CN102676480B - Method for producing extracellular pullulanase by applying auto-induction culture medium and dual-temperature control strategy - Google Patents

Abstract

The invention provides a method for producing extracellular pullulanase by applying an auto-induction culture medium and a dual-temperature control strategy, belonging to the technical field of pullulanase production through microbial fermentation. The method has the following beneficial effects: pullulanase coding genes from Klebsiella variicola CCTCC M2012108 are inserted into an expression vector pET28a(+) to construct recombinant plasmids and E.coli is converted to obtain a recombination strain E.coli BL21(DE3)/pET28a(+)-pulA containing the target pullulanase gene; the auto-induction culture medium is utilized to culture the recombinant E.coli BL21(DE3)/pET28a(+)-pulA and ferment the recombinant E.coli BL21(DE3)/pET28a(+)-pulA to generate enzyme by adopting the dual-temperature control mode of firstly culturing at 37 DEG C for 2-4 hours and then continuing culture at 25 DEG C for 48-72 hours; and after adopting the optimized fermentation conditions of the auto-induction culturemedium and dual-temperature, the extracellular pullulanase activity can reach 60-70U/mL. The method provides an effective strategy for producing extracellular pullulanase with recombinant E.coli and has great significance in the production process for developing novel recombinant pullulanase in future and application value of pullulanase.

Description

A kind of method of using self-induction substratum and the outer Pullulanase of two temperature regulating strategy production born of the same parents

Technical field

A kind of method of using self-induction substratum and the outer Pullulanase of two temperature regulating strategy production born of the same parents belongs to the technical field that microbial fermentation is produced Pullulanase.

Background technology

Pullulanase (pullulanase, EC 3.2.1.41) is that specificity is decomposed α-1 in Propiram, starch, amylopectin and the respective branch oligose, a kind of debranching factor of 6-glycosidic link.Compare with other Propiram lytic enzymes, Pullulanase is hydrolyzed α in the Propiram-1 specifically, and the 6-glycosidic link generates take trisaccharide maltose as main end products.This character has determined that it improving the action effect of amylase to starch, improve starch utilization ratio, reducing the grain consumption, improve the quality of products and develop and quite huge value is arranged aspect the new product, has important purposes and good market outlook in starch processing industry, food, washing composition, weaving etc.

Pullulanase be Bender the earliest Aureobasidium pullulans ( Aureobasidium pullulans) fermented liquid in find and its zymologic property be studied.After this, researcher separates microorganism and the plant that has obtained large volume production Pullulanase from occurring in nature.The enzymatic property difference in these sources is larger, as derives from Klebsiella planticola, optimal pH is 5.0, and optimum temperuture is at 40 ℃; And deriving from the Pullulanase (claim R-enzyme) of plant, the suitableeest pH and temperature are respectively 5.6 and 37 ℃.However, from the late 1970s that is found to of Pullulanase, the research of Pullulanase is in the evaluation of the screening of microbes producing cellulase and zymologic property always, the large-scale production of Pullulanase do not occur.Until early 1980s, Denmark Novozymes Company from Malay soil, screen a strain can produce the genus bacillus of Pullulanase ( Bacillus acidopullulytics).The Pullulanase that this bacterial strain is produced have heat-resisting acidproof characteristic (60 ℃ pH4.5) relatively are suitable for the needs of mashing industry.After this, Novozymes Company drops into huge fund this Pullulanase is studied, and releases simultaneously commercial Pullulanase (commodity are called Promozyme) in nineteen eighty-three in Japan and European market.At present, Novozymes Company has occupied the Pullulanase market in China and even the world.Until the nineteen ninety-five outstanding person can company of section utilize the Bacillus licheniformis heterogenous expression Bacillus deramificansPullulanase, its zymologic property with B. acidopullulyticsPullulanase is close, thereby makes outstanding person's energy section become the second largest manufacturer of Pullulanase.

Because Pullulanase suitability for industrialized production bacterial strain is less, the research of China on Pullulanase mainly concentrates on the aspects such as screening, mutagenesis and optimization of producing the Pullulanase bacterial strain, and effect is all not ideal.Obtain microorganism that a strain produce Pullulanase on-the-spot the separation such as the people such as the Xiao Min of Shandong University in 1998 from liquor fermentation, the Pullulanase thermotolerance of its production is relatively poor, rapid inactivation after temperature is greater than 50 ℃, but pH stability is better, can be in the medium-term and long-term preservation of refrigerator under neutral and slightly acidic condition.The people such as calendar year 2001 Tang Baoying separate the genus bacillus that obtains strain product Pullulanase from soil, it produces Pullulanase preferably (75 ℃ of zymologic properties, pH 4.6), but the enzymatic productivity of wild strain is not high, only have 1.6 U/mL, finally just make it produce enzyme level through selection by mutation and bring up to 8.8 U/mL.Along with Development of Molecular Biology, people begin to utilize genetic engineering means to express external source Pullulanase gene in recent years, wait such as summer in 2006 son virtue and utilize escherichia expression system to express the Pullulanase gene of Thermotoga maritima, live but only obtain intracellular enzyme; Zhang Mingyan in 2008 etc. will derive from Bacillus licheniformisThe Pullulanase encoding gene be cloned in the intestinal bacteria and express, but final enzyme is lived and is also only had 5.6 U/mL; 2010, Wang Shujun etc. utilized colibacillary expression system to realize deriving from the ancient bacterium in deep-sea Thermococcus siculiThe heterogenous expression of the Pullulanase encoding gene of HJ21, but the effect of heterogenous expression is relatively poor, and it is alive only to obtain intracellular enzyme.In sum, in the microbial fermentation production process of Pullulanase, no matter be wild mushroom or engineering bacteria, the main problem that exists is exactly that extracellular enzyme is alive lower, has caused the production cost of Pullulanase to rise, and forms the obstacle of suitability for industrialized production.

Self-induction (auto-induction) is to utilize carbon source conversion in the substratum by the propositions such as Studier of the Brooker Hei Wen National Laboratory of New York E Pudun a kind of the earliest and method that induction exogenous gene is expressed, namely at first support Escherichia coli Growth to saturated take glucose as carbon source, treat that glucose consumption totally, another kind of composition lactose sees through enzyme (lactose permease) and beta-galactosidase enzymes in the gene product of lac Y and lac Z (under the assistance of β-galactosidase), lactose passes cytolemma and Partial Conversion is the method for neolactose unlatching T7 expression system in the substratum.Lactose is except as the inductor, and its meta-bolites glucose and semi-lactosi (semi-lactosi also will change into glucose under the effect of gal operon) also can be used as the carbon source of bacterial growth.With traditional IPTG(isopropyl-β-D-thiogalactoside(IPTG)) induction method compares, self-induction is expressed process after inoculation does not need the growing state of cell is monitored, thereby reduced in culturing process the processing of culture, be very easy to high flux screening.And, substitute the high IPTG of price with cheap, nontoxic lactose and can reduce widely production cost, in the fermentative production of recombinant protein, have great significance.In addition, secretion inducing for recombinant protein is expressed, adopt two temperature stage by stage regulating strategy successfully be used for the activity expression of glucanotransferase secretory protein, increase biomass as under 37 ℃ of conditions, cultivating first, then reduce the temperature to 25 ℃ of synthesis rates that slow down recombinant protein to promote it to secrete to born of the same parents.Yet the self-induction substratum is mainly used in the expression of the isotopic labeling recombinant protein of nuclear magnetic resonance spectroscopy at present, there is no the report that the self-induction substratum is used for restructuring Pullulanase fermentative production.And, the federation policies of self-induction training method and two temperature regulation and control is used for restructuring Pullulanase fermentative production also rarely has report.

The present invention is successfully applied to self-induction substratum and two temperature control methods cultivation and the enzyme protein expression of Pullulanase genetic engineering bacterium, compare with the IPTG induction method with traditional LB substratum, recombinate the outward fermenting enzyme work of Pullulanase of born of the same parents rises to 70 U/mL from 11 U/mL.

Summary of the invention

The technical problem that (1) will solve

The purpose of this invention is to provide a kind of method of using self-induction substratum and the outer Pullulanase of two temperature regulating strategy production born of the same parents.The object of the invention not only is the outer Pullulanase by microorganism strains fermentative production born of the same parents, but the Pullulanase gene is recombinant expressed in intestinal bacteria, and utilize self-induction substratum and two temperature control methods further to improve recombination bacillus coli production born of the same parents outward ability and the output of Pullulanase, thereby production technique and the using value of development of new restructuring Pullulanase.

(2) technical scheme

The present invention at first will derive from a black seat klebsiella ( Klebsiella variicola) the Pullulanase gene of CCTCC NO:M 2012108 carries out recombinant expressedly in intestinal bacteria, on this basis, self-induction substratum and two temperature control methods is used for the cultivation of recombinant bacterium and the fermentative production of restructuring Pullulanase.

One, the acquisition of Pullulanase gene

Black seat klebsiella ( Klebsiella variicola) CCTCC NO:M 2012108 substratum: Zulkovsky starch 10 g/L, peptone 15 g/L, yeast extract paste 5 g/L, KH ₂PO ₄5 g/L, MgSO ₄7H ₂O 0.1 g/L, NH ₄Cl 0.5 g/L, FeSO ₄7H ₂O 0.1 g/L, pH 6.5.

To deceive a klebsiella ( Klebsiella variicola) CCTCC NO:M 2012108 bacterial classification inoculations are in the substratum liquid amount is 10% 250 mL shaking flasks, in 37 ℃, 200 rpm shaking culture, 72 h.After cultivate finishing, that thalline is centrifugal and use physiological saline washed twice, collecting cell to utilize genome DNA extracting reagent kit Genomic DNA Extraction Miniprep System(VIOGENE company) the extraction genome.

Synthetic primer 1:5'-CGG CTA GCA TGC TCA GAT ATA CCT GTC ATG G-3', synthetic primer 2:5'-CCG GAA TTC CGT TTA CTG CTC ACC GGC G-3'.Primer 1 contains NheThe I restriction enzyme site, primer 2 contains EcoRThe I restriction enzyme site.

PCR reaction system: ddH ₂O 37 μ L, 10 * Reaction Buffer, 5 μ L, dNTP(25 mmol/L) 0.5 μ L, primer 1(50 pmol/ μ L) 1 μ L, primer 2 (50 pmol/ μ L) 1 μ L, genomic dna 5 μ L, Taq DNA polymerase(5 U/ μ L) 0.5 μ L.

PCR reaction process: 94 ℃ of denaturation 5 min; 98 ℃ of 10 s, 55 ℃ of l min, 72 ℃ of 4 min carries out 30 circulations; 72 ℃ are extended 10 min.

Utilize 3S Spin Agarose Gel DNA Purification Kit(Shanghai Shenergy Biocolor BioScience ﹠ Technology Company) the purify DNA segment.

Sodium acetate solution (3 mol/L, pH 5.2) and 2 times of volume dehydrated alcohols of adding 1/10 volume in the dna solution ,-20 ℃ precipitate 1 hour.12000 rpm are in 4 ℃ of centrifugal 30 min.Add 75% ethanol, 500 μ L washing, 12000 rpm are dissolved in after aseptic operating platform dries up in an amount of TE damping fluid in 4 ℃ of centrifugal 30 min, use immediately or-20 ℃ of preservations.

Two, the structure that contains the recombination bacillus coli of Pullulanase gene

The enzyme of goal gene and plasmid pET28a (+) is cut

Utilize plasmid extraction kit Mini-Plasmid Rapid Isolation Kit(Beijing vast Tyke biological gene technology company limited) extraction plasmid pET28a (+).

Order according to water, damping fluid, PCR product or plasmid DNA, enzyme is added in the Eppendorf pipe, build the pipe lid, vibration makes the abundant mixing of liquid, placing interior centrifugal 2 s of whizzer that liquid is concentrated on manages at the end, 37 ℃ of water-bath 3 h, the Loading Buffer of adding 1/10 maybe places pipe 65 ℃ of insulation 10 min in pipe, stops endonuclease reaction.Enzyme is cut product and carried out the agarose gel electrophoresis analysis and cut glue recovery purpose fragment, and is concentrated.

Reaction composition: 10 * H Buffer, 4 μ L, DNA 10 μ L, NheI 2 μ L, EcoRI 2 μ L, ddH ₂O supplies 40 μ L with system.

Goal gene is connected with plasmid pET28a's (+)

Foreign gene is connected with plasmid pET28a (+), and reaction composition is as follows: plasmid pET28a (+) 0.8 μ L, foreign gene 4.2 μ L, Ligation Solution 5 μ L, ddH ₂O supplies 10 μ L with system.Mix connecting fluid, be placed on connection 12 h in 16 ℃ of incubators.

Utilize CCTCC NO:M 2012108 Pullulanase encoding genes that restriction enzyme NheI and EcoRI obtain amplification and carrier pET28a (+) to carry out double digestion and process, dna segment connects the recombinant plasmid pET28a (+) that obtains with the goal gene segment-pul A by sticky end after processing;

The recombinant plasmid transformed intestinal bacteria

100 μ L intestinal bacteria E. coliAdd 10 μ L in BL21 (DE3) the competent cell suspension and connect product, mixing leaves standstill 30 min in the ice bath gently.Change in 42 ℃ of water-baths thermal shock 90 s over to.Fast transfer cools off 2 min to ice bath.Add 700 μ L LB liquid nutrient mediums, 37 ℃ of 100 rpm shaking table incubation cultivated 1 h.Centrifugal 2 min of bacterium liquid 3000 rpm after cultivating abandon supernatant 600 μ L, are applied on the LB flat board that contains 50 μ g/mL kantlex behind the residue bacterium liquid mixing, are inverted for 37 ℃ and cultivate.Acquisition contains the recombination bacillus coli of purpose Pullulanase gene E. coliBL21 (DE3)/pET28a (+)-pul A.

Abduction delivering is cultivated

LB substratum: Tryptones 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, pH7.0.Add kantlex (50 μ g/mL) before using when needing, solid medium adds 1.5% agar powder.

Picking positive colony list colony inoculation contains in the LB liquid nutrient medium of 50 μ g/mL kantlex in 3 mL, and in 37 ℃, 200 rpm shaking culture are spent the night.Get 1 mL nutrient solution and transfer and contain in the LB liquid nutrient medium of 50 μ g/mL penbritins in 50 mL, in 37 ℃, 200 rpm shaking culture to OD ₆₀₀Be about 0.6.Add inductor IPTG in the culture to final concentration 0 ~ 1 mmol/L, under 30 ~ 37 ℃ of culture temperature, carried out inducing culture 12 hours.After cultivating end, fermented liquid must be fermented supernatant for bacterial strain extracellular enzyme mensuration alive after 10000 rpm centrifugal 10 minutes.Utilize LB substratum and IPTG induction mode, obtaining born of the same parents' Pullulanase vigor of recombinating outward is 11 U/mL.

The Pullulanase measuring method

Get 100 μ L with the acetate buffer of 100 mM, pH 5.0 suitably the enzyme liquid of dilution be mixed in mutually in 50 ℃ of water-baths with isopyknic 10 g/L Propirams that are dissolved in 100 mM, pH 5.0 acetate buffers and be incubated 30 minutes.Add DNS reagent 300 μ L and shake up, place boiling water to boil 15 minutes, take out with after the rapid cooling of the flowing water that flows, in 540 nm wavelength places, with 0.5 cm cuvette, the absorbance of assaying reaction liquid.

The enzyme unit definition of living: under the condition of above-mentioned appointment, it is 1 enzyme unit (U) that lives that per minute catalytic decomposition pulullan polysaccharide generates required awake of the reducing sugar that is equivalent to 1 μ mol glucose.

Three, the self-induction of recombination bacillus coli is cultivated and two temperature regulation and control enzymatic production

Self-induction substratum: alpha-lactose 10-20g/L, glucose 0.5-1 g/L, glycerine 5 g/L, KH ₂PO ₄6.8 g/L, MgSO ₄0.24 g/L, peptone 10 g/L, Na ₂HPO ₄12H ₂O 17.9 g/L, Na ₂SO ₄0.71 g/L, NH ₄Cl 2.67 g/L, trace element solution 200 μ L/L dispose with ultrapure water.

Trace element solution:

50 μ M FeCl ₃, 20 μ M CaCl ₂2H ₂O, 10 μ M MnCl ₂4H ₂O, 10 μ M ZnSO ₄7H ₂O, 2 μ M CoCl ₂6H ₂O, 2 μ M CuCl ₂, 2 μ M NiCl ₂, 2 μ M Na ₂Mo ₇O ₂₄, 2 μ M Na ₂SeO ₃, 2 μ M H ₃B ₄O ₇, dispose with ultrapure water.

With recombination bacillus coli E. coliBL21 (DE3)/pET28a (+)-pul A activation culture of on the LB solid culture that contains 50 μ g/mL kantlex, spending the night, the single colony inoculation of picking contains the liquid LB substratum of 50 μ g/mL kantlex in 37 ℃ in 3 mL from solid plate, 200 rpm shaking culture are monitored OD in the substratum at any time ₆₀₀Changing conditions.Treat OD ₆₀₀Reach at 2 ~ 3 o'clock, be inoculated in the self-induction substratum that 50 mL contain 50 μ g/mL kantlex by 5% ~ 10% inoculum size.Self-induction after the inoculation is cultivated based on 37 ℃, and 200 rpm cultivated after 2 ~ 4 hours, the temperature of cultivating is reduced to 25 ℃ continues to cultivate 48 ~ 72 hours.After cultivating end, the centrifugal removal thalline of 10000 rpm, collecting fermented supernatant fluid is the crude enzyme liquid of Pullulanase.

The optimization of self-induction medium component and fermentation condition

Carbon source, nitrogenous source and medium additives have been investigated respectively to engineering bacteria E. coliThe impact of BL21/pET28a (+)-pulA growth and secretion restructuring Pullulanase, determined that finally the optimization self-induction substratum of the outer Pullulanases of fermentative production born of the same parents forms:

Alpha-lactose 20 g/L, glucose 1 g/L, glycerine 5 g/L, KH ₂PO ₄6.8 g/L, MgSO ₄0.24 g/L, peptone, 10 g/L, Na ₂HPO ₄12H ₂O 17.9g/L, Na ₂SO ₄0.71 g/L, NH ₄Cl 2.67 g/L, Tween-80 9 g/L, trace element solution 200 μ L/L.

Investigate respectively initial pH, liquid amount, temperature, incubation time to extracellular enzyme impact alive, finally determined engineering bacteria E. coliThe culture condition that BL21/pET28a (+)-pulA produces enzyme is: initial pH5.0 ~ 8.0, liquid amount 10% ~ 20%, culture temperature be 37 ℃ cultivate first 4 hours after, change again 25 ℃ over to and cultivated 56 hours.

After adopting optimization self-induction substratum and two temperature fermentation condition, the outer Pullulanase enzyme work of born of the same parents can reach 60 ~ 70 U/mL.

(3) beneficial effect

Successfully cloned black seat klebsiella ( Klebsiella variicola) CCTCC NO:M 2012108 Pullulanase encoding genes, this full length gene 3309 bp, 1102 amino-acid residues of encoding, the nucleotides sequence of its gene is classified as: SEQ ID NO:1, its amino acid consist of SEQ ID NO:2.

To deceive a klebsiella ( Klebsiella variicola) CCTCC NO:M 2012108 Pullulanase encoding genes insert among the expression vector pET28a (+) and be transformed into corresponding expressive host E. coliSuccessfully make up the recombinant bacterial strain with goal gene among the BL21 (DE3) E. coliBL21 (DE3)/pET28a (+)-pul A.

Self-induction substratum and two temperature control methods are used for recombination bacillus coli E. coliBL21 (DE3)/pET28a (+)-cultivation of pul A and the enzymatic production of Pullulanase, behind optimization self-induction substratum and the two temperature fermentation condition, the outer Pullulanase enzyme work of born of the same parents can reach 60 ~ 70 U/mL.These work produce the ability of the outer Pullulanase of born of the same parents for recombination bacillus coli and the raising of output provides available strategy, and are significant for production technique and the using value of the novel restructuring Pullulanase of Future Development.

The biological material specimens preservation: black seat klebsiella ( Klebsiella variicola) SHN-1; Depositary institution: Chinese Typical Representative culture collection center, write a Chinese character in simplified form CCTCC, the address: Wuhan, China Wuhan University, deposit number CCTCC NO:M2012108, preservation date are on April 11st, 2012.

Embodiment

Embodiment 1

Self-induction substratum: alpha-lactose 10 g/L, glucose 0.5 g/L, glycerine 5 g/L, KH ₂PO ₄6.8 g/L, MgSO ₄0.24 g/L, peptone 10 g/L, Na ₂HPO ₄12H ₂O 17.9 g/L, Na ₂SO ₄0.71 g/L, NH ₄Cl 2.67 g/L, trace element solution 200 μ L/L.

With recombination bacillus coli E. coliThe bacterial classification of BL21 (DE3)/pET28a (+)-pul A lines on the LB solid medium that contains 50 μ g/mL kantlex overnight incubation in 37 ℃ of incubators.The single colony inoculation of picking places 37 ℃, 200 rpm shaking tables concussion cultivation, to OD in the 250 mL triangular flasks that 50 mL LB substratum (50 μ g/mL kantlex) is housed from flat board ₆₀₀Reach about 2.0 ~ 3.0.This seed culture fluid is inoculated in the 250 mL triangular flasks that the above-mentioned self-induction substratum of 50 mL is housed with 5% inoculum size, cultivates 60 hours (this moment OD in 37 ℃ ₆₀₀Reach about 15), finish fermentation.

Cultivate and enzymatic production technique by this recombinant bacterium, the enzyme work of the outer Pullulanase of born of the same parents reaches 15 U/mL in the fermentation supernatant.

Embodiment 2

Self-induction substratum: alpha-lactose 20 g/L, glucose 1 g/L, glycerine 5 g/L, KH ₂PO ₄6.8 g/L, MgSO ₄0.24 g/L, peptone 10 g/L, Na ₂HPO ₄12H ₂O 17.9 g/L, Na ₂SO ₄0.71 g/L, NH ₄Cl 2.67 g/L, trace element solution 200 μ L/L.

With recombination bacillus coli E. coliThe bacterial classification of BL21 (DE3)/pET28a (+)-pul A lines on the LB solid medium that contains 50 μ g/mL kantlex overnight incubation in 37 ℃ of incubators.The single colony inoculation of picking places 37 ℃, 200 rpm shaking tables concussion cultivation, to OD in the 250 mL triangular flasks that 50 mL LB substratum (50 μ g/mL kantlex) is housed from flat board ₆₀₀Reach about 2.0 ~ 3.0.This seed culture fluid is inoculated in the 250 mL triangular flasks that the above-mentioned self-induction substratum of 50 mL is housed with 5% inoculum size, cultivates 60 hours (this moment OD in 25 ℃ ₆₀₀Reach about 15), finish fermentation.

Cultivate and enzymatic production technique by this recombinant bacterium, the enzyme work of the outer Pullulanase of born of the same parents reaches 35 U/mL in the fermentation supernatant.

Embodiment 3

Self-induction substratum: alpha-lactose 20 g/L, glucose 1 g/L, glycerine 5 g/L, KH ₂PO ₄6.8 g/L, MgSO ₄0.24 g/L, peptone 10 g/L, Na ₂HPO ₄12H ₂O 17.9 g/L, Na ₂SO ₄0.71 g/L, NH ₄Cl 2.67 g/L, trace element solution 200 μ L/L.

With recombination bacillus coli E. coliThe bacterial classification of BL21 (DE3)/pET28a (+)-pul A lines on the LB solid medium that contains 50 μ g/mL kantlex overnight incubation in 37 ℃ of incubators.The single colony inoculation of picking places 37 ℃, 200 rpm shaking tables concussion cultivation, to OD in the 250 mL triangular flasks that 50mL LB substratum (50 μ g/mL kantlex) is housed from flat board ₆₀₀Reach about 2.0 ~ 3.0.This seed culture fluid is inoculated in the 250 mL triangular flasks that the above-mentioned self-induction substratum of 50 mL is housed with 5% inoculum size, after cultivating 4 hours in 37 ℃, 200 rpm shaking tables, again the temperature of shaking table is turned down 25 ℃ and continued to cultivate 56 hours (this moment OD ₆₀₀Reach about 15), finish fermentation.

Cultivate and enzymatic production technique by this recombinant bacterium, the enzyme work of the outer Pullulanase of born of the same parents reaches 50 U/mL in the fermentation supernatant.

Embodiment 4

Self-induction substratum: alpha-lactose 20 g/L, glucose 1 g/L, glycerine 5 g/L, KH ₂PO ₄6.8 g/L, MgSO ₄0.24 g/L, peptone 10 g/L, Na ₂HPO ₄12H ₂O 17.9 g/L, Na ₂SO ₄0.71 g/L, NH ₄Cl 2.67 g/L, Tween-80 9 g/L, trace element solution 200 μ L/L.

With recombination bacillus coli E. coliThe bacterial classification of BL21 (DE3)/pET28a (+)-pul A lines on the LB solid medium that contains 50 μ g/mL kantlex overnight incubation in 37 ℃ of incubators.The single colony inoculation of picking places 37 ℃, 200 rpm shaking tables concussion cultivation, to OD in the 250 mL triangular flasks that 50 mL LB substratum (50 μ g/ml kantlex) is housed from flat board ₆₀₀Reach about 2.0 ~ 3.0.This seed culture fluid is inoculated in the 250 mL triangular flasks that the above-mentioned self-induction substratum of 25 mL is housed with 5% inoculum size, after cultivating 4 hours in 37 ℃, 200 rpm shaking tables, again the temperature of shaking table is turned down 25 ℃ and continued to cultivate 56 hours (this moment OD ₆₀₀Reach about 15), finish fermentation.

Cultivate and enzymatic production technique by this recombinant bacterium, the enzyme work of the outer Pullulanase of born of the same parents reaches 70 U/mL in the fermentation supernatant.

<210>SEQ ID NO: 1

<211>3309

<212>DNA

＜213〉a black seat klebsiella ( Klebsiella variicola) CCTCC NO:M 2012108

<400>1

atgctcagat atacctgtca tgccctattt cttggatcgt tagtattatt gagtggctgt 60

gataacagct cttcctcttc accctctggc tcaccgggtt caccaggcaa tcctggcaac 120

ccaggcacgc ccggtacgcc cgacccgcag gatgtggtcg tccgcttacc ggacgttgcc 180

gtccctggcg aagcggcgca ggcttccgcc aaccaggctg tcattcacct tgtcgatatc 240

gccggcatca ccagcagcac gccggccgac tatgcgacta aaaacctcta tctatggaat 300

aacgaaacct gtgatgcgct gagcgcgccg gtggcggact ggaatgatgt cagcactacg 360

ccgaccggca gcgacaaata tggcccttac tgggtgatcc cgctgactaa agagagcgga 420

tgtatcaacg ttatcgtccg cgatggcacc aataagctta tcgacagcga cctgcgcgtc 480

tcttttggcg atttcaccga tcgcacggta tcggtcatcg ccggcaacag cgcggtctat 540

gactcccgcg ccgacgcctt ccgcgccgcc tttggcgtgg cgctggccga tgcgcactgg 600

gtcgataaaa ccaccctgct gtggccgggc ggcgaaaata aacccattgt gcgcctctat 660

tacagccaca gcagtaaggt ggccgccgac agtaacggcg aatttagcga taaatatgtc 720

aagctgaccc ccactactgt cagccagcag gtgagcatgc gcttcccgca tctcgccagc 780

tatcctgcct ttaagctgcc ggatgatgtc aacgtcgatg aattgctgca gggcgagacg 840

gtggcgatat ccgccgaaag cgacgggatc cttagctccg ccacccaggt gcagaccgcc 900

ggcgtgctgg acgataccta tgccgctgcc gccgaggcgc tgagctatgg cgcccagcta 960

actgatagcg gcgtgacctt ccgcgtctgg gcgcccacgg cgcagcaggt tgagctggtg 1020

gtctacagcg ccgacaagaa ggtggtggcc agccatccga tgacccgcga cagcgcctcc 1080

ggcgcctggt cctggcaggg cgggagcgac ctgaagggcg cgttctaccg ctacgcgatg 1140

accgtctacc acccgcagtc gcgtaaagtc gagcagtacg aagtgaccga tccctacgcc 1200

catagtttgt cgaccaactc ggagtacagc caggtggtcg atctcaacga cagcgcgctg 1260

aagccggaag gctgggacgg gctgacgatg ccgcacgcgc agaaaaccaa agccgacctg 1320

gcgaaaatga cgatccacga gtcgcatatt cgcgatctct ctgcctggga tcaaaccgtt 1380

cccgccgaac tgcgcggtaa gtatctggcg ctcaccgccc aggagagcaa tatggtccag 1440

catctgaaac agctgtcggc ctcgggagtg acccatattg agctgctgcc ggtcttcgat 1500

ctggcgacgg tcaatgagtt cagcgacaaa gtcgccgata ttcagcagcc gttcagtcgc 1560

ctgtgcgaga tcaacagcgc ggtgaagagc agcgagttcg cgggctattg cgacagcggt 1620

tcgacggtcg aagaggtgct gacccagctg aagcagaacg acagcaagga taacccgcag 1680

gtgcaggcgt tgaatacgct ggtggcgcag accgactcct ataactgggg ctacgatccg 1740

ttccactaca cggtgccgga aggatcctac gccaccgatc cggaaggcac ggcgcgcatt 1800

aaagagttcc gcaccatgat tcaggcgatc aagcaggatc tgggaatgaa cgtcattatg 1860

gacgtggtgt acaaccacac caacgccgcc ggcccgaccg accgcacctc ggtgctggat 1920

aagatcgtcc cctggtacta tcagcgcctg aatgaaacca ccggcagcgt ggaatcggcc 1980

acctgttgct ccgactcggc gccagagcac cggatgttcg ccaagcttat cgccgattca 2040

ctggcggtat ggaccaccga ttataagatc gatggcttcc gcttcgacct gatgggctat 2100

catccgaaag cgcagatcct ctcggcctgg gagcgcatta aagcgctgaa cccggacatt 2160

tatttctttg gtgaaggttg ggattccaac cagagcgatc gctttgaaat tgcctcgcaa 2220

atcaatctga aaggcaccgg gatcggcacg ttctccgatc gtctgcgcga cgccgtgcgc 2280

ggtggcgggc cgttcgattc cggcgatgca ttgcgccaga atcagggggt gggcagcggc 2340

gccggcgttc agccgaatga gctgaccagc atgaccgacg atcaggcgcg ccacctcgcc 2400

gatctgaccc gtctgggcat ggccggtaac ctcgcggact tcgtgctgat cgacaaagac 2460

ggcgcggtga agaaaggcag cgagattgac tataacggcg cgcctggcgg ctatgcggct 2520

gacccgacgg aagtcgtgaa ttatgtgtca aaacacgaca accaaacgct gtgggacatg 2580

atcagctata aagctgctca ggaggcggat ctcgataccc gcgtccggat gcaggcggtg 2640

tcgctggcga cggtgatgct cggccagggg atcgcctttg accagcaggg ctcggagctg 2700

ctgcgctcta aatcttttac ccgcgattcg tatgattccg gtgactggtt taaccgcgtg 2760

gattactccc tgcaggacaa caactacaac gtcggtatgc cgcgcagcag cgatgatggc 2820

agcaactatg acattatcgc ccgggtgaaa gacgcggtgg ctactccggg tgaagcggag 2880

ctcaagcaga tgaccgcgtt ttatcaggag ctgaccgcgc tgcgtaaatc gtctccgctg 2940

tttacccttg gtgacggcgc gacggtgatg aagcgcgtgg acttccgcaa caccggcgcc 3000

gatcagcaga cgggtctgct ggtgatgacc atcgatgatg ggatgcaggc tggcgccagt 3060

ctggacagcc gtgtcgacgg catcgtggtg gcgatcaacg ccgcaccgga aagccggacg 3120

ctgcaggact tcgccggcac atcgctgcag ctgagcgcca tccagcaggc ggcaggcgac 3180

cgttcgctgg cgagcggcgt gcaggttgcc gctgacggct cggtcacgct gccggcctgg 3240

tcggtagcgg tcctcgagct gccacagggg gaatcgcagg gcgctggcct gccggtgagc 3300

agtaaataa 3309

<210>SEQ ID NO: 2

<211>1102

<212>PRT

＜213〉a black seat klebsiella ( Klebsiella variicola) CCTCC NO:M 2012108

<400>2

Met Leu Arg Tyr Thr Cys His Ala Leu Phe Leu Gly Ser Leu Val

1 5 10 15

Leu Leu Ser Gly Cys Asp Asn Ser Ser Ser Ser Ser Pro Ser Gly

20 25 30

Ser Pro Gly Ser Pro Gly Asn Pro Gly Asn Pro Gly Thr Pro Gly

35 40 45

Thr Pro Asp Pro Gln Asp Val Val Val Arg Leu Pro Asp Val Ala

50 55 60

Val Pro Gly Glu Ala Ala Gln Ala Ser Ala Asn Gln Ala Val Ile

65 70 75

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

80 85 90

Tyr Ala Thr Lys Asn Leu Tyr Leu Trp Asn Asn Glu Thr Cys Asp

95 100 105

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

110 115 120

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

125 130 135

Thr Lys Glu Ser Gly Cys Ile Asn Val Ile Val Arg Asp Gly Thr

140 145 150

Asn Lys Leu Ile Asp Ser Asp Leu Arg Val Ser Phe Gly Asp Phe

155 160 165

Thr Asp Arg Thr Val Ser Val Ile Ala Gly Asn Ser Ala Val Tyr

170 175 180

Asp Ser Arg Ala Asp Ala Phe Arg Ala Ala Phe Gly Val Ala Leu

185 190 195

Ala Asp Ala His Trp Val Asp Lys Thr Thr Leu Leu Trp Pro Gly

200 205 210

Gly Glu Asn Lys Pro Ile Val Arg Leu Tyr Tyr Ser His Ser Ser

215 220 225

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

230 235 240

Lys Leu Thr Pro Thr Thr Val Ser Gln Gln Val Ser Met Arg Phe

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315

Tyr Gly Ala Gln Leu Thr Asp Ser Gly Val Thr Phe Arg Val Trp

320 325 330

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

335 340 345

Lys Lys Val Val Ala Ser His Pro Met Thr Arg Asp Ser Ala Ser

350 355 360

Gly Ala Trp Ser Trp Gln Gly Gly Ser Asp Leu Lys Gly Ala Phe

365 370 375

Tyr Arg Tyr Ala Met Thr Val Tyr His Pro Gln Ser Arg Lys Val

380 385 390

Glu Gln Tyr Glu Val Thr Asp Pro Tyr Ala His Ser Leu Ser Thr

395 400 405

Asn Ser Glu Tyr Ser Gln Val Val Asp Leu Asn Asp Ser Ala Leu

410 415 420

Lys Pro Glu Gly Trp Asp Gly Leu Thr Met Pro His Ala Gln Lys

425 430 435

Thr Lys Ala Asp Leu Ala Lys Met Thr Ile His Glu Ser His Ile

440 445 450

Arg Asp Leu Ser Ala Trp Asp Gln Thr Val Pro Ala Glu Leu Arg

455 460 465

Gly Lys Tyr Leu Ala Leu Thr Ala Gln Glu Ser Asn Met Val Gln

470 475 480

His Leu Lys Gln Leu Ser Ala Ser Gly Val Thr His Ile Glu Leu

485 490 495

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

500 505 510

Val Ala Asp Ile Gln Gln Pro Phe Ser Arg Leu Cys Glu Ile Asn

515 520 525

Ser Ala Val Lys Ser Ser Glu Phe Ala Gly Tyr Cys Asp Ser Gly

530 535 540

Ser Thr Val Glu Glu Val Leu Thr Gln Leu Lys Gln Asn Asp Ser

545 550 555

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

560 565 570

Thr Asp Ser Tyr Asn Trp Gly Tyr Asp Pro Phe His Tyr Thr Val

575 580 585

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

590 595 600

Lys Glu Phe Arg Thr Met Ile Gln Ala Ile Lys Gln Asp Leu Gly

605 610 615

Met Asn Val Ile Met Asp Val Val Tyr Asn His Thr Asn Ala Ala

620 625 630

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

635 640 645

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

650 655 660

Thr Cys Cys Ser Asp Ser Ala Pro Glu His Arg Met Phe Ala Lys

665 670 675

Leu Ile Ala Asp Ser Leu Ala Val Trp Thr Thr Asp Tyr Lys Ile

680 685 690

Asp Gly Phe Arg Phe Asp Leu Met Gly Tyr His Pro Lys Ala Gln

695 700 705

Ile Leu Ser Ala Trp Glu Arg Ile Lys Ala Leu Asn Pro Asp Ile

710 715 720

Tyr Phe Phe Gly Glu Gly Trp Asp Ser Asn Gln Ser Asp Arg Phe

725 730 735

Glu Ile Ala Ser Gln Ile Asn Leu Lys Gly Thr Gly Ile Gly Thr

740 745 750

Phe Ser Asp Arg Leu Arg Asp Ala Val Arg Gly Gly Gly Pro Phe

755 760 765

Asp Ser Gly Asp Ala Leu Arg Gln Asn Gln Gly Val Gly Ser Gly

770 775 780

Ala Gly Val Gln Pro Asn Glu Leu Thr Ser Met Thr Asp Asp Gln

785 790 795

Ala Arg His Leu Ala Asp Leu Thr Arg Leu Gly Met Ala Gly Asn

800 805 810

Leu Ala Asp Phe Val Leu Ile Asp Lys Asp Gly Ala Val Lys Lys

815 820 825

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

830 835 840

Asp Pro Thr Glu Val Val Asn Tyr Val Ser Lys His Asp Asn Gln

845 850 855

Thr Leu Trp Asp Met Ile Ser Tyr Lys Ala Ala Gln Glu Ala Asp

860 865 870

Leu Asp Thr Arg Val Arg Met Gln Ala Val Ser Leu Ala Thr Val

875 880 885

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

890 895 900

Leu Arg Ser Lys Ser Phe Thr Arg Asp Ser Tyr Asp Ser Gly Asp

905 910 915

Trp Phe Asn Arg Val Asp Tyr Ser Leu Gln Asp Asn Asn Tyr Asn

920 925 930

Val Gly Met Pro Arg Ser Ser Asp Asp Gly Ser Asn Tyr Asp Ile

935 940 945

Ile Ala Arg Val Lys Asp Ala Val Ala Thr Pro Gly Glu Ala Glu

950 955 960

Leu Lys Gln Met Thr Ala Phe Tyr Gln Glu Leu Thr Ala Leu Arg

965 970 975

Lys Ser Ser Pro Leu Phe Thr Leu Gly Asp Gly Ala Thr Val Met

980 985 990

Lys Arg Val Asp Phe Arg Asn Thr Gly Ala Asp Gln Gln Thr Gly

995 1000 1005

Leu Leu Val Met Thr Ile Asp Asp Gly Met Gln Ala Gly Ala Ser

1010 1015 1020

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

1025 1030 1035

Pro Glu Ser Arg Thr Leu Gln Asp Phe Ala Gly Thr Ser Leu Gln

1040 1045 1050

Leu Ser Ala Ile Gln Gln Ala Ala Gly Asp Arg Ser Leu Ala Ser

1055 1060 1065

Gly Val Gln Val Ala Ala Asp Gly Ser Val Thr Leu Pro Ala Trp

1070 1075 1080

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

1085 1090 1095

Gly Leu Pro Val Ser Ser Lys***

1100 1102

Claims (2)

1. a method of using self-induction substratum and the outer Pullulanase of two temperature regulating strategy production born of the same parents is characterized in that recombination bacillus coli E. coliBL21 (DE3)/pET28a (+)-pul A is inoculated in the self-induction substratum, and employing two temperature control methods, be about to self-induction after the inoculation cultivate cultivate 2 ~ 4 hours based on 37 ℃, 200 rpm after, the temperature of cultivating is reduced to 25 ℃ to be continued to cultivate 48 ~ 72 hours, after cultivating end, collect fermented supernatant fluid and obtain the outer Pullulanase of born of the same parents; Step is:

(1) will derive from a black seat klebsiella ( Klebsiella variicola) the Pullulanase gene of CCTCC NO:M 2012108 carries out recombinant expressed in intestinal bacteria:

The acquisition of a, Pullulanase gene

CCTCC NO:M 2012108 substratum: Zulkovsky starch 10 g/L, peptone 15 g/L, yeast extract paste 5 g/L, KH ₂PO ₄5 g/L, MgSO ₄7H ₂O 0.1 g/L, NH ₄Cl 0.5 g/L, FeSO ₄7H ₂O 0.1 g/L, with the ultrapure water preparation, pH 6.5;

CCTCC NO:M 2012108 bacterial classification inoculations are in the substratum liquid amount is 10% 250 mL shaking flasks, in 37 ℃, 200 rpm shaking culture, 72 h; After cultivate finishing, that thalline is centrifugal and use physiological saline washed twice, collecting cell to utilize the genome DNA extracting reagent kit Genomic DNA Extraction Miniprep System extraction genomic dna of VIOGENE company;

Black seat klebsiella ( Klebsiella variicola) CCTCC NO:M 2012108 Pullulanases, the nucleotides sequence of its gene is classified as: SEQ ID NO:1, its amino acid consist of SEQ ID NO:2;

Synthetic primer 1:5'-CGG CTA GCA TGC TCA GAT ATA CCT GTC ATG G-3', primer 1 contains NheThe I restriction enzyme site;

Synthetic primer 2:5'-CCG GAA TTC CGT TTA CTG CTC ACC GGC G-3', primer 2 contains EcoThe RI restriction enzyme site;

PCR reaction system: ddH ₂O 37 μ L, 10 * Reaction Buffer, 5 μ L, the dNTP 0.5 μ L of 25 mmol/L, 50 pmol/ μ L primer 1:1 μ L, 50 pmol/ μ L primer 2s: 1 μ L, genomic dna 5 μ L, the Taq DNA polymerase 0.5 μ L of 5 U/ μ L;

PCR reaction process: 94 ℃ of denaturation 5 min; 98 ℃ of 10 s, 55 ℃ of 1 min, 72 ℃ of 4 min carries out 30 circulations; 72 ℃ are extended 10 min;

Utilize the 3S Spin Agarose Gel DNA Purification Kit purify DNA segment of Shanghai Shenergy Biocolor BioScience ﹠ Technology Company:

Sodium acetate solution and 2 times of volume dehydrated alcohols of adding 3 mol/L, the pH 5.2 of 1/10 volume in the dna solution ,-20 ℃ precipitate 1 hour; In 4 ℃, centrifugal 30 min of 12000 rpm; Add 75% ethanol, 500 μ L washing, in 4 ℃, centrifugal 30 min of 12000 rpm, be dissolved in after aseptic operating platform dries up in an amount of TE damping fluid, use immediately or-20 ℃ of preservations;

B, contain the structure of the recombination bacillus coli of Pullulanase gene

Utilize restriction enzyme NheI and EcoRI carries out the double digestion processing to CCTCC NO:M 2012108 Pullulanase encoding genes and the carrier pET28a (+) that amplification obtains, dna segment connects the recombinant plasmid pET28a (+) that obtains with the goal gene segment-pul A by sticky end after processing, with the recombinant plasmid transformed intestinal bacteria E. coliBL21 (DE3) competent cell obtains to contain the recombination bacillus coli of purpose Pullulanase gene by the LB plate screening that contains 50 μ g/mL kantlex E. coliBL21 (DE3)/pET28a (+)-pul A; Step is:

The enzyme of goal gene and plasmid pET28a (+) is cut:

Utilize plasmid extraction kit Mini-Plasmid Rapid Isolation Kit, vast Tyke, Beijing biological gene technology company limited provides, and extracts plasmid pET28a (+);

Order according to water, damping fluid, PCR product or plasmid DNA, enzyme is added in the Eppendorf pipe, build the pipe lid, vibration makes the abundant mixing of liquid, placing interior centrifugal 2 s of whizzer that liquid is concentrated on manages at the end, 37 ℃ of water-bath 3 h, the Loading Buffer of adding 1/10 maybe places pipe 65 ℃ of insulation 10 min in pipe, stops endonuclease reaction; Enzyme is cut product and carried out the agarose gel electrophoresis analysis and cut glue recovery purpose fragment, and is concentrated;

Reaction composition: 10 * H Buffer, 4 μ L, DNA 10 μ L, NheI 2 μ L, EcoRI 2 μ L, ddH ₂O supplies 40 μ L with system;

Goal gene is connected with plasmid pET28a's (+):

CCTCC NO:M 2012108 Pullulanase encoding genes are connected with plasmid pET28a (+), and reaction composition is as follows: plasmid pET28a (+) 0.8 μ L, foreign gene 4.2 μ L, Ligation Solution 5 μ L; Mix connecting fluid, be placed on connection 12 h in 16 ℃ of incubators;

The recombinant plasmid transformed intestinal bacteria:

100 μ L intestinal bacteria E. coliAdd 10 μ L in BL21 (DE3) the competent cell suspension and connect product, mixing leaves standstill 30 min in the ice bath gently; Change in 42 ℃ of water-baths thermal shock 90 s over to; Fast transfer cools off 2 min to ice bath; Add 700 μ L LB liquid nutrient mediums, 37 ℃ of 100 rpm shaking table incubation cultivated 1 h; Centrifugal 2 min of bacterium liquid 3000 rpm after cultivating abandon supernatant 600 μ L, are applied on the LB flat board that contains 50 μ g/mL kantlex behind the residue bacterium liquid mixing, are inverted for 37 ℃ and cultivate; Acquisition contains the recombination bacillus coli of purpose Pullulanase gene E. coliBL21 (DE3)/pET28a (+)-pul A;

(2) self-induction of recombination bacillus coli is cultivated and two temperature regulation and control enzymatic production

Self-induction substratum: alpha-lactose 10-20 g/L, glucose 0.5-1 g/L, glycerine 5 g/L, KH ₂PO ₄6.8 g/L, MgSO ₄0.24 g/L, peptone 10 g/L, Na ₂HPO ₄12H ₂O 17.9 g/L, Na ₂SO ₄0.71 g/L, NH ₄Cl 2.67 g/L, trace element solution 200 μ L/L prepare with ultrapure water;

Trace element solution: contain 50 μ M FeCl ₃, 20 μ M CaCl ₂2H ₂O, 10 μ M MnCl ₂4H ₂O, 10 μ M ZnSO ₄7H ₂O, 2 μ M CoCl ₂6H ₂O, 2 μ M CuCl ₂, 2 μ M NiCl ₂, 2 μ M Na ₂Mo ₇O ₂₄, 2 μ M Na ₂SeO ₃With 2 μ M H ₃B ₄O ₇, prepare with ultrapure water;

The culture condition that produces enzyme is: with recombination bacillus coli E. coliBL21 (DE3)/pET28a (+)-pul A activation culture of on the LB solid medium that contains 50 μ g/mL kantlex, spending the night, the single colony inoculation of picking contains the liquid LB substratum of 50 μ g/mL kantlex in 37 ℃, 200 rpm shaking culture in 3 mL from solid plate, monitors at any time OD in the substratum ₆₀₀Changing conditions, treat OD ₆₀₀Reach at 2 ~ 3 o'clock, be inoculated in the self-induction substratum that 50 mL contain 50 μ g/mL kantlex by 5% ~ 10% inoculum size, with the self-induction after the inoculation cultivate cultivate 2 ~ 4 hours based on 37 ℃, 200 rpm after, the temperature of cultivating is reduced to 25 ℃ to be continued to cultivate 48 ~ 72 hours, after cultivating end, the centrifugal removal thalline of 10000 rpm, collecting fermented supernatant fluid is the crude enzyme liquid of Pullulanase.

2. described application self-induction substratum and two temperature regulating strategy are produced born of the same parents' method of Pullulanase outward according to claim 1, it is characterized in that the optimization self-induction substratum composition of the outer Pullulanase of fermentative production born of the same parents replaces with:

Alpha-lactose 20 g/L, glucose 1 g/L, glycerine 5 g/L, KH ₂PO ₄6.8 g/L, MgSO ₄0.24 g/L, peptone 10 g/L, Na ₂HPO ₄12H ₂O 17.9g/L, Na ₂SO ₄0.71 g/L, NH ₄Cl 2.67 g/L, Tween-80 9 g/L, trace element solution 200 μ L/L prepare with ultrapure water;

Engineering bacteria E. coliThe optimum culture condition that BL21/pET28a (+)-pulA produces enzyme is: initial pH5.0 ~ 8.0, and liquid amount 10% ~ 20%, culture temperature is after 37 ℃, 200 rpm are cultivated first 4 hours, to change 25 ℃ over to again and cultivated 56 hours;

After adopting optimization self-induction substratum and two temperature fermentation condition, the outer Pullulanase enzyme work of born of the same parents reaches 60 ~ 70 U/mL.