CN107475224A - A kind of new thermophilic Pullulanase, preparation method and applications - Google Patents

Abstract

The invention belongs to Pullulanase preparing technical field, and in particular to a kind of new thermophilic Pullulanase, preparation method and applications patent application matters.By cloning specific Thermophilic BacteriaThermosipho melanesiensis（DSM1202）Pullulanase gene, and then obtain thermophilic Pullulanase using carrier conversion heterologous strain structure recombinant strains, final heterologous induced expression.Preliminary characterization analysis shows, the appropriate pH 5.6 ~ 6.0 of prepared thermophilic Pullulanase, preference temperature is 75 DEG C ~ 85 DEG C, with higher heat endurance, catalytic efficiency and the higher affinity to pulullan substrate, it is worth with certain production application, and preferably application effect also provides for other bacterial strain screenings and biology enzyme preparation and preferably uses for reference reference.

Description

A kind of new thermophilic Pullulanase, preparation method and applications

Technical field

The invention belongs to Pullulanase preparing technical field, and in particular to a kind of new thermophilic Pullulanase, preparation method And its apply patent application matters.

Background technology

Starch is the biomass resource enriched on the earth, and it is widely present in the seed of plant, root tuber, stem tuber.Starch In addition to directly as raw-food material, also other valuable materials can be generated by the method for chemistry or biology, including：Amino Acid, glucose syrup, organic acid, alcohol etc..Native starch is by amylose（amylose）And amylopectin（amylopectin） Composition, wherein, amylose is the polysaccharide being formed by connecting by 250-300 D-Glucose molecule with α-Isosorbide-5-Nitrae-glycosidic bond, and it contains Amount accounts for the 15%-25% of starch total amount；And amylopectin is hyperbranched structure, by 24-30 glucose residue with α -1, 4- glycosidic bonds, which join end to end, to be formed, and just has a branch every about 20 glucose molecules, in bifurcation by α -1,6- glycosidic bonds Connection, wherein α -1, the content of 6- glycosidic bonds accounts for 5%, and the content of amylopectin accounts for the 75%-85% of starch total amount.So And most amylolytic enzyme（Such as alpha-amylase, beta amylase, glucoamylase）α -1,6- glycosidic bonds are not risen The complete decomposition that can not make starch, therefore the hydrolysis effect of α -1,6- glycosidic bond is used in combination in effect, alpha-amylase and carbohydrase Rate directly influences the utilization rate of starch.

Pullulanase is a kind of important starch debranching enzymes, and the whole side chain of amylopectin can be cut with selectivity, so as to Amylose is formed, therefore Pullulanase has a very important role in starch processing industry.Acted on according to Pullulanase Substrate specificity, Pullulanase can be divided into two classes：I type Pullulanases（EC.3.2.1.41）General Shandong can be hydrolyzed with selectivity α -1 in blue sugar and amylopectin, 6- glycosidic bonds, produces maltotriose（maltotriose）And amylose；II type Propiram Enzyme（EC.3.2.1.1/41）, also known as starch Pullulanase can not only hydrolyze the α -1,6- in pulullan and amylopectin Glycosidic bond, while also hydrolyzable α-Isosorbide-5-Nitrae-glycosidic bond therein.

The application of Pullulanase industrially is very extensive, but the heat endurance of most of Pullulanase and specific enzyme activity are not Height, or the environment of acidic high-temperature is not adapted to, and research of the China to Pullulanase starts from the 1970s, rising Step is than later.But domestic market is big to the demand of Pullulanase, import is relied primarily on, it is expensive.This is certain The development of domestic relevant industries is limited in degree.Therefore, Pullulanase of the exploitation with independent intellectual property right just seems particularly It is important.

The content of the invention

The application purpose is that provide one passes through institute after heterogenous expression using the thermophilic Pullulanase gene of specific bacterial strain The heat-resisting Pullulanase prepared, the enzyme have preferable heat endurance, catalytic efficiency and the affinity higher to pulullan substrate Power, there is certain application prospect during amylorrhexis.

Details are as follows for the technical scheme that the application is taken.

The preparation method of thermophilic Pullulanase, by cloning the Pullulanase gene of specific thermophilus strain, and then utilize load Body conversion heterologous strain structure recombinant strains, final heterologous induced expression obtain thermophilic Pullulanase, specifically included as follows Step：

（1）Clone the Pullulanase gene of thermophilus strain；

The Thermophilic Bacteria that numbering with German strain collections DSMZ preservations is DSM12029Thermosipho melanesiensisBased on, its genome is extracted, as template, designs primer, PCR amplifications obtain Pullulanase gene When TM-pulA, PCR are expanded, primer sequence specific design is as follows：

Sense primer FTM：5 '-CTAGCTAGCATGAAAAGATTGTTAGTGTTTTTCTTTGTTTTGTTATC-3 ', 5 ' ends GCTAGC partial sequences are NheI restriction enzyme sites；

Anti-sense primer RTM：5 '-TCCGCTCGAGTTTGTTCTTGTAAAAAACATATGCAGAAATTCCTTC-3 ', 5 ' ends CTCGAG partial sequences are XhoI restriction enzyme sites；

After electrophoresis detection is carried out to pcr amplification product and purifies acquisition Pullulanase gene TM-pulA, 4 DEG C save backup；

After clone's gained further sequencing analysis of Pullulanase gene TM-pulA, its base sequence is as shown in SEQ ID NO.1；

（2）Double digestion, and connect structure and obtain TM-pulA-pET21a expression vectors；

To step（1）In after purification Pullulanase gene TM-pulA carry out NheI and XhoI double digestions, and reclaim digestion products；

NheI and XhoI double digestions are carried out to pET21a plasmids simultaneously, and reclaim digestion products；

Utilize T4 ligases（T4 DNA ligase）To the enzyme of Pullulanase gene TM-pulA and the pET21a plasmid reclaimed Cut product to be attached, structure obtains TM-pulA-pET21a expression vectors；

（3）Heat-shock transformed and Screening and Identification；

By step（2）In connection product（Constructed TM-pulA-pET21a expression vectors）Converted with heat shock method to Escherichia coli Rosetta（DE3）Competent cell, competent cell is coated on the resistance LB flat boards containing chloramphenicol after conversion is recovered （The μ g/mL of chloramphenicol concentration final concentration 0.034）, 12 ~ 16 h are cultivated to carry out resistance screening；

The positive monoclonal bacterium colony on resistance LB flat boards is selected, carries out electrophoresis detection checking, and finally extraction plasmid is sequenced Checking is correct to ensure to convert and recombinate；

（4）Induced expression simultaneously produces enzyme liquid；

To step（3）The middle correct recombinant clone of identification is inoculated into the resistance LB culture mediums containing chloramphenicol（Chloramphenicol Concentration is 0.034 μ g/mL）, cultivate to OD₆₀₀Add IPTG to final concentration of 0.2 mmol/L when about 0.6,30 DEG C, 220 R/min cultivates 6h or so to carry out induced expression,

After induced expression terminates, bacterium solution is taken, thalline is collected by centrifugation, distilled water carries out ultrasonication after being resuspended, and centrifuges and receives after crushing It is crude enzyme liquid to collect supernatant, further, can carry out Co to crude enzyme liquid²⁺Chelating sepharose electrophoresis is pure to purify acquisition Pullulanase.

Utilize thermophilic Pullulanase prepared by the thermophilic Pullulanase preparation method.

Application of the thermophilic Pullulanase in amylorrhexis, during concrete application, appropriate pH 5.6 ~ 6.0 is most suitable pH5.8；Preference temperature is 75 DEG C ~ 85 DEG C, and optimum temperature is 80 DEG C.

For prepared by Pullulanase and improve, main in the prior art and Normal practice is screened from nature Production Pullulanase producing strains, and then obtain bacterial strain progress further screening or cultivating system optimization to publishing screening.It is but this Screening mode tool bears the character of much blindness and randomness, and screens and obtain with preferable heat endurance and preferable active Propiram Enzyme bacterium producing multi enzyme preparation possibility is relatively low, and prepared acquisition Pullulanase can not often adapt to specified temp and pH in actual production Demand, and then limit the improvement of associated production technology.

With technique for gene engineering progress, preliminary pre-sifted is carried out to related gene using bioinformatics method, can be significantly Reduce blindness and the screening cycle of related strain screening operation.The present invention be in this thinking, from database KEGG and Screening determines Thermophilic Bacteria in NCBI Thermosipho melanesiensis（DSM12029）.

To Thermophilic BacteriaThermosipho melanesiensis（DSM12029）Pullulanase gene cloned and different After the expression of source, preliminary characterization analysis shows, the appropriate pH 5.6 ~ 6.0 of prepared thermophilic Pullulanase, preference temperature 75 DEG C ~ 85 DEG C, there is higher heat endurance, catalytic efficiency and the higher affinity to pulullan substrate, have certain Production application value, and preferably application effect is also prepared to provide preferably to use for reference for other bacterial strain screenings and biology enzyme and joined Examine.

Brief description of the drawings

Fig. 1 is Pullulanase gene TM-pulA and the electrophoretogram of two step PCR primers, and wherein M is λ-Hind III Marker, 1, be TM-pulA genes PCR primer, 2 be pET21a plasmids, and 3 be TM-pulA double through NheI and XhoI Digestion；4 be pET21a plasmids through NheI and XhoI double digestions；

Fig. 2 is recombinant plasmid TM-pulA-pET21a electrophoretogram, and wherein M is the marker of λ-Hind III, and 1 is TM-pulA- PET21a recombinant plasmids；

The SDS-PAGE detection figures that Fig. 3 is pure enzyme TM-pulA, wherein M is protein marker, and arrow is signified in 1,2,3 It is pure enzyme TM-pulA；

The optimal pH measure figure that Fig. 4 is TM-pulA；

The optimum temperature measure figure that Fig. 5 is TM-pulA；

Fig. 6 is TM-pulA half-life period to determine figure；

Fig. 7 is the measure figure of TM-pulA enzyme kinetic analysis parameter (pulullan is as substrate).

Embodiment

Explanation is further explained to the application with reference to embodiment.It is just following first before specific embodiment is introduced It is related to the briefly introduction of the backgrounds such as part biological material, experiment reagent in embodiment to be described as follows.

Biomaterial：

Thermophilc anaerobeThermosipho melanesiensis, it is preserved in Germany Microbiological Culture Collection Center DSMZ, preservation DSM12029 is encoded to, is bought and obtained by inventor；

Escherichia coli Rosetta（DE3）, laboratory is often used transformed bacteria, obtained for open channel；

Relevant primer synthesizes and sequencing, and completion is provided by Jin Wei intelligence Science and Technology Ltd.；

Experiment reagent：

LB culture mediums（1 L）：The g of tryptone 10, the g of sodium chloride 10, the g of yeast extract 5, solid medium it is possible to additionally incorporate 1.5% agar powder, 121 DEG C, 30 min high pressure steam sterilizations；

Pulullan, Tokyo HuaCheng Industry Co., Ltd's product；

Yeast extract（Yeast Extract）, tryptone（Trytone）Purchased from OXID companies；

The super fidelity dna polymerases of restriction enzyme Nhe I and Xho I, T4 DNA Ligase, Q5 are purchased from NEB companies；

The DNA Marker of λ-Hind III are purchased from Shanghai offshore protein Science and Technology Ltd.；

Protein molecular weight Marker is purchased from Smobio Science and Technology Ltd.s；

Ago-Gel DNA QIAquick Gel Extraction Kits, DNA product purification kit, small amount plasmid extraction kit are purchased from Beijing rope Lai Bao Science and Technology Ltd.s；

Co²⁺Chelating sepharose TALON Metal Affinity Resin are purchased from TaKaRa companies.

Embodiment 1

Thermophilic Pullulanase provided herein, is made by the steps acquisition.

（1）Clone the Pullulanase gene of thermophilus strain；

The Thermophilic Bacteria that numbering with German strain collections DSMZ preservations is DSM12029Thermosipho melanesiensisBased on, it is standby to extract its genome.

It is as follows to design primer sequence：

Sense primer F_TM：5 '-CTAGCTAGCATGAAAAGATTGTTAGTGTTTTTCTTTGTTTTGTTATC-3 ', 5 ' ends GCTAGC partial sequences are NheI restriction enzyme sites；

Anti-sense primer R_TM：5 '-TCCGCTCGAGTTTGTTCTTGTAAAAAACATATGCAGAAATTCCTTC-3 ', 5 ' ends CTCGAG partial sequences are XhoI restriction enzyme sites；

Using extraction Thermophilic Bacteria DSM12029 genomes as template, utilize above-mentioned sense primer F_TMWith anti-sense primer R_TMEnter performing PCR Amplification, amplification program are designed as：

98 DEG C of min of pre-degeneration 3；98 DEG C of 40 s of denaturation, 66 DEG C of 15 s of annealing, 72 DEG C of extension s of 1 min 20, totally 35 circulate； 72 DEG C of 4 min of extension；

Electrophoresis detection is carried out to pcr amplification product（As a result it is as shown in Figure 1）And after purifying acquisition Pullulanase gene TM-pulA, 4 DEG C save backup.

（2）Double digestion, and connect structure and obtain TM-pulA-pET21a expression vectors；

To step（1）In after purification Pullulanase gene TM-pulA carry out NheI and XhoI double digestions, 37 DEG C of h of digestion 3, then 65 DEG C of 20 min of inactivation, digestion products reclaim digestion products, 4 DEG C save backup after purification after agarose gel electrophoresis detects Or directly carry out follow-up connection experiment.

NheI and XhoI double digestions are carried out to pET21a plasmids simultaneously, 37 DEG C of h of digestion 3, then 65 DEG C inactivate 20 min, Digestion products reclaim digestion products, 4 DEG C save backup or directly subsequently connected after purification after agarose gel electrophoresis detects Connect experiment.

Utilize T4 ligases（T4 DNA ligase）To Pullulanase gene TM-pulA and the pET21a plasmid reclaimed Digestion products（The TM-pulA and pET21a of linearisation）It is attached, 16 DEG C of connection 16h, then 65 DEG C of 20 min of inactivation（Even Thing of practicing midwifery directly carries out subsequent transformation experiment, or 4 DEG C save backup）, structure acquisition TM-pulA-pET21a expression vectors.

（3）Heat-shock transformed and Screening and Identification；

By step（2）In connection product（Constructed TM-pulA-pET21a expression vectors）Converted with heat shock method to Escherichia coli Rosetta（DE3）Competent cell, concrete operations are：

Take step（2）In the μ L of connection product 10 containing TM-pulA-pET21a expression vectors be added to 200 μ L Escherichia coli Rosetta（DE3）In competent cell, gently after piping and druming uniformly, ice puts 30 min, then 42 DEG C of s of heat shock 90, then ice puts 90 s；

Competent cell after recovery is coated on the resistance LB flat boards containing chloramphenicol（The μ of chloramphenicol concentration final concentration 0.034 g/mL）, 14 h or so are cultivated to carry out resistance screening.

The positive monoclonal bacterium colony on resistance LB flat boards is selected, after being expanded, carries out detection checking, concrete operations are：

By positive monoclonal colony inoculation into LB liquid medium（Containing the μ g/mL of chloramphenicol 0.034）, 37 DEG C, 220 r/min Cultivate 12 ~ 16 h or so；100 μ L zymotic fluids are taken, 12000 r/min centrifuge 1 min and collect thalline；Then it is fast that 100 μ L are added Examine liquid（Referring to Zhang Guimin etc., a kind of method of easy quick screening recon, 2005, Hubei University's journal (natural science Version)）, after fully mixing, 12000 r/min centrifuge 10 min；Then 6 ~ 7 μ L of supernatant are taken to carry out agarose gel electrophoresis checking, position Put is correctly positive colony（The result is as shown in Figure 2）；Plasmid is further extracted to correct positive colony to be sequenced Checking is correct to ensure to convert and recombinate.

（4）Induced expression simultaneously produces enzyme liquid；

Concrete operations are：To step（3）The middle correct recombinant clone of identification is inoculated into the resistance LB cultures containing chloramphenicol In base（Chloramphenicol concentration is 0.034 μ g/mL）, 37 DEG C of cultures to OD₆₀₀IPTG to final concentration of 0.2 is added when about 0.6 Mmol/L, 30 DEG C, 220 r/min culture 6h or so to be to carry out induced expression；

After induced expression terminates, bacterium solution is taken, 4 DEG C, 6000 r/min, 10 min of centrifugation, collects thalline, adding distilled water makes thalline Fully mix, placement carries out ultrasonic disruption on ice（3s × 4s × 99 time）, then 4 DEG C, 6000 r/min, 10 min of centrifugation, collect Supernatant is dispensed into 1.5 mL centrifuge tube, then 4 DEG C, 12000 r/min, 30 min of centrifugation, and it is crude enzyme liquid to collect supernatant； Further, Co is utilized²⁺Chelating sepharose obtains the Pullulanase TM-pulA of purifying to purifying the crude enzyme liquid（Detection knot Fruit is as shown in Figure 3）.

Embodiment 2

To be had gained some understanding to the zymologic property of the Pullulanase TM-pulA prepared by embodiment 1, Propiram is determined using DNS methods The enzyme activity of enzyme（Detected by taking crude enzyme liquid prepared by embodiment 1 as an example）, inventor to its optimal pH, optimum temperature, half-life period, Enzyme kinetic analysis etc. performance is detected, and related experiment is briefly discussed below.

The measure of optimum pH

PH gradients, which are respectively configured, is：5.0th, 5.2,5.4,5.6,5.8,6.0,6.2,6.4 citrate phosphate buffer；

Under condition of ice bath, in every 100 μ L enzyme liquids, 100 μ L, 1% pulullan are added（Mass fraction）PHs different with 100 μ L Citrate phosphate buffer；

15 min are reacted under the conditions of 70 DEG C, are eventually adding 600 μ L DNS solution terminating reactions, boil 10 min colour developings, cooling 5 mL are settled to distilled water afterwards, are mixed evenly, light absorption value is surveyed under 540 nm wavelength；

Using the enzyme liquid of inactivation as control, aforesaid operations are repeated；

Each reaction sets 3 repetitions, averages as final result.

As a result as shown in figure 4, measurement result shows, prepared Pullulanase TM-pulA optimal pH is 5.8.

The measure of optimum temperature

According to above-mentioned optimal pH course of reaction, under the conditions of pH5.8, exist respectively：55℃、60℃、65℃、70℃、75℃、80 DEG C, 85 DEG C, 90 DEG C, 95 DEG C reacted, compareed with the enzyme liquid of inactivation；When specific course of reaction determines with reference to above-mentioned optimal pH Operation；

As a result as shown in figure 5, measurement result shows, prepared Pullulanase TM-pulA optimum temperature is 80 DEG C, illustrates that this is general Shandong orchid enzyme is resistant to higher reaction temperature.

The measure of half-life period

Pullulanase TM-pulA crude enzyme liquids prepared by embodiment 1 are incubated at 70 DEG C, respectively in the 0th h of insulation, 1 h, 2 H, 3 h, 4 h, 5 h, 6 h, 7 h take out 450 μ L samples be placed in mixture of ice and water, after all take out after, pH5.8,80 DEG C Under the conditions of reacted, specific course of reaction with reference to above-mentioned optimal pH determine when operation；Measured with being incubated 0h enzyme liquid Enzyme activity as 100%, compareed with the enzyme liquid of inactivation.

As a result it is as shown in Figure 6.Measurement result shows, half inactivations of the prepared Pullulanase TM-pulA under the conditions of 70 DEG C Time is 4.75 h, illustrates that the enzyme has higher heat endurance.

Enzyme kinetic analysis property determines

Compound concentration is respectively：2 mg/mL、4 mg/mL、6 mg/mL、8 mg/mL、10 mg/mL、12 mg/mL、14 mg/ ML, 16 mg/mL pulullan solution are as substrate；

PH5.8, reacted under the conditions of 80 DEG C, compareed with the enzyme liquid of inactivation；Specific course of reaction is surveyed with reference to above-mentioned optimal pH The operation of timing.

The OD values measured are substituted into glucose Standard for Sugars curve, calculate Pullulanase TM-pulA hydrolysis various concentrations substrates Obtained reduced sugar quality, so as to calculate different concentration of substrate（[S]）The reaction speed of lower enzyme（v）, according to Hanes-Woolf Method is mapped, and makees scatter diagram with [S]/v- [S], and carry out linear fit and obtain a straight line, slope 1/V_max, with reference axis transverse axis Intercept be-K_m；Mapping is as shown in Figure 7.

Final calculation result shows：Pullulanase TM-pulA Km, Vmax, Kcat and Kcat/Km value is respectively 4.68 mg/mL、0.0085 μmol·mL^-1·s^-1、71.18 s^-1、15.21.As a result it is higher to show that Pullulanase TM-pulA has Substrate affinity and catalytic efficiency.

SEQUENCE LISTING

<110>Henan Yangshao Biochemical Engineering Co., Ltd.

<120>A kind of new thermophilic Pullulanase, preparation method and applications

<130> none

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 2529

<212> DNA

<213> Thermosipho melanesiensis

<400> 1

atgaaaagat tgttagtgtt tttctttgtt ttgttatctt ttattgcaat ttcatctaca 60

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tggcctatga aacccatagc aatggaagga aaatcctatc aatttacaga tcaagataat 180

tttggtgtag tagctaagat aacacttgac ctggatcttg aacaggtggg aataatcgta 240

aggctcaacg attggcaagc aaaagatgta gcaaaagata ggtttataga cataaaagat 300

ggaaaagcgg aggtgtggat tttgcaagga gtcgaggaaa tttacacaaa aaaacctgat 360

acaaggccaa gggtattttt cgcagctgca aaagcagata atgtaattga agcatattta 420

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gagggcttct ctccgtctac tgtttatatg ttagatgttt tagacaaatt ttattttgaa 660

ggtgagctcg gttacatata ctccaaaaac aggaccatat ttaggacctg gtctcctgtt 720

tcaaaatctg cacaagtttt attgtataga aacggagaag acaaaaaacc atacaaaata 780

gttaatatga aatacatggg aaatggtgta tgggaggcca gggttgatgg tgacttagat 840

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aatcctgatg gttggttcaa tgataaaaga ccatacatga aaacacccga agatgcaata 1020

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tatctcttca ctgtacctga aggaagatac tcaacagatc caaaaaatcc atacgctaga 1320

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gatatggtct tcccacatac atttggaata ggtgaacttt ccgcctttga tcaaacagta 1440

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aatgtgatag caacggaaag accgatgatg agaaaataca tagtagatac aattttgtat 1560

tggattaatg aatatcacgt ggatggattt agatttgatc aaatgggcct aatagataaa 1620

aaaactatgc tagaaattga aagaaaagca cacaaaattg ataagacgat tatattatat 1680

ggtgaacctt ggggaggttg gggagcacca attaggtttg gtaaatctga cgttgcacat 1740

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gaaaaggtaa aaggattttt aatgggggct attggaaaag aaacacgcgt taaaagagga 1860

gttgttggaa gtattcaata taattcacga ataaaaagct ttgcggcaga tcctgaagaa 1920

actattaatt acgttgcatg tcatgataat catacattgt gggataaaaa ttaccttgcg 1980

gcaaaatacg ataaaaaata taaatggaca gatgaaatgt taagaaacgc acaaaaactt 2040

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gactatcaaa gaaaatacga atatttagat gtatttgaat attacaaagg acttattaaa 2220

ctaagaaagg ggcacccggc atttagaatg acaaatgcac aagaaataaa agaacacatc 2280

aaatttttac ccagcagaaa aagaagaata gtttcttttg tcatatccaa tcacgcaaga 2340

aatgacgaat ggaaagatat tttggtaata tataatggaa atgttgattc tgtagaatat 2400

gaacttcctg aaggcgaatg gaatatgata gtaaatggga aaattgcagg aactgacatt 2460

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aacaaataa 2529

Claims (6)

1. a kind of preparation method of new thermophilic Pullulanase, it is characterised in that specifically comprise the following steps：

（1）Clone the Pullulanase gene of thermophilus strain；

The Thermophilic Bacteria that numbering with German strain collections DSMZ preservations is DSM12029Thermosipho melanesiensisBased on, its genome is extracted, as template, designs primer, PCR amplifications obtain Pullulanase gene When TM-pulA, PCR are expanded, primer sequence specific design is as follows：

Sense primer FTM：5′-CTAGCTAGCATGAAAAGATTGTTAGTGTTTTTCTTTGTTTTGTTATC-3′；

Anti-sense primer RTM：5′-TCCGCTCGAGTTTGTTCTTGTAAAAAACATATGCAGAAATTCCTTC-3′；

After electrophoresis detection is carried out to pcr amplification product and purifies acquisition Pullulanase gene TM-pulA, 4 DEG C save backup；

（2）Double digestion, and connect structure and obtain TM-pulA-pET21a expression vectors；

To step（1）In after purification Pullulanase gene TM-pulA carry out NheI and XhoI double digestions, and reclaim digestion products；

NheI and XhoI double digestions are carried out to pET21a plasmids simultaneously, and reclaim digestion products；

It is attached using the digestion products of Pullulanase gene TM-pulA and pET21a plasmid of the T4 ligases to being reclaimed, Structure obtains TM-pulA-pET21a expression vectors；

（3）Heat-shock transformed and Screening and Identification；

By step（2）In connection product converted with heat shock method to Escherichia coli Rosetta competent cells, and carry out resistance sieve Choosing；Positive monoclonal bacterium colony is selected to be verified；

（4）Induced expression simultaneously produces enzyme liquid；

To step（3）The middle correct recombinant clone of identification is inoculated into LB culture mediums, cultivates simultaneously induced expression；Induced expression After end, thalline is crushed using supercritical ultrasonics technology, and it is the crude enzyme liquid containing Pullulanase to centrifuge extraction supernatant.

2. the preparation method of thermophilic Pullulanase as claimed in claim 1, it is characterised in that step（4）In, use is final concentration of 0.2 mmol/L IPTG carries out induced expression.

3. the preparation method of thermophilic Pullulanase as claimed in claim 1, it is characterised in that step（4）In, crude enzyme liquid is carried out Co²⁺Chelating sepharose electrophoresis obtains pure Pullulanase to purify.

4. utilize thermophilic Pullulanase prepared by any one of the claim 1 ~ 3 thermophilic Pullulanase preparation method.

5. application of the thermophilic Pullulanase in amylorrhexis described in claim 4, it is characterised in that appropriate pH 5.6 ~ 6.0, fit Suitable temperature is 75 DEG C ~ 85 DEG C.

6. application of the thermophilic Pullulanase in amylorrhexis as claimed in claim 5, it is characterised in that optimal pH 5.8, it is most suitable Temperature is 80 DEG C.