggagctgcac gaccgggggc tccgggtcat tatggacgtg 840

gtgtataacc acgtatatga cgggtacctg acccacttca gcaaactagt tcccggttat 900

tacttgcgct acaaaccgga tggaactttt tcaaatggtg cgttttgcgg aaacgagtgt 960

gcctcggagc ggccgatgat gtcaaagtat atcgttgatt cagtacttca ctgggttcgc 1020

gagtaccata tcgatggctt ccggtttgac ctgatgggcc tgatcgatat aagtactatg 1080

aacgagattc gacggcagct tcaggagata gacccttcgt tgatgctgct cggcgaaggt 1140

tggatcatgg atacggttct tccggaagct gcgagggcca atcagactaa tgcggctcag 1200

ctgccgggta tcggtttctt taatgacgga cttcgggacg cggtcaaagg ggatattttt 1260

cagtttgaaa aaaccgggtt catcagtggg ggaggcggct ttgaggagag cgtcaagcgt 1320

ggcgtcgtcg gaggtatcga ttatggcggc acaatccggc aatttgccgt agaccctgga 1380

cagtcggtga actatgtcga gtgccacgac aaccacacat tgtgggacaa aatcgtgctg 1440

tctactcccg gagtgaatga cgaacatcgc cgtgcgatgc accgccttgc ctcagccatc 1500

gtgatgacta gccaggggat tccgtttatc catgccggac aggagtttat gcgaacgaaa 1560

gacggcgtgg aaaacagcta caaatcccca attgagatca actggctcga ttgggagcgc 1620

tgcgcagcac accagtatga cgtagcctat atgcggagcc tgatcgagct gcgcaaggcg 1680

catcgggcgt ttcgcctgcg aacggcggag gagattcggg agcatttaca gtttgaagat 1740

gctccgcctc ataccgtagc ctatacgctg cgggatcatg ccggaggcga tgcagctcgc 1800

cacttgtatg tgctctacaa cgcggcgtca ccggaggcgg tcaccttgcg cctaccagag 1860

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ctagtcgtcc aaggcatcgg tatggtcgtg ctgagcatcg cggagtag 1968

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

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Thr Lys Phe Arg Leu Trp Ala Pro Thr Ala Ser Glu Ala Lys Val Ile

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Phe Tyr Lys Thr Trp Asp Gly Glu Pro Glu Arg Glu Leu Ser Met Lys

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Arg Asp Val Gln Gly Thr Trp Ile Leu Thr Val Met Glu Asp Cys Ala

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

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Ala Ala Asp Pro Tyr Ala Lys Ala Val Gly Val Asn Gly Asp Lys Ala

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Val Val Leu Tyr Leu Arg Ser Thr Asp Pro Glu Gly Trp Asn Lys Glu

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Lys Pro Leu Phe Asp Ser Ser Val Asp Ala Val Ile Tyr Glu Leu His

145 150 155 160

Val Arg Asp Leu Ser Ile His Pro Ser Ser Gly Ile Asp Pro Gln Asn

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Gln Gly Lys Phe Leu Gly Leu Ala Glu Ser Gly Thr Lys Gly Pro Gly

180 185 190

Gly Ile Ala Thr Gly Leu Asp His Ile Ala Gly Leu Gly Val Thr His

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Val Gln Leu Leu Pro Ile Phe Asp Tyr Ala Thr Glu Ser Val Asp Glu

210 215 220

Gln Lys Leu Asp Gln Pro His Tyr Asn Trp Gly Tyr Asp Pro Lys Asn

225 230 235 240

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

245 250 255

Leu Arg Ile Thr Glu Leu Lys Arg Met Ile Gln Glu Leu His Asp Arg

260 265 270

Gly Leu Arg Val Ile Met Asp Val Val Tyr Asn His Val Tyr Asp Gly

275 280 285

Tyr Leu Thr His Phe Ser Lys Leu Val Pro Gly Tyr Tyr Leu Arg Tyr

290 295 300

Lys Pro Asp Gly Thr Phe Ser Asn Gly Ala Phe Cys Gly Asn Glu Cys

305 310 315 320

Ala Ser Glu Arg Pro Met Met Ser Lys Tyr Ile Val Asp Ser Val Leu

325 330 335

His Trp Val Arg Glu Tyr His Ile Asp Gly Phe Arg Phe Asp Leu Met

340 345 350

Gly Leu Ile Asp Ile Ser Thr Met Asn Glu Ile Arg Arg Gln Leu Gln

355 360 365

Glu Ile Asp Pro Ser Leu Met Leu Leu Gly Glu Gly Trp Ile Met Asp

370 375 380

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

385 390 395 400

Leu Pro Gly Ile Gly Phe Phe Asn Asp Gly Leu Arg Asp Ala Val Lys

405 410 415

Gly Asp Ile Phe Gln Phe Glu Lys Thr Gly Phe Ile Ser Gly Gly Gly

420 425 430

Gly Phe Glu Glu Ser Val Lys Arg Gly Val Val Gly Gly Ile Asp Tyr

435 440 445

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

450 455 460

Tyr Val Glu Cys His Asp Asn His Thr Leu Trp Asp Lys Ile Val Leu

465 470 475 480

Ser Thr Pro Gly Val Asn Asp Glu His Arg Arg Ala Met His Arg Leu

485 490 495

Ala Ser Ala Ile Val Met Thr Ser Gln Gly Ile Pro Phe Ile His Ala

500 505 510

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

515 520 525

Ser Pro Ile Glu Ile Asn Trp Leu Asp Trp Glu Arg Cys Ala Ala His

530 535 540

Gln Tyr Asp Val Ala Tyr Met Arg Ser Leu Ile Glu Leu Arg Lys Ala

545 550 555 560

His Arg Ala Phe Arg Leu Arg Thr Ala Glu Glu Ile Arg Glu His Leu

565 570 575

Gln Phe Glu Asp Ala Pro Pro His Thr Val Ala Tyr Thr Leu Arg Asp

580 585 590

His Ala Gly Gly Asp Ala Ala Arg His Leu Tyr Val Leu Tyr Asn Ala

595 600 605

Ala Ser Pro Glu Ala Val Thr Leu Arg Leu Pro Glu Leu Gly Glu Trp

610 615 620

Gln Val Arg Tyr Gly Gly Glu Phe Val Gln Thr Leu Ser Gly Asn Gln

625 630 635 640

Leu Val Val Gln Gly Ile Gly Met Val Val Leu Ser Ile Ala Glu

645 650 655

<210> 3

<211> 26

<212> DNA

<213>artificial synthesized (artificial sequence)

<400> 3

gctttcatya ckgcstattg caaagt 26

<210> 4

<211> 26

<212> DNA

<213>artificial synthesized (artificial sequence)

<400> 4

taattcttcg gatcdtascc ccagtt 26

<210> 5

<211> 26

<212> DNA

<213>artificial synthesized (artificial sequence)

<400> 5

tggaacgagg ccgccgaycc ntaygc 26

<210> 6

<211> 29

<212> DNA

<213>artificial synthesized (artificial sequence)

<400> 6

aggcccatca ggtcgaacck raanccrtc 29

<210> 7

<211> 26

<212> DNA

<213>artificial synthesized (artificial sequence)

<400> 7

gacgtgtaya accaygtnta tgacgg 26

<210> 8

<211> 23

<212> DNA

<213>artificial synthesized (artificial sequence)

<400> 8

tacccyagwa tgaakagacc cgc 23

<210> 9

<211> 34

<212> DNA

<213>artificial synthesized (artificial sequence)

<400> 9

cgcggatcct tgtcagtaca gaaggaaagc aatg 34

<210> 10

<211> 30

<212> DNA

<213>artificial synthesized (artificial sequence)

<400> 10

cccaagcttc tactccgcga tgctcagcac 30

Claims

1. a kind of encoding gene of the novel Pullulanase from series bacillusPulA,It is characterized in that, nucleotide sequence As shown in SEQ ID NO.1.

2. a kind of novel Pullulanase from series bacillus, which is characterized in that amino acid sequence such as SEQ ID NO.2 institute Show.

3. the novel Pullulanase according to claim 2 from series bacillus, which is characterized in that described novel general Shandong orchid enzyme is type Pullulanase.

4. a kind of recombinant plasmid, which is characterized in that insert nucleotide sequence described in SEQ ID NO.1 in claim 1.

5. a kind of recombinant plasmid according to claim 4, which is characterized in that the recombinant plasmid pass through bypulACoding The multiple cloning sites of gene cloning to expression vector pET-28a (+) are obtained, and pET-28a (+)-is denoted aspulA。

6. a kind of engineering strain, which is characterized in that contain recombinant plasmid as claimed in claim 4.

7. engineering strain according to claim 6, which is characterized in that host cell is e. coli bl21 (DE3).

8. according to claim 1 from the encoding gene of the novel Pullulanase of series bacilluspulA, claim 4 The recombinant plasmid, engineering strain as claimed in claim 6 derive from class gemma bar described in the preparation claim 2 The application of the novel Pullulanase of bacterium.

9. application according to claim 8, which is characterized in that comprise the following steps that

(1) Pullulanase genepulAClone；

(2) construction recombination plasmid；

(3) by recombinant plasmid transformed host cell described in step (2), recombinant bacterial strain is obtained, i.e., the described engineering strain；

(4) expression and purifying of the novel Pullulanase from series bacillus.

10. a kind of application from the novel Pullulanase of series bacillus in Fermentation Engineering described in claim 2.