CN102604861B - Pullulanase, pullulanase producing strain and application of pullulanase - Google Patents
Pullulanase, pullulanase producing strain and application of pullulanase Download PDFInfo
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- 241000607534 Aeromonas Species 0.000 claims description 12
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Abstract
The invention belongs to the technical field of biological enzymes, and in particular relates to a pullulanase, a pullulanase producing strain and application of the pullulanase. The pullulanase is derived from Aeromonas sp. As in bacteria. The pullulanase produced by the strain is pullulanase type I, and can specifically hydrolyze alpha-1,6 glycosidic bond in pullulan or starch. The pullulanase is expressed under an induction condition in which 1.5% soluble starch is used as a substrate. Under a condition of medium pH of 7.5 and culture temperature of 30 DEG C, shake-flask fermentation is carried out for 48h to produce the pullulanase at a level up to 24.8U/mL. For the pullulanase produced by the strain, the optimum reaction temperature is 60 DEG C, the optimum pH is 6.0, and the relative enzyme activity in the pH range of 6.0-9.0 is greater than 90 percent. After treatment for 50min at 60 DEG C, the residual activity of the pullulanase reaches about 50 percent. The pullulanase type I provided by the invention can be applied to industries such as starch processing, environment protection, foods, medical care and the like.
Description
Technical field
The invention belongs to the biological enzyme technology field, be specifically related to screening and the evaluation of a kind of novel Pullulanase and bacterium producing multi enzyme preparation thereof.The present invention also provides this Pullulanase production method, enzymatic property and application.
Background technology
Starch is the photosynthetic product of green plants, consists of amylose starch and amylopectin.Wherein amylose starch is mainly by α-Isosorbide-5-Nitrae glucose glycoside key is formed by connecting, and amylopectin also contains branched chain, and what connect bifurcation is α-1,6 glucose glycoside key.Starch is one of industrial raw material of current widespread use the most, such as preparing glucose syrup, high maltose syrup etc.Yet owing in starch, containing 4%～5% α-1,6 glycosidic link of having an appointment, use thoroughly hydrolyzed starch of α-amylase (hydrolyzing alpha-Isosorbide-5-Nitrae glycosidic link), affected the raising of Starch Hydrolysis degree.
I type Pullulanase (EC 3.2.1.41) is with α-1,6 glucose glycoside key (the Doman-Pytka M of internal-cutting way hydrolysis pulullan or starch
Et al., Critical Reviews in Microbiology, 2004, Vol 30, and Issue 2, and 107-121), so I type Pullulanase has very important industrial application value.The industrial thoroughly hydrolyzed starch of α-amylase and Pullulanase synergy that utilizes, prepare high glucose slurry, superhigh maltose syrup, raising beer fermentation degree etc.; In addition, Pullulanase is directly acted on to starch and can produce high amylose starch, slowly digestible starch, these products all have important effect in food, environmental protection, medical industry.
Pullulanase has improved the utilization ratio of starch resource greatly, however the product enzyme enzyme lower (general 2～5 U/mL) alive of current separating obtained product Pullulanase bacterial strain both at home and abroad, and expensive, limited it in industrial application.These bacterium producing multi enzyme preparations be mainly derived from bacillus (
Bacillus) and klebsiella spp (
Klebsiella), the method by experiment that has no is produced the Pullulanase bacterial strain from Aeromonas, screening.The present invention from paddy soil, screening a strain, have first the pulullan polysaccharide degradation capability the Aeromonas bacterial strain (
Aeromonas sp. As), its Pullulanase produced can be hydrolyzed α-1,6 glycosidic link in pulullan polysaccharide and starch, can not hydrolyzing alpha-Isosorbide-5-Nitrae glycosidic link, identify that it is I type Pullulanase.This enzyme can be applicable to take starchiness in the processing industry of raw material.
Summary of the invention
First purpose of the present invention is to provide a kind of enzyme enzyme high product Pullulanase bacterial strain alive that produces.
Second purpose of the present invention is to provide this Pullulanase strain fermentation method.
The present invention utilizes the sole carbon source method from paddy soil, having obtained a kind of strains A s that produces Pullulanase, this culture presevation number is CGMCC NO.5490, preservation date is on November 28th, 2011, and depositary institution is China Committee for Culture Collection of Microorganisms's common micro-organisms center, is numbered 3G2.16S rDNA(SEQ ID NO 1 to this bacterial strain) phylogenetic tree is analyzed (see figure 1), shows the Aeromonas (called after of this bacterium source in bacterium
Aeromonas sp.As).Its 16S rDNA sequence with
AeromonaspunctataThe bacterial classification similarity is 99%.This bacterial strain microscopic morphology is tyrothricin, and bacterium colony is that circle is larger on the LB flat board, and micro white is transparent, the smooth of the edge, bacterium colony intermediate projections, thalline thickness (see figure 2).The present invention is the product Pullulanase bacterial strain that the method for domestic and international first passage experiment screens the Aeromonas source.
The present invention also provides the fermentation process (namely producing the method for Pullulanase with the Pullulanase bacterial strain) of the bacterial strain of above-mentioned product Pullulanase, concrete steps are as follows: after the As bacterial strain is carried out to separation and purification, (this nutrient media components is picking list colony inoculation: Zulkovsky starch 1-2% in 15-25 mL seed culture medium, peptone 0.5-1.0 %, yeast powder 0.1-0.3 %, MgSO
47H
2O 0.02-0.05 %, KH
2PO
40.05-0.1 %, all the other are water; PH 6.5-7.5), 28-32 ℃, 16～18 h are cultivated in concussion; Get subsequently 1-2 mL and transfer in 25-50 mL fermention medium that (this nutrient media components is: Zulkovsky starch 1-2 %, peptone 0.5-1.5 %, yeast powder 0.5-1.0 %, MgSO
47H
2O 0.02-0.05 %, KH
2PO
40.05-0.1 %, all the other are water; PH 6.5-7.5), 36-60 h is cultivated in 28-32 ℃ of concussion.The enzyme work of the I type Pullulanase that its shake flask fermentation produces is that 24.8 U/mL(are shown in Fig. 3).
It is that the Amicon super filter tube of 50 KDa concentrates and dams 50 mL fermented supernatant fluids that the present invention uses the molecular retention amount, and to change the background damping fluid be the Tris-HCl (pH8.0) of 20 mM.The enzyme liquid of getting after concentrated preliminary purification carries out follow-up enzymatic property analysis.By the method for thin-layer chromatography (TLC) and chromatography of ions (HPAEC), identify that Pullulanase that this bacterial strain produces is I type Pullulanase (seeing Fig. 5, Fig. 6).The enzymatic reaction temperature of this I type Pullulanase can be 40 ℃-70 ℃, and optimum temperuture is 60 ℃; Enzymatic reaction pH can be pH5-9, and optimum pH is 6.0, and enzyme activity all is greater than 90% in pH 6.0～9.0 scopes; Process 50 min for 60 ℃, enzyme activity residual approximately 50%.This Pullulanase is about 20% of its hydrolysis pulullan polysaccharide to the degradation capability of yam starch.I type Pullulanase provided by the invention can be applicable to the industries such as starch processing, environmental protection, food, health care.
The accompanying drawing explanation
Fig. 1: the 16S rDNA Phylogenetic Analysis figure that produces Pullulanase strains A s.According to bacterial 16 S rDNA criteria for classification, 16S rDNA sequence similarity degree is more than or equal to 99% can tentatively think the same bacterium.Pass through pcr amplification
Aeromonas sp. As strain gene group 16S rDNA sequence, microorganism 16S rDNA sequence known in the sequence of acquisition and GenBank database is carried out sequence analysis, result show its with
AeromonaspunctataThe bacterial classification similarity is 99%.In phylogenetic tree, form same bunch with Aeromonas bacterial strain 16S rDNA sequence, and with
AeromonaspunctataBacterial classification has the highest similarity.
Fig. 2: bacterium producing multi enzyme preparation
Aeromonas sp. As colonial morphology and microscopic morphology figure.This bacterial strain microscopic morphology is tyrothricin; The bacterium colony thickness easily grows up to lawn on the LB flat board, the bacterium colony circle, and micro white is transparent, the smooth of the edge, bacterium colony intermediate projections.This colonial morphology is consistent with Aeromonas ne ar feature with the feature of microscopic morphology.
Fig. 3: fermentation time pair
Aeromonas sp. As produces the impact of Pullulanase.By right
Aeromonas sp. the optimization of As strain fermentation condition (pH is 7.5, and culture temperature is 30 ℃), select different incubation times to measure the fermented supernatant fluid enzyme and live.Result in figure shows that while cultivating 48 h, its yield of enzyme is the highest, and the work of supernatant liquor Pullulanase reaches 24.8 U/mL, and the thick specific activity of enzyme that calculates this fermented liquid by determination of protein concentration is about 40 U/mg.
Fig. 4: SDS-PAGE analyzes
Aeromonas sp. the purification result of As fermented supernatant fluid Pullulanase.
Aeromonas sp. As is after not amyloid LB substratum fermentation 48 h, and there is the protein band (swimming lane 2) of big or small approximately 60 KDa, 50 KDa and 38 KDa in its supernatant liquor, by the enzyme activity determination to fermented supernatant fluid, finds that it does not have the Pullulanase enzyme and lives.After using amyloid fermention medium shake-flask culture As bacterial strain 24 h, except above-mentioned 3 main albumen, also at 140 KDa places, detect a protein expression band (swimming lane 3); Ferment after 48 h, the protein band at 140 KDa places is deepened, and the enzyme activity that records Pullulanase in fermented liquid improves (swimming lane 4).Take subsequently the method for ultrafiltration to carry out preliminary two-step purifying to crude enzyme liquid, using respectively molecular weight cut-off is 30 KDa(swimming lanes 5) and 50 KDa(swimming lanes 6) the Amicon super filter tube fermented supernatant fluid is concentrated and dams.After two-step purifying, 140 KDa protein bands obviously thicken, and the protein ratio enzyme is lived and further improved.The band albumen of results suggest 140 KDa may be
Aeromonas sp. Pullulanase that As produces.
Fig. 5:
Aeromonas sp. Pullulanase that As produces hydrolysis pulullan polysaccharide product thin layer chromatography analysis figure.According to IUBMB(International Union of Biochemistry and Molecular Biology) the two class criteria for classifications set up (are divided into multiclass to the Propiram degrading enzyme, I type Pullulanase (Type I pullulanase wherein, EC 3.2.1.41) single-minded hydrolysis pulullan α-1,6 glycosidic link produces trisaccharide maltose; The α of the new single-minded hydrolysis pulullan polysaccharide of Pullulanase (Neopullulanase, EC3.2.1.135)-Isosorbide-5-Nitrae glycosidic link produces panose.Dissimilar Propiram degrading enzyme can be hydrolyzed different glycosidic links, and generates different products, and therefore the application in industry-by-industry also is not quite similar; And I type Pullulanase is in industrial being most widely used.The present invention analyzes the product of As Pullulanase that bacterial strain produces hydrolysis pulullan polysaccharide by thin-layer chromatography (TLC) and ion chromatography (HPAEC, as Fig. 5).In figure, swimming lane 1 is position after glucose (C1), maltose (C2), trisaccharide maltose (C3) chromatography; Swimming lane 2 is unhydrolysed pulullan polysaccharide; Swimming lane 3 is to use
Aeromonas sp. the product obtained after the Pullulanase hydrolysis pulullan polysaccharide that the As bacterial strain produces.This figure shows that the product that As bacterial strain Pullulanase hydrolysis pulullan polysaccharide obtains is trisaccharide.
Fig. 6:
Aeromonas sp. the Pullulanase hydrolysis pulullan polysaccharide product ion stratographic analysis figure that As produces.In order further to identify the catalysis classification of this Propiram degrading enzyme, the present invention takes the method for chromatography of ions to identify the product of its hydrolysis pulullan polysaccharide.The trisaccharide product that this Propiram degrading enzyme hydrolysis pulullan polysaccharide produces, its chromatography of ions retention time are about 16 min(as figure A), with the retention time consistent (if schemed B) of the standard substance of trisaccharide maltose.By the standard addition method analysis, result shows the peak of the chromatographic peak of this enzymic hydrolysis pulullan trisaccharide product and trisaccharide maltose standard substance overlap (seeing figure C), form simple spike, the result shows that the product of this Pullulanase hydrolysis pulullan polysaccharide is trisaccharide maltose, so As Pullulanase that bacterial strain produces belongs to I type Pullulanase.
Fig. 7:
Aeromonas sp. the Pullulanase optimal reactive temperature that As produces is measured figure.Crude enzyme liquid after 5 μ L preliminary purifications and 95 μ L are contained to (pH 6.0) in 20 mM citric acids of 1% pulullan polysaccharide-trisodium citrate damping fluid to be mixed, respectively 30,40,50,60, at 70 and 80 ℃ of temperature, reaction 10 min, then measure the Pullulanase vigor by the DNS method, and what wherein enzyme activity was the highest is made as 100%.The result of Fig. 7 shows that this Pullulanase can carry out enzymatic reaction at 40-70 ℃, and enzyme activity is more than 40%, and optimal reactive temperature is 60 ℃.
Fig. 8:
Aeromonas sp. the optimal pH of the Pullulanase that As produces is measured figure.Preparation contains the damping fluid of the pH 4.0～10.0 of 1% pulullan polysaccharide respectively, and wherein pH 4.0～6.0 is 20 mM citric acids-trisodium citrate damping fluid, and pH 7.0 is the NaH of 20 mM
2PO
4-Na
2HPO
4Damping fluid, pH 8.0～10.0 are the Tris-HCl damping fluid of 20mM.Get the damping fluid 95 μ L that contain substrate together with enzyme liquid after 5 μ L purifying in water-bath 60 ℃ of reaction 10 min.Measure the Pullulanase vigor, what wherein enzyme activity was the highest is made as 100%.Fig. 7 shows, lives all more than 90% at the scope endoenzyme of pH 6.0～9.0, and pH 5.0～9.0 enzymes are lived all more than 70%, and the optimal pH of this enzyme is 6.0.
Fig. 9:
Aeromonas sp. the thermal stability determination figure of the Pullulanase that As produces.Appropriate enzyme liquid is placed in 60 ℃ and 65 ℃ of lower waters bath with thermostatic control, and every 10 min sampling and measuring residual enzyme vigor, the enzyme activity when being incubated 0 min counts 100%.The result of Fig. 8 shows, under 60 ℃ of conditions, processes 50 min, the enzyme activity of this Pullulanase residual approximately 50%.
Figure 10:
Aeromonas sp. the substrate specificity of the Pullulanase that As produces is measured.Pulullan polysaccharide, yam starch, amylose starch, alpha-cylodextrin, beta-cyclodextrin solution with 20 mM citric acids-trisodium citrate damping fluid (pH 6.0) preparation 1%, getting 5 μ L enzyme liquid mixes with 95 μ L substrates, in 60 ℃ of reaction 10 min, measure the hydrolyzing activity of strains A Pullulanase that s produces to different substrates with the DNS reducing sugar method.The result of Figure 10 shows
Aeromonas sp. the amylatic ability of Pullulanase that As produces is about 20% of hydrolysis pulullan polysaccharide, can not be hydrolyzed amylose starch (consisting of α-Isosorbide-5-Nitrae glycosidic link), alpha-cylodextrin and beta-cyclodextrin.
Embodiment
Experiment material used and related operating method are as follows, and the detailed description place can be with reference to " molecular cloning " (J.Sambrook, et al., second edition, 1996) of cold spring laboratory:
1. bacterial strain, carrier: coli strain Top10, plasmid pPMD18-T are this laboratory and preserve, identical with the character of respective substance in existing document.
2. substratum
The LB substratum: 1.0% Polypeptone(BBI), 0.5% Yeast Extract(Oxoid), 0.5% NaCl, pH 7.0.
Dull and stereotyped enrichment medium: pulullan polysaccharide 3.0 g, (NH
4)
2SO
45.0 g, KH
2PO
40.5g, MgSO
47H
2O 0.1 g, agarose 20.0 g, add water to 1000 mL, pH value 5.0.
The Isolation and Culture base: peptone 10.0 g, extractum carnis 3.0 g, NaCl 5 g, add water to 1000 mL, pH value 6.0.
Produce the sub-substratum of enzyme: Zulkovsky starch 1.2 %, peptone 0.8 %, yeast powder 0.2 %, MgSO
47H
2O 0.05 %, KH
2PO
40.1 %, pH 7.0.
Produce the enzymic fermentation substratum: Zulkovsky starch 1.5 %, peptone 1.0 %, yeast powder 0.5 %, MgSO
47H
2O 0.05 %, KH
2PO
40.1 %, pH 7.0.
3. experiment reagent: the polysaccharide such as pulullan polysaccharide, yam starch, amylose starch are purchased from Sigma company; The reagent that molecular biology needs is purchased from TaKaRa company; Primer is synthetic to be completed by the English Weihe River, Shanghai Jie Ji Bioisystech Co., Ltd with DNA sequencing.DNS(3, the 5-dinitrosalicylic acid) reagent (first liquid: dissolve 6.9 g crystalline phenols in 15.2 mL 10%NaOH, with distilled water diluting to 69 mL, then add 6.9 g NaHSO
3Second liquid: 255 gram Seignette salts add in 300 mL 10%NaOH, then add 880 mL 1%3,5-dinitrosalicylic acid solution; First liquid and second liquid-phase mixing are namely obtained to yellow DNS reagent, be stored in brown bottle, at room temperature place and used afterwards in 7～10 days).
4. Pullulanase enzyme activity determination method: the present invention uses the DNS reducing sugar method to measure the enzyme activity of Pullulanase.Concrete grammar is as follows: get 5 μ L enzyme liquid and add 95 μ L to contain the 1%(mass volume ratio) the citric acid-trisodium citrate damping fluid of pulullan polysaccharide in (pH 6.0,20 mM), 60 ℃ of reaction 10 min, add 100 μ L DNS termination reactions, boils 10 min colour developings; The enzyme liquid that the 5 μ L of take boil deactivation is blank, and 570 nm measure light absorption value, according to typical curve, calculates the amount that produces reducing sugar, and 3 parallel sample are done in every group reaction.Enzyme unit definition alive: per minute discharges 1 μ mol reducing sugar (with glucose meter) and is defined as an enzyme activity unit (IU).
Embodiment 1: the screening, the Isolation and Identification that produce the Pullulanase bacterial strain
Getting 10 g soil samples joins in the triangular flask that 90 mL sterilized waters and granulated glass sphere are housed, 30 min vibrate, after sample is fully broken up, draw after 100 μ L nutrient solutions dilute to coat and take pulullan polysaccharide on the dull and stereotyped enrichment medium of sole carbon source, in 30 ℃ of incubators, cultivate 48 h.From each 2 of picking different shape list bacterium colonies on enrichment medium, be connected on the strains separation substratum, and carry out repeatedly streak culture to single bacterium colony of picking.Subsequently, the access of the bacterium colony after the plate streaking purifying is produced in the sub-substratum of enzyme, after 30 ℃ of shaking culture 24 h, make bacteria suspension.Bacteria suspension is produced to enzymic fermentation substratum, 30 ℃ of shaking culture 48 h with 4% inoculum size access; Take out supernatant liquor and add the sodium citrate buffer solution (pH 7.0) that contains 1% pulullan polysaccharide, 37 ℃ of reaction 30 min, in the rough determination fermented liquid supernatant, the enzyme of Propiram degrading enzyme is lived.
Bacterial strain with pulullan degrading activity is carried out to the genome extracting, pcr amplification 16S rDNA.Extension increasing sequence is 16S rDNA universal primer 27F(AGAGTTTGATCMTGGCTCAG) and 1492R(TACGGYTACCTTGTTACGACTT).The As bacterial strain 16S rDNA sequence (SEQ ID NO 1) obtained, the RDP database (
Http:// rdp.cme.msu.edu/) in classify.Result screening obtains produces Pullulanase strains A s, through the analysis of 16S rDNA phylogenetic tree, colonial morphology, microscopic morphology analysis (seeing Fig. 1, Fig. 2) show this bacterial strain belong to Aeromonas (
Aeromonas sp.).The present invention is that by experiment method obtains the Pullulanase bacterial strain from screening Aeromonas first.
Embodiment 2:
Aeromonas sp. the As fermentation condition optimization
The Pullulanase of As bacterial strain belongs to Inducing enzyme, need under the substrate for induction condition, express, and therefore, when optimizing As bacterial strain product Pullulanase condition, adopts Zulkovsky starch as producing the enzyme induction thing.After the As bacterial strain was carried out to separation and purification, picking list colony inoculation was in being equipped with 25 mL seed culture mediums, and 30 ℃, 16～18 h are cultivated in concussion; Get subsequently 2 mL and transfer and be equipped with in 50 mL fermention mediums, 48 h are cultivated in 30 ℃ of concussions, get supernatant and measure enzyme activity.Adjust fermention medium pH value (pH6.5 and pH7.5) and leavening temperature (30 ℃ and 37 ℃), measure Pullulanase enzyme activity in fermented liquid every 12 h, set up strains A s high yield Pullulanase condition.
Embodiment 3:
Aeromonas sp. the preliminary purification of Pullulanase in the As fermented supernatant fluid
Aeromonas sp. the As bacterial strain, after fermentation culture 48 h, is got 20 μ L fermented supernatant fluids and is used 12% SDS-PAGE to detect the protein expression band.Get 30 mL fermented supernatant fluids, with Amicon super filter tube (molecular weight 30 KDa) ultrafiltration and concentration to 1 mL, damping fluid is 20 mM Tris-HCl(pH 8.0), get 20 μ L and carry out the SDS-PAGE detection, detect simultaneously the 1st Pullulanase after ultrafiltration than enzyme (U/mg albumen) alive.It is the Amicon super filter tube of 50 KDa that the 2nd ultrafiltration purification selected molecular weight cut-off, 20 mM Tris-HCl(pH 8.0), final volume after ultrafiltration is 800 μ L, takes out 20 μ L and carries out SDS-PAGE detection (as Fig. 4), measures simultaneously the ratio enzyme of Pullulanase and lives.
Embodiment 4:
Aeromonas sp. the As bacterial strain produces the zymetology classification analysis of Pullulanase
Because the application of dissimilar Pullulanase is not identical; Wherein, I type Pullulanase is industrial most widely used.In order to identify
Aeromonas sp. the As bacterial strain produces the zymetology classification of Pullulanase, and the present invention uses TLC and HPAEC method to analyze the product of As bacterial strain Pullulanase hydrolysis pulullan polysaccharide, and concrete operations are as follows:
Get the substrate that enzyme liquid after 5 μ L preliminary purifications joins 95 μ L (contain 20 mM citric acids of 1% pulullan polysaccharide-trisodium citrate damping fluid, pH 6.0), 60 ℃ of reaction 3 h, get 2～3 μ L hydrolysis after the sample point sample in Silica gel 60 F
254On plate, after drying up, silica-gel plate is placed in butanols/ethanol/water (5:3:2) exhibition layer liquid and opens up 2～3 times.After the exhibition layer finishes, on silica-gel plate, spray methyl alcohol/vitriol oil (7:3) nitrite ion, dry colour developing 5 min in 110 ℃.
By 2000 times of above-mentioned enzymolysis solution dilutions, get 1 mL loading chromatographic column, use Dionex 600 ion chromatography system to detect the composition of enzymolysis solutions, dilute 1% the trisaccharide maltose of 2000 times as standard substance.The HPAEC elution requirement: with 0～250 mM sodium-acetate gradient elution 30 min, flow velocity is 1 mL/min in 100 mM NaOH.
Embodiment 5:
Aeromonas sp. the As bacterial strain produces the enzymatic property analysis of Pullulanase
(1) optimal reactive temperature is measured: the enzyme liquid after 5 μ L purifying and 95 μ L are contained to (20 mM in the citric acid of 1% pulullan polysaccharide-trisodium citrate damping fluid, pH 8.0) mix, respectively 30,40, reaction 10 min at 50,60,70 and 80 ℃ of temperature, then by the DNS method, measure the Pullulanase vigor, wherein enzyme activity is the highest is made as 100%(such as Fig. 7).
(2) optimum pH is measured: preparation contains the damping fluid of the pH 3.0～10.0 of 1% pulullan respectively, and wherein pH 4.0～6.0 is 20mM citric acid-trisodium citrate damping fluid, and pH 7.0 is the NaH of 20mM
2PO
4-Na
2HPO
4Damping fluid, pH 8.0～10.0 are the Tris-HCl damping fluid of 20mM.Get the damping fluid 95 μ L that contain substrate together with enzyme liquid after 5 μ L purifying in water-bath 60 ℃ of reaction 10 min.Measure the Pullulanase vigor, wherein enzyme activity is the highest is made as 100%(such as Fig. 8).
(3) thermal stability analysis: appropriate enzyme liquid is placed in 60 ℃ and 65 ℃ of lower waters bath with thermostatic control, and every 10 min sampling and measuring residual enzyme vigor, the enzyme activity when being incubated 0 min is counted 100%(such as Fig. 9).
(4) substrate specificity analysis: with pulullan polysaccharide, yam starch, amylose starch, alpha-cylodextrin, the beta-cyclodextrin solution of 20 mM citric acids-trisodium citrate damping fluid (pH 6.0) preparation 1%, get 5 μ L enzyme liquid and react 10 min with 60 ℃ of 95 μ L substrates, measure the hydrolyzing activity of strains A Pullulanase that s produces to different substrates with the DNS reducing sugar method.
Visible, I type Pullulanase provided by the invention can be applicable to the industries such as starch processing, environmental protection, food, health care.
<110 > Fudan University
<120 > a kind of Pullulanase and producing bacterial strain thereof and application
<130> 001
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 956
<212> DNA
<213>
<400> 1
attactgggc gtaaagcgca cgcaggcggt tggataagtt agatgtgaaa gccccgggct 60
caacctggga attgcattta aaactgtccg gctagagtct tgtagagggg ggtagaattc 120
caggtgtagc ggtgaaatgc gtagagatct ggaggaatac cggtggcgaa ggcggccccc 180
tggacaaaga ctgacgctca ggtgcgaaag cgtggggagc aaacaggatt agataccctg 240
gtagtccacg ccgtaaacga tgtcgatttg gaggctgtgt ccttgagacg tggcttccgg 300
agctaacgcg ttaaatcgac cgcctgggga gtacggccgc aaggttaaaa ctcaaatgaa 360
ttgacggggg cccgcacaag cggtggagca tgtggtttaa ttcgatgcaa cgcgaagaac 420
cttacctggc cttgacatgt ctggaatcct gcagagatgc gggagtgcct tcgggaatca 480
gaacacaggt gctgcatggc tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg 540
caacgagcgc aacccctgtc ctttgttgcc agcacgtaat ggtgggaact caagggagac 600
tgccggtgat aaaccggagg aaggtgggga tgacgtcaag tcatcatggc ccttacggcc 660
agggctacac acgtgctaca atggcgcgta cagagggctg caagctagcg atagtgagcg 720
aatcccaaaa agcgcgtcgt agtccggatt ggagtctgca actcgactcc atgaagtcgg 780
aatcgctagt aatcgcgaat cagaatgtcg cggtgaatac gttcccgggc cttgtacaca 840
ccgcccgtca caccatggga gtgggttgca ccagaagtag atagcttaac cttcgggagg 900
gcgtttacca cggtgtgatt catgactggg gtgaagtcgt acaggggtac ccgtaa 956
Claims (2)
1. a strain Aeromonas bacterial strain, is characterized in that producing Pullulanase, and culture presevation number is CGMCC NO.5490, called after: Aeromonas As(
Aeromonas sp.As);
Described Pullulanase produces by shake flask fermentation, is I type Pullulanase, and its enzyme work is 24.8 U/mL.
2. the method for producing Pullulanase with the described Aeromonas bacterial strain of claim 1, it is characterized in that concrete steps are as follows: after the Aeromonas bacterial strain is carried out to separation and purification, picking list colony inoculation is in 15-25 mL seed culture medium, and 28-32 ℃, shake and cultivate 16～18 h; Get subsequently 1-2 mL and transfer in 25-50 mL fermention medium, 36-60 h is cultivated in 28-32 ℃ of concussion; Wherein, described seed culture medium component is: Zulkovsky starch 1-2%, peptone 0.5-1.0 %, yeast powder 0.1-0.3 %, MgSO
47H
2O 0.02-0.05 %, KH
2PO
40.05-0.1 %, all the other are water, pH 6.5-7.5; Described fermention medium component is: Zulkovsky starch 1-2 %, peptone 0.5-1.5 %, yeast powder 0.5-1.0 %, MgSO
47H
2O 0.02-0.05 %, KH
2PO
40.05-0.1 %, all the other are water; PH 6.5-7.5.
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| CN106084016B (en) * | 2016-03-07 | 2020-03-20 | 南宁邦尔克生物技术有限责任公司 | Signal peptide mutant capable of improving expression quantity of recombinant pullulanase and application thereof |
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