CN105112433A - Novel coding gene of Type-I pullulanase, and recombinant expression and application thereof - Google Patents

Abstract

The invention relates to a novel coding gene of Type-I pullulanase, and recombinant expression and application thereof. A PCR (polymerase chain reaction) technique is utilized to obtain a full-length sequence of a T.flagellatus sp. strain (CGMCC NO.1.12170) pullulanase gene pulB, the full length of the gene is 1728bp, and the nucleotide coding sequence is disclosed as SEQ ID NO:1. The coding gene is used for coding 575 amino acids, the predicted protein size is 66kDa, and the amino acid sequence is disclosed as SEQ ID NO:2. The recombinant pullulanase can be used for specifically cutting off alpha-1,6-glycosidic bond in an amylopectin branch point, thereby cutting off the whole side branch and forming amylose. The coding gene is capable of generating high-glucose syrup and ultrahigh-malt syrup under the synergistic actions of alpha-amylase, beta-amylase and the like, enhancing the degree of fermentation in beer and decomposing the branched chain with the minimum unit, and is applicable to the industries of starch processing, environmental protection, food, medical care and the like.

Description

A kind of encoding gene of new I type Pullulanase and recombinant expressed and application thereof

Technical field

The invention belongs to genetically engineered and protein engineering field, be specifically related to the cloning and expression that a kind of bacillus of expanding produces Pullulanase bacterial strain Pullulanase encoding gene.The present invention also provides structure and the restructuring Pullulanase preparation method of this Pullulanase encoding gene recombinant plasmid.

Background technology

Pullulanase (Pullulanase, EC3.2.1.41) is a kind of debranching factor in alpha-amylase family GH13, and it can α-1,6-glycosidic link in narrow spectrum hydrolysis Propiram, starch and glycogen.The characteristic that Pullulanase can decompose side chain determines its widespread use in starch processing industry, such as: in starch hy-drolysis process, energy specificity cuts the α-1 in amylopectin tapping point, 6-glycosidic link, thus cut whole side shoot, form amylose starch, the latter has good water resisting property and film-forming properties, be expected to produce edible packing membrane, solve " white pollution " problem; I type Pullulanase can act synergistically with α-amylase, beta-amylase etc. and produce that high glucose is starched, fermentation degree in superhigh maltose syrup, raising beer; Pullulanase and α-amylase and saccharifying enzyme synergy, can play a role in the liquefaction being raw material production alcohol with non-grain cassava and saccharifying, the substrate molecule structure that Pullulanase requires is minimum, the side chain of least unit can decompose by it, maximally utilise starch material, accelerate saccharifying, thus effective raising starch utilization ratio and hydrolysis efficiency.In fields such as food, medical cosmetic, analytical chemistry, Pullulanase can reverse reaction malt-base-alpha-cylodextrin, compares the cyclodextrin of current widespread use, without any toxicity and hemolytic; In addition, adopt Pullulanase that gelatinization burin-in process again after all kinds of starch debranching can be increased the content of resistant starch, the latter has important physiological function to aspects such as blood sugar for human body level, obesity, cancer, lipid metabolism and energy.Therefore, Pullulanase has important use and good market outlook on starch processing industry, medicine food.

Pullulanase is obtained by gas bacillus (Aeorbaetereaerogenes) fermentation in 1966 by Bender and Wallenefls at first ^[1].After this, the scientific research personnel of various countries, through studying extensively and profoundly, obtains this enzyme from different regional microorganisms.The YoshiyukiTakaskai of Japan in 1975 finds that deep shape mutation (BacilluscereusVar.mycodes) of wax-like bud pole bacterium can produce Pullulanase ^[2], its best use of condition is pH6.0 ~ 6.5, temperature 50 C.Last century early eighties, Novo company of Denmark obtain acidophilia decompose pulullan polysaccharide genus bacillus (Bacillusacidopullulyticus) ^[3], the Pullulanase heat-resisting (60 DEG C) that this bacterium produces, acidproof (pH4.5).The said firm drops into huge fund and develops commodity Pullulanase Pormozyme, is the Pullulanase that most widely used and output is maximum at present.1986, the YuhsiykuiTakaskai of Japan reported the Bacillus subtilis TU that a strain can produce heat-resisting Pullulanase, and the best use of pH of enzyme is 7.0 ~ 7.5, the best use of temperature 60 C ^[4].Last century the nineties, Deweer ^[5]find Pullulanase producing strains Bacillusnaganoensis; Tomimura ^[6]filter out Bacillusderamificans, the zymologic property of the Pullulanase that this two strains bacterium produces is similar to the zymologic property of B.Acidopullulyticus.The discovery of this two strains Pullulanase producing strains, has widened the application of Pullulanase further.At present, the product Pullulanase bacterium that China researchist is separated is mainly derived from bacillus and klebsiella spp ^[7,9], produce enzyme enzyme and live lower, produce enzyme level and be generally 2 ~ 5U/mL, these bacterium producing multi enzyme preparations are mostly pathogenic bacteria, and its fermented liquid can not be directly used in industry to be added, and particularly foodstuffs industry, therefore Pullulanase gene is recombinant expressed most important.

Along with the development of genetic engineering technique, utilize genetic engineering technique to build genetic engineering bacterium, to improve the output of target protein, and reduce the cost of downstream process, this is worldwide paid attention to more and more widely and is applied.1984, Japanese Scientists entered intestinal bacteria be able to effective expression wherein the Pullulanase transgenosis in Klebsiellaaerogenes, but the enzyme running water of this colibacillus flat not high and in Nonsele ctive culture media phenotype extremely unstable.1985, Takiazwa was cloned into multi-copy vector pBR322 Pullulanase gene (comprising structure gene and operator gene), and obtain the engineering bacteria of putting down high 20 ~ 40 times than wild strain enzyme running water, this engineering bacteria can keep high enzyme level two weeks.In above-mentioned research, most enzyme is all in born of the same parents, is not secreted into outside born of the same parents.1999, the people such as TegauewMartin are from Bacillusnanganoensis (ATCC53909) (USPTO6,300,115) Pullulanase gene has been isolated in, and expression is obtained in prokaryotic expression system bacillus subtilis, the restructuring Pullulanase produced has good application characteristic ^[10].It is pH5.0 that this enzyme records optimal reaction 60 DEG C time, and under pH4.5 condition, record optimum temperuture is 60 DEG C; At pH4.5,60 DEG C are incubated the remnant enzyme activity that 55h still has 50%, have good thermostability.Henceforth many Pullulanase genes obtain cloning and expressing; after particularly entering the nineties; these work progress speed are accelerated greatly, in succession have many heat-resisting Pullulanase genes to be cloned, to check order and express in large intestine and Bacillus subtilus, and patent protection ^[11].Had dozens of Pullulanase gene to carry out heterogenous expression, main expression system is escherichia expression system.Table 1-1 lists using gene engineering technique in recent years and expresses the situation of the Pullulanase gene of different sources.As can be seen from table 1-1, although genetic engineering technique achieves some achievements in the heterogenous expression research of Pullulanase, on the whole, the expression level of Pullulanase is not high.China just researchs and develops Pullulanase from the seventies, but be still only limitted to laboratory study up till now and produce enzyme enzyme live lower, Pullulanase is expressed by genetic engineering means, improve Pullulanase expression amount, will also become the main development direction of Pullulanase fundamental research and applied research.

Table 1 ?1 Pullulanase gene engineering expression situation

Table1‐2Theconditionofpullulanaseexpressionbygeneengineering

As can be seen from these reports above, still have much in the exploration of Pullulanase research field about genetic engineering technique at present, which demonstrate the validity utilizing genetic engineering means to express to improve Pullulanase.But domestic so far also to clone Pullulanase gene from expansion bacillus, builds genetically engineered system, obtain the report of new Pullulanase ^[8].

In addition, starch adds the temperature of reaction that man-hour takes 50 ~ 55 DEG C usually, and this just proposes higher requirement to Pullulanase physicochemical stability at such a temperature.Screen the Propiram degrading enzyme that novel high enzyme is alive, have certain thermostability, there is important application value.

The seminar of applicant had previously screened the expansion Bacillus strain Tumebacillusflagellatussp. that Pullulanase is produced in a strain from the waste water of tapioca (flour) factory, its culture presevation number is: CGMCCNO.1.12170, preservation date is on April 11st, 2012, and depositary institution is China Committee for Culture Collection of Microorganisms's common micro-organisms center.

Summary of the invention

First object of the present invention is to provide the Propiram degrading enzyme encoding gene that enzyme work is high, acid resistance is good.

Second object of the present invention is to provide the recombinant plasmid of the activated above-mentioned Pullulanase gene fragment of tool.

3rd object of the present invention is to provide enzymatic property and the function and application of above-mentioned restructuring Pullulanase.

Propiram degrading enzyme encoding gene provided by the invention, is utilize T.flagellatussp. bacterial strain (CGMCCNO.1.12170) to produce, is designated as pulB.

The present invention utilizes round pcr to obtain the full length sequence of T.flagellatussp. bacterial strain (CGMCCNO.1.12170) Pullulanase gene pulB, full length gene 1728bp, its nucleotide coding sequence is as shown in SEQIDNO:1, and the nucleotideBLAST in NCBI does not search homologous sequence.This sequence encoding 575 amino acid, its aminoacid sequence as shown in SEQIDNO:2, predicted protein size 66kDa.Aminoacid sequence and the highest protein of lane database similarity are the Cyclomaltodextrinase from Thermicanusaegyptius.From the result of blastX, the Identities of comparison is 54%, Positives is 69%.Therefore, this enzyme belongs to new enzyme.Predict at the multiple parameters of Expasy website (http://www.expas.org/tools), analyze discovery by identifying the structural domain of protein and noting the instrument SMART (simplemodulararchitectureresearchtool) analysed, this enzyme has diastatic structural domain (Fig. 1).Protein structure prediction and domain analyses show that the PulB albumen of T.flagellatussp. has 4 conserved domains, are respectively the PUD (CBM2) shown in Fig. 2, CBM48, GH13 and DUF (Domainofunknownfunction).Wherein PUD structural domain is the Pullulanase substrate-binding domain of prediction; CBM48 is present in the enzyme of multiple decomposition side chain, such as: Pullulanase, isoamylase etc.; GH13 structural domain is the α-amylase catalyst structure domain existed in Pullulanase, can be hydrolyzed α-1, the 6-glycosidic link in pulullan polysaccharide, amylopectin; The Unknown Function of DUF structural domain, has all found this conserved domain in bacterium and fungi.

PulB hydrolyzable pulullan, amylopectin etc., produce the oligosaccharides of the different head of α, belongs to multifunctional amylase.Multifunctional amylase is the new class member of α-amylase family (α-amylasefamily), comprise new Pullulanase, maltogenic amylase and ring maltodextrin enzyme, they structurally have the region of 4 high conservatives that alpha-amylase family has, include catalytic site and substrate binding site, have stable (α/β) ₈folding bucket structural domain, catalytic site is Asp ... Glu ... Asp pattern, but functionally have again the catalytic activity of glycoside hydrolase and glycosyltransferase concurrently, although and different multifunctional amylases has certain similarity on structure and fuction, the enzyme of different sources also exists difference in the specificity etc. of substrate specificity and hydrolysis.

The present invention also provides the cloning process of above-mentioned Pullulanase full-length gene (pulB), namely with T.flagellatussp. bacterial strain (CGMCCNO.1.12170) genomic dna for template, by the gene order-checking result of this laboratory to this bacterial strain, through bioinformatic analysis, design the primer being with suitable restriction enzyme site, and obtain this enzyme gene (pulB) total length (Fig. 3) by round pcr.

The present invention also provides the recombinant plasmid of a kind of nucleotide coding sequence containing Pullulanase gene fragment.This plasmid is pET22b-pulB.By being cloned into the multiple clone site of carrier by ordinary method containing Pullulanase gene segment encodes sequence of the present invention, above-mentioned recombinant plasmid (see Fig. 4) can be formed.

The present invention also provides the method for recombinant expressed tool activated I type Pullulanase, and expression system used can be escherichia expression system, also can be yeast expression system.

In order to obtain the activated Pullulanase of tool, the present invention adopts escherichia expression system, this Recombinant protein expression plasmid is pPET-pulB, Host Strains is BL21 (DE3), this recombinant expression protein C holds fusion 6 × His label, and this recombinant protein utilizes ni-sepharose purification to obtain the restructuring Pullulanase consistent with predicted protein molecular weight after IPTG induction.As shown in Figure 5: wherein M is protein standard molecular weight, the contrast that swimming lane 1 is done for the empty plasmid not containing object fragment, the PulB that swimming lane 2 ~ 4 is expressed for different time after IPTG induction.To be that the Pullulanase of PluB after the PluB purifying after purifying is more alive than enzyme be about 58U/mg to swimming lane 5 ~ 7 respectively.

The optimal pH of the present invention to restructuring Pullulanase measures.Preparation is containing the 20mM citrate-phosphate sodium damping fluid of pH3.0 ~ 7.0 of 1% pulullan polysaccharide respectively.Get damping fluid 400 μ L containing substrate together with the enzyme liquid after 100 μ L purifying in a water bath 50 DEG C react 10min.Measure Pullulanase vigor, what wherein enzyme activity was the highest is set to 100%.Fig. 6 (left side) shows, the optimal pH of this enzyme is 5.0, and when pH4.6, pH4.8 and pH5.4, the enzyme activity of Pullulanase is respectively 5%, 25% and 75% under optimal pH condition.

The pH stability of the present invention to restructuring Pullulanase measures.A certain amount of pure enzyme through 4 DEG C, different pH buffer retains its enzyme activity of mensuration after 24h, draw pH beta stability line, see shown in Fig. 6 (right side).As seen from the figure, Pullulanase all has good stability in the scope of pH4.5 ~ 5.5, and enzyme activity all remains on more than 60%.

The present invention measures restructuring Pullulanase optimum temperuture.Enzyme liquid after 100 μ L purifying and 400 μ L are mixed containing in the citrate phosphate buffer (pH5.0) of 1% pulullan polysaccharide, respectively 30,35,40,45,50,55, react 10min at 60 and 65 DEG C of temperature, then measure Pullulanase vigor by DNS method, what wherein enzyme activity was the highest is set to 100%.The result of Fig. 7 (left side), the optimal reactive temperature of restructuring Pullulanase is 55 DEG C.See shown in Fig. 7 (left side).

The thermostability of the present invention to restructuring Pullulanase measures.Under appropriate enzyme liquid being placed on different (temperature 30 DEG C ~ 65 DEG C), water bath with thermostatic control is incubated 1h, and sampling and measuring residual enzyme vigor, counts 100% with enzyme activity when being incubated 0min.The result of Fig. 7 (right side) shows, under 55 DEG C of conditions, process 1h, and the enzyme of PulB is lived and substantially remained unchanged.Because starch adds the temperature of reaction that man-hour takes 50 ~ 55 DEG C usually, the thermostability at this temperature of this enzyme meets the demand of industrial development.See shown in Fig. 7 (right side).

According to the two class criteria for classifications that IUBMB (InternationalUnionofBiochemistryandMolecularBiology) sets up, Pullulanase is divided into two classes, wherein I type Pullulanase (TypeIpullulanase, EC3.2.1.41) single-minded hydrolysis pulullan α-1,6-glycosidic link produces trisaccharide maltose; α-1, the 6-glycosidic link that II type Pullulanase (TypeIIpullulanase, EC3.2.1.135) can be hydrolyzed pulullan polysaccharide simultaneously and α-Isosorbide-5-Nitrae-glycosidic link produce the linear polymer that is connected with α-Isosorbide-5-Nitrae-glycosidic link and trisaccharide maltose.Dissimilar Pullulanase and other lytic enzyme act synergistically, and generate different products, and the application therefore in industry-by-industry is also not quite similar, I type Pullulanase being most widely used industrially.

For identifying the catalysis classification of this Propiram degrading enzyme, the present invention is analyzed the product that PulB is hydrolyzed pulullan polysaccharide by high performance liquid chromatography (HPLC), and stratographic analysis figure as shown in Figure 8.The trisaccharide product that this Propiram degrading enzyme hydrolysis pulullan polysaccharide produces, its liquid chromatography retention time is about 8.003min (as Fig. 8 A).Analyzed by standard addition method, result shows that the chromatographic peak of the product of this enzymic hydrolysis pulullan is consistent with the retention time at the peak of trisaccharide maltose standard substance, and what show PulB is hydrolysis pulullan polysaccharide by internal-cutting way, and the end product obtained is trisaccharide.Therefore the catalysis classification of PulB belongs to I type Pullulanase.

Because the side chain of least unit can decompose by the blue enzyme in Shandong, contribute to starch and be finally hydrolyzed into monose, play an important role at enzymolysis process such as manufacture medical glucose, high maltose syrup and starch processing liquefying-saccharifyings.Usual amylorrhexis process comprises liquefaction and saccharification two steps: liquefaction process is add α-amylase in the slurries of 30% ~ 35% (m/v) at starch solid substance, at 80 ~ 105 DEG C of temperature, be hydrolyzed into dextrin; Saccharifying generally uses saccharifying enzyme to be hydrolyzed at 60 ~ 65 DEG C of temperature, makes the starch of partial hydrolysis and dextrin resolve into glucose.Saccharifying enzyme energy hydrolyzing alpha-Isosorbide-5-Nitrae-glycosidic link, but low to α-1,6-glycosidic link functioning efficiency, furthermore α-1,6-glycosidic link content is up to 4-5%.Therefore, in this process, whether very crucial beyond doubt for the production of starchy material hydrolysis product monose the thorough hydrolysis of α-1,6-glycosidic link is.Therefore, be necessary to study restructuring Pullulanase PulB and the cooperative saccharification effect of saccharifying enzyme to liquefied starch.As shown in Figure 9.Wherein, A is the glucose products content of liquefied starch different time under Glucoamylase hydrolysis effect; B is the glucose products content of liquefied starch different time under saccharifying enzyme and Pullulanase synchronious hydrolysis.The result of this figure shows, uses two kinds of enzyme synergetic hydrolysis starch can prepare the product of more glucose content, shortens saccharification time.

The present invention's high performance liquid chromatography (HPLC) analyzes PulB and saccharifying enzyme to the hydrolysate after the cooperative saccharification effect of liquefied starch.As shown in Figure 10.Wherein: (A) is the liquefied starch (saccharification 0h sample) before saccharification, and before saccharification, sample glucose content is lower as seen from the figure, mainly the oligosaccharide compositions of the different polymerization degree such as disaccharides, trisaccharide; (B) be the product of single interpolation Glucoamylase hydrolysis liquefied starch 6h gained; As we know from the figure, after saccharification 6h, in liquefier, obtain the glucose component of higher concentration, but also have three a small amount of sugar components.Because saccharifying enzyme is lower to α-1,6-hydrolysis of glycoside bond efficiency, infer that this three sugar component may be panose or different panose (being made up of α-1,6-glycosidic link and α-Isosorbide-5-Nitrae-glycosidic link); (C) be that interpolation saccharifying enzyme and Pullulanase analyze collection of illustrative plates to liquefied starch synergetic hydrolysis 6h after product simultaneously; Result shows to use two kinds of enzymes to have better effect simultaneously, and gained sugar product is all glucose, three unnecessary sugar components do not detected.Therefore, in industrial application, use two kinds of enzymes better, more efficiently to act on by having to the preparation of the products such as glucose syrup simultaneously.

To sum up, the present invention obtains the intestinal bacteria recombinant plasmid of PulB gene core structural domain, recombinant expression protein about 58U/mg more alive than enzyme.Recombinant expressed Pullulanase optimal pH is 5.0, and optimal reactive temperature is 55 DEG C; At 55 DEG C of process 1h, the residual activity of this Pullulanase reaches more than 90%.Identify that this recombinant expressed Pullulanase is I type Pullulanase by efficient liquid phase chromatographic analysis, α-1, the 6-glucose glycoside key in pulullan polysaccharide or starch can be hydrolyzed, can not hydrolyze amylose and cyclodextrin.

Restructuring Pullulanase of the present invention can be applicable to the industries such as starch processing, environmental protection, food, health care.

Accompanying drawing explanation

Fig. 1: with the functional domain of the PulB of SMART analyses and prediction.Redness represents the functional domain of α-amy.According to completing T.flagellatussp. bacterial strain (CGMCCNO.1.12170) genomic dna sequencing result, design the primer amplification Pullulanase encoding gene (pulB) of T.flagellatussp.This full length gene 1728bp, 575 amino acid of encoding, albumen predicted molecular weight is 66kDa.Find that the product of this genes encoding is the highest with the amino acid sequence similarity from the Cyclomaltodextrinase of Thermicanusaegyptius with Blastx software retrieval GenBank pool of amino acids, Identities is 54%, Positives is 69%, shows that obtaining Pullulanase gene is new gene; With the unit construction of this gene encoding production of SMART tool analysis, be family GH13 hydrolysis of glycoside bond enzyme α-amylase functional domain from the 133-490 amino acids of N end.

Fig. 2: the unit construction of the PulB utilizing Expasy website to predict and protein structure domain.Protein structure prediction and domain analyses show that the PulB albumen of T.flagellatussp. has 4 conserved domains, are respectively the PUD (CBM2) shown in Fig. 2, CBM48, GH13 and DUF (Domainofunknownfunction).Wherein PUD structural domain is the Pullulanase substrate-binding domain of prediction; CBM48 is present in the enzyme of multiple decomposition side chain, such as: Pullulanase, isoamylase etc.; GH13 structural domain is α-amylase catalyst structure domain, can be hydrolyzed α-1,6 glycosidic link in pulullan polysaccharide, amylopectin; The Unknown Function of DUF structural domain.

Fig. 3: the PCR primer electrophoretic analysis figure of the pulB gene utilizing round pcr to increase, diagram PCR primer DNA fragmentation size be about 1.7kb, with expection consistent.

Fig. 4: the recombinant plasmid pPET-pulB of structure and enzyme are cut, PCR verifies electrophoretic analysis figure.Enzyme cuts the fragment all occurring with PCR checking expecting, construction of recombinant plasmid success is described.M1:DL2000 molecular weight marker in figure; M2: λ/HindIII molecular weight marker; CK: the blank taking empty plasmid as template; 1-3: recombinant plasmid pET-pulB; The pET-pulB of 4-9:BamHI and HindIII double digestion; 10-15: the pcr amplification product taking transformant as template.

Fig. 5: the SDS-PAGE electrophoretic analysis of the protein of e. coli protein expression product and purifying

Escherichia expression system is after IPTG abduction delivering, there is more obvious protein characteristic band to occur in 66kD position, this protein size and gene order infer that the Theoretical molecular size of gained conforms to, after ni-sepharose purification, obtain the albumen of single band, this protein is the Pullulanase (PulB) of heterogenous expression.M in figure: protein molecular weight standard; Before 1:BL21/pET-pulBIPTG induction; After 2-3:BL21/pET-pulBIPTG induction; 4:BL21/pET-22b; 5-6: the pulB protein of purifying.

Fig. 6: restructuring Pullulanase optimal pH and pH Stability Determination diagram.

Fig. 7: the optimum temperuture of restructuring Pullulanase and thermal stability determination diagram.

Fig. 8: restructuring Pullulanase PulB is hydrolyzed high performance liquid chromatography (HPLC) analysis chart of the reaction product of pulullan and amylose starch.The 1% pulullan substrate (without enzymic hydrolysis) of (A) pH5.0 in figure; (B) PulB is hydrolyzed pulullan product; (C) 1.0% amylopectin substrate of pH5.0; (D) PulB hydrolyze amylose product; (E) maltose standard specimen.

Fig. 9: the impact of different metal ion pair restructuring Pullulanase PulB

Figure 10: restructuring Pullulanase PulB and the cooperative saccharification effect of saccharifying enzyme to liquefied starch illustrate.In figure, A is the independent role of saccharifying enzyme; B is that restructuring Pullulanase PulB and saccharifying enzyme act synergistically.

Figure 11: HPLC analysis PulB and saccharifying enzyme are to the hydrolysate after liquefied starch cooperative saccharification.In figure, (A) is the liquefied starch not adding saccharifying enzyme; (B) be the hydrolysate adding separately saccharifying enzyme; (C) be restructuring Pullulanase PulB and the synergistic hydrolysate of saccharifying enzyme.

Embodiment

Experiment material used and related operating method as follows, place is not described in detail in detail can with reference to " the molecule of Pehanorm Brooker

Cloning experimentation guide " third edition, Beijing: Science Press, 2002.

1. bacterial strain, carrier: coli strain E.coliBL21 (DE3) and Top10, plasmid pPMD18-TVector, pET-22b, be this laboratory and preserve, identical with the character of respective substance in existing document.

2. substratum

LB substratum (g/L): Tryptone (Oxoid) 10, YeastExtract (Oxoid) 5, NaCl5, pH7.0.

3. experiment material: the polysaccharide such as pulullan polysaccharide, Zulkovsky starch, amylose starch are purchased from Shanghai traditional Chinese medicines group; The reagent that molecular biology needs is purchased from TaKaRa company; Primer synthesis and DNA sequencing are completed by Shanghai biotechnology company limited.DNS (3,5-dinitrosalicylic acid) reagent: Seignette salt 18.2g, is dissolved in 50ml distilled water, heating, 3 are added successively in hot solution, 5-dinitrosalicylic acid 0.63g, NaOH2.1g, phenol 0.5g, be stirred to molten, be settled to 100mL with distilled water after cooling, store in brown bottle, at room temperature place (within 7 ~ 10 days, using afterwards).

4. E. coli system Expression and purification zymoprotein: by the pET-22b Plastid transformation containing enzyme pulB gene in escherichia coli expression bacterium BL21 (DE3), coat LB+Amp flat board, 37 DEG C of inversions are cultured to transformant and occur.With toothpick picking single colony inoculation in 5mLLA (LB nutrient solution+100ug/ml penbritin) nutrient solution, 37 DEG C, 220rpm overnight incubation.Get 1.0mL next day and spend the night bacterium in 100mLLA, 37 DEG C, 220rpm cultivates, as strain density (OD ₆₀₀) reach 0.4 ~ 0.6, add the IPTG that final concentration is 1mM, 30 DEG C, 220rpm continues cultivation 12 hours.Collected by centrifugation supernatant, loads onto clear liquid with dialysis tubing, external application PEG8000, placement refrigerator concentrates, when supernatant volume be about original ten/for the moment, take out supernatant in 4 DEG C, the centrifugal 10min of 8000rpm, concentrated supernatant Ni-NTA post binding purposes zymoprotein, with the imidazoles eluted protein of gradient concentration.

5. Pullulanase enzyme activity determination method: the present invention uses DNS reducing sugar method to measure the enzyme activity of Pullulanase.Concrete grammar is as follows: the enzyme liquid 100 μ L getting suitably dilution, be added into 400 μ L Acetic acid-sodium acetate damping fluid (pH4.8, containing 1% pulullan) in, 10min is reacted under 60 DEG C of conditions, 5min is boiled in boiling water bath after adding 500 μ LDNS termination reactions, cooling, with the absorbance of microplate reader detection reaction liquid under 540nm wavelength.The enzyme liquid of deactivation is boiled for blank with 100 μ L.Calculate the amount producing reducing sugar according to typical curve, 3 parallel sample are done in every group reaction.

Enzyme unit definition alive: at corresponding conditions, per minute decomposes the reduced carbohydrate that pulullan discharges, and namely its reducing power is equivalent to the enzyme amount needed for 1 μm of ol glucose, represents with 1U.

Embodiment 1: obtain TumebacillusflagellatesGST4 Pullulanase full-length gene pulB by PCR method.

According to TumebacillusflagellatesGST4 gene order-checking result (deriving from the information to obtaining after bacterium producing multi enzyme preparation genome sequencing), by gene information Epidemiological Analysis, PCR primer PulB-F and PulB-R is as follows in design:

PulB-F:5 ’ – CGC gGATCCcAATCAGGAAGCTATTTTTCA-3 ' (forward primer, underscore is restriction site BamH I), i.e. SEQIDNO:3.

PulB-R:5 ’ – ACC aAGCTTcACCGTTCCGCCGCTCA-3 ' (reverse primer, underscore is restriction site HindIII), i.e. SEQIDNO:4.

With T.flagellatesGST4 genome DNA for template, carry out pcr amplification with above-mentioned primer.PCR reaction system composition and PCR response procedures are: contain in 50 μ L reaction solutions: 5 × PSPCR damping fluid 10 μ L, dNTPs200 μM, STb gene template 10ng, each 3.2pmol, the PrimeSTARDNA polysaccharase of primer 5 units.Amplification program is: 95 DEG C of denaturation 2min; 98 DEG C, 10sec; 53.6 DEG C, 15sec; 72 DEG C, 2min, totally 30 circulations extend; 72 DEG C, 10min.

Add after A through fragment ends after pcr amplification product reclaims and be connected in pMD18-T carrier, transformation of E. coli Top10 bacterial strain, obtain plasmid pMDT-pulB, the raw work order-checking through Shanghai, obtain the base sequence of gene pulB, long 1728bp, as SEQIDNO:1.Its base sequence is encoded 575 amino acid, and its sequence is as SEQIDNO:2.Utilize Blastn and the Blastp software of the database of NCBI (http://www.ncbi.nlm.nih.gov/) to carry out protein homology search, utilize Expasy website (http://www.expasy.org/) to carry out protein structure domain analysis (Fig. 1 and Fig. 2).

Embodiment 2: the structure of recombinant plasmid and e. coli bl21/pET-pulB genetic engineering bacterium

With plasmid pMDT-pulB for template, be below primer:

PulB-F:5 ’ – CGC gGATCCcAATCAGGAAGCTATTTTTCA-3 ' (forward primer, underscore is restriction site BamH I), i.e. SEQIDNO:3.

PulB-R:5 ’ – ACC aAGCTTcACCGTTCCGCCGCTCA-3 ' (reverse primer, underscore is restriction site HindIII), i.e. SEQIDNO:4.

Carry out pcr amplification, PCR reaction system composition and PCR response procedures are: contain in 50 μ L reaction solutions: 5 × PSPCR damping fluid 10 μ L, dNTPs200 μM, STb gene template 10ng, each 3.2pmol, the PrimeSTARDNA polysaccharase of primer 5 units.Amplification program is: 95 DEG C of denaturation 2min; 98 DEG C, 10sec; 53.6 DEG C, 15sec; 72 DEG C, 2min, totally 30 circulations extend; 72 DEG C, 10min.The pulB gene fragment obtained as shown in Figure 3 is detected through agarose electrophoresis.

PCR primer is after BamH I/HindIII double digestion, and connect under ligase enzyme effect with the carrier pET-22b large fragment through same double digestion, the enzyme of plasmid is cut with linked system as follows:

Enzyme Qie Wendu 37 DEG C, the time is 3h, and digestion products mixes after purifying respectively according to a certain percentage, mixes, spend the night in 16 DEG C of connections with the SolutionI of isopyknic LigationKit (TaKaRa company).

10 μ L are connected product conversion host e. coli competent cell E.coliTop10 by next day, place 30min on ice, 42 DEG C of water-bath thermal shock 90s, add the SOC substratum of 600 μ L42 DEG C preheatings, in 37 DEG C, 200r/min preculture 30min, get be applied in right amount containing ammonia benzyl resistance LB dull and stereotyped, overnight incubation are inverted by 37 DEG C of thermostat containers.

Carry out enzyme through colony lift and plasmid extraction to cut and PCR checking, obtain positive recombinant plasmid (Fig. 4).This Recombinant protein expression plasmid is pET-pulB, and this recombinant expression protein C end has 6 × His label of amalgamation and expression.

Embodiment 3: restructuring Pullulanase expression and purification

Express: by the method transformation of E. coli BL21 (DE3) of prokaryotic expression plasmid pET-pulB by " Molecular Cloning: A Laboratory guide ", coat LB+Amp flat board, 37 DEG C of inversions are cultured to transformant and occur.From transformant, select 5 positive transformants with toothpick, be seeded in 5mLLA (LB nutrient solution+100ug/ml penbritin) nutrient solution together with the recipient bacterium negative control containing empty plasmid, 37 DEG C, 220rpm overnight incubation.Get 1.0mL next day and spend the night bacterium in 100mLLA, 37 DEG C, 220rpm cultivates, as strain density (OD ₆₀₀) reach 0.4 ~ 0.6, add the IPTG that final concentration is 1mM, 30 DEG C, 220rpm continues cultivation 12 hours.Collected by centrifugation supernatant, clear liquid is loaded onto with dialysis tubing, external application PEG8000, placement refrigerator concentrates, when supernatant volume be about original ten/for the moment, take out supernatant in 4 DEG C, the centrifugal 10min of 8000rpm, concentrated supernatant is ascended the throne crude enzyme liquid, and the Ni-NTA post binding purposes zymoprotein for next step carries out purifying.

Purifying: IMAC post is containing chelating sepharose (FF) medium of 5mL column volume, medium uses milli-Q water after NiCl load balance, with 25mL level pad (300mmol/LNaCl, 5 ~ 10mmol/L imidazoles, 50mMpH7.4 phosphoric acid buffer) balance pillar, crude enzyme liquid is adjusted to upper prop after the pH identical with level pad, with 25mL (300mmol/LNaCl, 20mmol/L imidazoles, 50mMpH7.4 phosphoric acid buffer) wash foreign protein, then 25mL (300mmol/LNaCl is used, 60mmol/L imidazoles, 50mMpH7.4 phosphoric acid buffer) wash foreign protein, target protein 25mL (300mmol/LNaCl, 250mmol/L imidazoles, 50mMpH7.4 phosphoric acid buffer) wash-out, collect the elutriant containing target protein matter, be 30 with molecular weight cut-off, the ultra-filtration centrifuge tube of the Millipore of 000 carries out buffer exchange, the pure enzyme of gained is dissolved in pH5.0, in 50-100mM sodium phosphate-citrate buffer solution, and add final concentration be 10% glycerine cook protective material, for carrying out SDS-PAGE analysis, enzyme activity determination and characterization analysis.

The recombinase of fermentation supernatant and purifying carries out the enzyme activity detection of SDS-PAGE electrophoretic analysis and Pullulanase all respectively, and each positive transformant of result display engineering strain BL21/pET-pulB all has target protein and expression amount is lived consistent with enzyme.Target protein bar after the expression and purification of recombinase is as Fig. 5.

Embodiment 4: the enzymatic property analysis of restructuring Pullulanase

1. the mensuration of the optimum pH of enzyme: measure the impact of pH on Pullulanase vigor in the damping fluid of a series of pH.Use the sodium phosphate-lemon damping fluid of 50mM, in the scope of pH3.6 ~ 6.0, in the different pH buffer of 0.2 unit, measure the vigor of Pullulanase.The damping fluid 400 μ L getting the different pH value containing substrate reacts 10min together with the enzyme liquid after 100 μ L purifying in 50 DEG C of water-baths, measure Pullulanase vigor, calculate the enzyme activity under each pH, with wherein enzyme activity the highest be set to 100%, be the optimum pH of this enzyme.As Fig. 6 (left side).

2. the pH stability analysis of enzyme: the damping fluid appropriate enzyme liquid being placed in respectively pH3.5 ~ 7.0, after enzyme liquid places 24h in 4 DEG C, remaining enzyme activity is measured under Pullulanase optimum condition, draw pH beta stability line with without being incubated comparing of enzyme activity, what wherein enzyme activity was the highest is set to 100%.As Fig. 6 (right side).

3. the mensuration of the optimum temperuture of enzyme: the sodium phosphate-lemon damping fluid (pH5.0) the enzyme liquid after 100 μ L purifying and 400 μ L being contained 1% pulullan polysaccharide under optimal pH condition mixes, respectively 30,35,40,45,50,55, react 10min at 60 and 65 DEG C of temperature, then measure Pullulanase vigor by DNS method, what wherein enzyme activity was the highest is set to 100%.As Fig. 7 (left side).

4. the temperature stability analysis of enzyme: under optimal pH condition, is placed on appropriate enzyme liquid after being incubated 1h in the water bath with thermostatic control of the differing temps in temperature 30 DEG C ~ 65 DEG C and measures residual enzyme activity, count 100% with enzyme activity when being incubated 0min.As Fig. 7 (right side).

Embodiment 5: the substrate specificity analysis of recombinant expressed Pullulanase

Pulullan polysaccharide, potato shallow lake Zulkovsky starch, amylose starch, amylopectin, alpha-cylodextrin, beta-cyclodextrin, the γ-cyclodextrin solution of 1% is prepared with the sodium phosphate-lemon damping fluid (pH5.0) of 50mM, get the enzyme liquid after 100 μ L purifying to mix with 400 μ L substrates, in 55 DEG C of reaction 10min, measure restructuring Pullulanase to the hydrolyzing activity of different substrate with DNS reducing sugar method.The ability of restructuring Pullulanase hydrolysis pulullan polysaccharide is set to 100%, calculates the ability of its polysaccharide such as hydrolyzed starch, glycogen.Result is as shown in table 1, and restructuring Pullulanase can special hydrolysis pulullan polysaccharide and starch, and its hydrolyzed starch ability is about 14% ~ 16% of hydrolysis pulullan polysaccharide, can not hydrolyze amylose and α-/β-/γ-cyclodextrin.

Table 1.PluAs-1 substrate specificity is analyzed

Embodiment 6: the zymetology catalytic type of restructuring Pullulanase is analyzed

Because Pullulanase is divided into two types, the application of dissimilar Pullulanase is not identical, wherein, and I type Pullulanase industrially most widely used.In order to identify the catalytic type of restructuring Pullulanase, the present invention uses the product of HPLC (high performance liquid chromatography) to restructuring Pullulanase hydrolysis pulullan polysaccharide to analyze, and concrete operations are as follows:

The enzyme liquid got after 100 μ Lization joins the substrate of 400 μ L (containing the 20mM sodium phosphate-lemon damping fluid of 1% pulullan polysaccharide, pH5.0), 55 DEG C of reaction 24h heat the enzyme that goes out and live, above-mentioned enzymolysis solution is diluted 2000 times, get 10 μ L and be splined on chromatographic column, use the composition wearing peace efficient liquid phase chromatographic analysis systems axiol-ogy enzymolysis solution, using dilute 2000 times 1% trisaccharide maltose as standard substance.HPLC test condition is: wear peace Utimat3000, automatic sampler, chromatographic column: AminexHPX-87H300 × 7.8mm (organic acid post), moving phase: 5mmol/LH2SO4, pH2.5, column temperature 45 DEG C, sample size 10 μ L, flow velocity 0.6mL/min, Composition distribution (Shodex), detector temperature is 45 DEG C.

HPLC analytical results as shown in Figure 8.Restructuring Pullulanase PulB can be hydrolyzed pulullan, amylopectin, has trisaccharide maltose to there is (amylopectin enzymic hydrolysis figure does not show), but do not have glucose to occur in these reaction product in hydrolysate, and can not hydrolyze amylose.Restructuring Pullulanase energy specific for hydrolysis α-1,6-glycosidic link is described, α-Isosorbide-5-Nitrae-glycosidic link can not be acted on, belong to I type Pullulanase.

Embodiment 7: metal ion is to the impact analysis of enzymic hydrolysis activity

Under the suitableeest pH5.0 and temperature 55 DEG C of conditions, a certain amount of purifying enzyme PulB acts on after 30 minutes in the pulullan solution of 1% of the metal ion containing 1mmol/L, measure the hydrolysis activity of PulB in various metal ion buffer solution, not add the enzyme liquid of metal ion in contrast.As shown in Figure 9, all metal ions does not all have obvious promoter action to Pullulanase PulB to result, Zn ²⁺, Pb ²⁺, Co ²⁺, Cu ²⁺obvious restraining effect is had, wherein Cu to enzyme activity ²⁺restraining effect can make the basic inactivation of enzyme, Ba ²⁺the restraining effect of living to enzyme is then relatively little, and Ca ²⁺, Mg ²⁺, K ⁺, Fe ²⁺, Mn ²⁺then enzyme is lived and have an impact (Fig. 9) hardly.

Embodiment 8: restructuring Pullulanase PulB and saccharifying enzyme are to the cooperative saccharification effect of liquefied starch

The potato Zulkovsky starch slurries of configuration 5%, the α-amylase adding 1.2U/g liquefies in 90 DEG C to farinaceous size, and hand mixing to iodine look is brown.The starch of post liquefaction utilizes saccharifying enzyme and Pullulanase PulB to carry out saccharification, and temperature of reaction is 50 DEG C, and every 12h sampling, use glucose determination reagent box (enzymatic measurement) measures the glucose yield in saccharifying.

By above-mentioned saccharification 6h sample, centrifugal rear dilution 10000 times, gets 10 μ L and is splined on chromatographic column.HPLC test condition is: wear peace Utimat3000, automatic sampler, chromatographic column: AminexHPX-87H300 × 7.8mm (organic acid post), moving phase: 5mmol/LH2SO4, pH2.5, column temperature 45 DEG C, sample size 10 μ L, flow velocity 0.6mL/min, Composition distribution (Shodex), detector temperature is 45 DEG C.

Result as shown in figure 11.

Reference

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[2]YoshiyukiTakaskai.Agr.Biol.Chem.[J]1976,40(8):1515-1530.

[3]Jensen,B.F.,andB.E.Norman.Baeillusacidopullulyticuspullullanase:Applicationandregulatoryaspectsforuseinthefoodindustry[J].ProcessBiochem.1984,19:351-369.

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[7]HyunHH，ZeikusJG.GeneralbiochemicalcharacterizationofthermostablepullulanaseandglucoamylasefromClostridiumthermohydrosulfuricum[J].ApplEnvironMicrobiol，1985，49(5)：1168-1173.

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[13]BertoldoC，ArmbrechtM，BeckerFetal..Cloning，sequencing，andcharacterizationofaheat-andalkali-stabletypeⅠpullulanasefromAnaerobrancagottschalkii[J].Appl.Environ.Microbiol.，2004，70(6):3407-3416.

[14]TomiyasuK，YatoK，YasudaM，etal..CloningandnucleotidesequenceofthepullulanasegeneofThermophilusHB8andproductionoftheenzymeinE.coli.[J].Biosci.Biotechnol.Biochem.,2001，65(9):2090-2094.

[15]KurikiT，ParkJH，OkadaS，etal..PurificationandcharacterizationofthermostablepullulanasefromBacillusstearothermophilusandmolecularcloningandexpressionofthegeneinBacillussubtili[J].Appl.Environ.Microbiol.,1988，54(11):2881-2883.

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Sequence table

<110> Guangxi Academy Of Sciences

The encoding gene of the I type Pullulanase that <120> mono-kind is new and recombinant expressed and application thereof

<160>4

<170>PatentInversion3.5

<210>1

<211>1728

<212>DNA

<213> expansion genus bacillus (Tumebacillusflagellatussp.) GST4

<400>1

atgaatcaggaagctatttttcacatgtctcacggcgcgtacgcgtacgcgttgggaccg60

catcatgcgttgatcaaacttcgcgccaagcgcggagacttgaaatcggtgcatatggtg120

cacgaagaccgcttcgaactgcccggctcttatgcaactcaagaattgaactacgcgggt180

tcagatgaattttatgattactttacagcggtggtggagacgagaacgcggaagctccgc240

taccgctttttgttggacaacgggccggagcagtattggtacggggagcgggcgctctcg300

aacatcgccgatttcgccggctggttccaactggcgtacttgccgaaccgcgacttgttc360

gtgattccggagtgggcgaagagtgcggtggtctatcagatcttcccggaccgcttccaa420

aacgggaatccggacaacgatccggaaggcgtgcgtccgtggggcgagttgccgacgccg480

catacgttttttggcggggatttgcaagggattttggacagactcccgtacttggagaac540

ttgggcgtcaacttgatctacatgacgccgatcttcttgtcgccgtccacgcacaagtat600

gacacggccgattactacgcgatcgacccgatgtttggcgatgtggagacgttgaagcgt660

ttggtggagaacgcgcacgttcgcggcatccgcgtgatgctcgacgccgtgttcaaccat720

tgcggtgcggagtttccgcctttccaagacgttttggcgaagggggaggcgtccgagtac780

gccgactggttccacattcacagcttcccggtcgatatgcaggaagtgaattacgagacg840

tttgccaaccatgtggcgtcgatgccgaaattgcgcacggagaatccaaacgtgcgcgat900

tatctgctggatgtggcggagttttgggtgaaggaagtagggatcgacggttggcgtctc960

gacgtggccaacgaagtcgatcacgcgttctggcgcgcgttccgcgatcgggtgcgcgag1020

gcgaacccggagacgttgatcatcggcgaggtgtggcatgactcgtcgccttggttgcaa1080

ggagatcaattcgacggcgtgatgaactacctgttccgcgatgcggtgattgagtttttt1140

gcaaagcggacgatcagcgcggatcgttttgatgcgatgctgaccaaaacgcggatgatg1200

tacaagcgtcaagcgaacttcatgatgttcaacttgctgggcagccatgacacggcgcgt1260

tttttgacgatctgcaacgggcgtgaagagcgcatgcgcttggcggtggtgttccagatg1320

acctatgtggggattccggaagtgtactacggcgacgaagtgggcatggtcggagagaac1380

gacccggattgccgccggacgatgatctgggaagaggagaagcagaaccgcgagttgttc1440

cgcctccatcaacagttgatctctgttcgaaaagcgcatccggctctgcaaaccggtctg1500

tatcgcgcggtggacaaagacgcgctgcacaatctctacgggtttgttcgcgagacggag1560

aacgagtcgatctacatcttgcttaacaacggcagcggcaaccactgggtgtcgcttccg1620

gaaggggtaagcggtcgggatctgttgaccgatcgggtgtacagcggcacgttcgatctg1680

gagtcgtacggtttccgcattctcctgttgagcggcggaacggtgtag1728

<210>2

<211>575

<212> amino acid

<213> expansion genus bacillus (Tumebacillusflagellatussp.) GST4

<400>1

MNQEAIFHMSHGAYAYALGPHHALIKLRAKRGDLKSVHMVHEDRFELPGSYATQELNYAG60

SDEFYDYFTAVVETRTRKLRYRFLLDNGPEQYWYGERALSNIADFAGWFQLAYLPNRDLF120

VIPEWAKSAVVYQIFPDRFQNGNPDNDPEGVRPWGELPTPHTFFGGDLQGILDRLPYLEN180

LGVNLIYMTPIFLSPSTHKYDTADYYAIDPMFGDVETLKRLVENAHVRGIRVMLDAVFNH240

CGAEFPPFQDVLAKGEASEYADWFHIHSFPVDMQEVNYETFANHVASMPKLRTENPNVRD300

YLLDVAEFWVKEVGIDGWRLDVANEVDHAFWRAFRDRVREANPETLIIGEVWHDSSPWLQ360

GDQFDGVMNYLFRDAVIEFFAKRTISADRFDAMLTKTRMMYKRQANFMMFNLLGSHDTAR420

FLTICNGREERMRLAVVFQMTYVGIPEVYYGDEVGMVGENDPDCRRTMIWEEEKQNRELF480

RLHQQLISVRKAHPALQTGLYRAVDKDALHNLYGFVRETENESIYILLNNGSGNHWVSLP540

EGVSGRDLLTDRVYSGTFDLESYGFRILLLSGGTV

<210>1

<211>30

<212>DNA

<213> synthetic

<400>1

cgcggatcccaatcaggaagctatttttca30

<210>1

<211>26

<212>DNA

<213> synthetic

<400>1

accaagcttcaccgttccgccgctca26

Claims (6)

1. derive from a Pullulanase encoding gene for expansion bacillus, it is characterized by described gene is encoding Type I Pullulanase gene, and its nucleotide sequence, as shown in SEQIDNO:1, is designated as pulb.

2. the Pullulanase encoding gene as described in right 1, is characterized in that the aminoacid sequence of described encoding Type I Pullulanase is as shown in SEQIDNo:2.

3. have the amino acid fragment of I type Pullulanase activity, it is characterized in that described fragment is the amino acid of SEQIDNO:2, be designated as PulB, the molecular size range of protein fragments is 66kDa.

4. the recombinant plasmid of the nucleotide sequence containing coding amino acid fragment as claimed in claim 3, is characterized in that being cloned into the multiple clone site of carrier pET-22b by described nucleotide sequence and obtaining, is designated as pET- pulb.

5. a recombinant expressed system as claimed in claim 3 with the amino acid fragment of I type Pullulanase activity, it is characterized in that, described system is escherichia expression system, or yeast expression system.

6. the application of I type Pullulanase fragment in environmental protection, food or health care of the recombinant expressed acquisition of recombinant plasmid according to claim 4.