CN105349508A - Application of novel fructosidase coding gene - Google Patents
Application of novel fructosidase coding gene Download PDFInfo
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- CN105349508A CN105349508A CN201510981576.5A CN201510981576A CN105349508A CN 105349508 A CN105349508 A CN 105349508A CN 201510981576 A CN201510981576 A CN 201510981576A CN 105349508 A CN105349508 A CN 105349508A
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- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
- C12N9/2431—Beta-fructofuranosidase (3.2.1.26), i.e. invertase
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- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01026—Beta-fructofuranosidase (3.2.1.26), i.e. invertase
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Abstract
The invention discloses an application of a novel fructosidase coding gene. Fructosidase has the amino acid sequence shown as SEQ.ID NO:1 in a sequence table. The coding gene has the nucleotide sequence shown as SEQ.ID NO:2 in the sequence table. The fructosidase originates from Lactobacillus plantarum, compared with fructosidase originating from other sources, the enzyme has the enzymatic activity of decomposing FOS (fructooligosaccharides) and freeing fructose radicals from the FOS as well as the inulinase activity and levanase activity; lactic acid bacteria without the capability of utilizing the FOS can have the capability of utilizing the FOS when the enzyme is heterogeneously expressed in the other lactic acid bacteria.
Description
The divisional application that the application is application number is 201310521419.7, denomination of invention is " a kind of Novel fruit Glycosylase and encoding gene and application " thereof, the applying date is the patent application on October 28th, 2013.
Technical field
The invention belongs to biological technical field, be specifically related to a kind of application of Novel fruit Glycosylase encoding gene.
Background technology
Oligofructose (Fructooligosaccharides, FOS) has another name called fructooligosaccharide, refers to that 1 ~ 9 fructosyl is connected to the mixture that the D-Fructose base of sucrose is formed with β-2,1 key or β-2,6 key.As a kind of by the prebiotic substance extensively approved, oligofructose directly can not be digested and assimilated by human body, when it arrives human gastrointestinal tract, the probiotics (as bifidus bacillus and part milk-acid bacteria) in enteron aisle can be stimulated selectively to breed, the short chain fatty acid that metabolism simultaneously produces, pH value in enteron aisle is reduced, suppresses the growth and breeding of intrinsic fungi etc. in exogenous pathogenic bacterium and enteron aisle, play the effect of regulating intestinal canal flora.
Why microorganism has utilizes the ability of oligofructose to be because it contains the enzyme that can decompose oligofructose glycosidic link, i.e. saccharase.Saccharase (β-fructofuranosidase, EC3.2.1.26), have another name called saccharase, be present in organic sphere widely, belong to Glycosylase 32 family, have the function of the β-fructofuranose glycosidic bond hydrolysis of non-reducing end in catalysis β-fructofuranoside molecule, what have also has transglycosylation, and as made fructosyl donor with sucrose, fructosyl can be shifted sugar industry oligofructose by it.The saccharase in Bacterium lacticum bifidus bacillus source is without transglycosylation, and its major function is that the carbon sources such as hydrolysis oligofructose utilize for thalline.Research finds, the different in kind of the saccharase of different sources, comprises substrate kinetics, substrate diversity, optimum reaction conditions etc., makes different strains utilize ability and the characteristic difference of oligofructose.In addition, many milk-acid bacterias with prebiotic function do not have the ability utilizing oligofructose, and the conventional bacterial strain lactobacillus bulgaricus as produced Yoghourt just can not utilize oligofructose, which limits it and grows in human gastrointestinal tract and maintain vigour.
Therefore, based on the demand of practical application, be necessary the fructosidase of development of new, if simultaneously its heterogenous expression can also be realized, make not have and utilize the probiotic bacterium of oligofructose ability to have the ability utilizing oligofructose, then there is larger actual application value.
Summary of the invention
Technical problem to be solved by this invention is abundant not enough for existing fructosidase type, the present situation of practical application request can't be met, and the application of a kind of Novel fruit Glycosylase, its encoding gene, the recombinant expression vector comprising this gene and transformant and described enzyme is provided, this fructosidase derives from plant lactobacillus, compared with the fructosidase of originating with other, this enzyme not only has and decomposes oligofructose and the enzymic activity of therefrom free fructosyl, and it also has that inulinase is active and levanase is active simultaneously; In other milk-acid bacterias, this enzyme of heterogenous expression can make not have and utilize the milk-acid bacteria of oligofructose to obtain the ability utilizing oligofructose.
One of technical scheme provided by the invention is: a kind of protein of separation, its
(1) aminoacid sequence is as shown in SEQ.IDNO:1 in sequence table; Or
(2) fusion rotein for being made up of the protein of aminoacid sequence as shown in SEQ.IDNO:1 in sequence table and protein purification label.
In the present invention, described protein is a kind of novel saccharase, has no before making the present invention and reports, it is active that it has efficient fructosidase, also has that inulinase is active and levanase is active concurrently simultaneously.Protein source of the present invention is in plant lactobacillus (Lactobacillusplantarum) ST-III, it can be obtained by the method for this area routine, as obtained by host cell suitable for the channel genes of code for said proteins can the transformant of protein as described in normal expression, then cultivate described transformant, from culture, be separated described protein.Described protein can also be synthesized into by artificial complete sequence.
As is known to the person skilled in the art: the aminoacid sequence of described protein suitably can introduce replacement, lack, change, insert or increase one or more amino acid and the homologue of described protein, as long as the homologue of this protein still can keep the function of described protein, namely keeping under the prerequisite that the fructosidase catalytic activity of described protein, inulinase are active and levanase is active, the aminoacid sequence that can carry out suitable degree changes.
In the present invention, described protein purification label is for described in the routine of this area, be preferably His purification tag, i.e. the fusion rotein of fusion rotein of the present invention preferably for being made up of the protein of aminoacid sequence as shown in SEQ.IDNO:1 in sequence table and His purification tag.
Two of technical scheme provided by the invention is: a kind of nucleic acid of separation, the protein of its encoding amino acid sequence as shown in SEQ.IDNO:1 in sequence table.
The preparation method of wherein said nucleic acid is the preparation method of this area routine, described preparation method preferably comprises: the nucleic acid molecule extracting the protein of naturally occurring encoding amino acid sequence as shown in SEQ.IDNO:1 sequence table from occurring in nature, or obtained the nucleic acid molecule of the protein of encoding amino acid sequence as shown in SEQ.IDNO:1 in sequence table by gene clone technology, or obtained the nucleic acid molecule of the protein of encoding amino acid sequence as shown in SEQ.IDNO:1 in sequence table by the method that artificial complete sequence synthesizes.
In the present invention, described nucleic acid preferably has the nucleotide sequence as shown in SEQ.IDNO:2 in sequence table, and more preferably, the nucleotide sequence of described nucleic acid is as shown in SEQ.IDNO:2 in sequence table.
As is known to the person skilled in the art: the nucleotide sequence of the protein of encoding amino acid sequence as shown in SEQ.IDNO:1 in sequence table suitably can introduce replacement, disappearance, change, the mode inserted or increase one or more base provides the homologue of a polynucleotide, as long as the protein of this homologue coding still can keep aminoacid sequence to form the function of the protein as shown in SEQ.IDNO:1 in sequence table, namely the fructosidase catalytic activity keeping aminoacid sequence to form protein shown in SEQ.IDNO:1 in as sequence table, under the prerequisite that inulinase is active and levanase is active, the nucleotide sequence that can carry out suitable degree changes.
Three of technical scheme provided by the invention is: a kind of recombinant expression vector comprising above-mentioned nucleic acid.
Wherein said recombinant expression vector obtains by this area ordinary method, that is: above-mentioned nucleic acid molecule is connected to structure on various expression vector and forms.Described expression vector is the various carriers of this area routine.Described carrier preferably comprises: various plasmid, clay, phage or virus vector etc., preferred plasmid, carrier of the present invention is more preferably plasmid pET-28a (+), and namely recombinant expression vector of the present invention is more preferably and above-mentioned nucleic acid is connected to the upper structure of plasmid pET-28a (+) and forms.
Four of technical scheme provided by the invention is: a kind of recombinant expressed transformant comprising above-mentioned recombinant expression vector.
The preparation method of wherein said recombinant expressed transformant is that this area is conventional, as obtained by being converted into by above-mentioned recombinant expression vector in host microorganism.Described host microorganism is the various host microorganisms of this area routine, as long as can meet, above-mentioned recombinant expression vector is stably copied voluntarily, and make the gene of entrained encoding amino acid sequence composition protein as shown in SEQ.IDNO:1 in sequence table can by effective expression.Wherein said host microorganism is preferably: intestinal bacteria (E.coli), is more preferably e. coli bl21 (DE3) or bacillus coli DH 5 alpha.Aforementioned recombinant expression vector is converted in E.coliBL21 (DE3), the preferred engineering strain of the present invention.Wherein said method for transformation is the method for transformation of this area routine, is preferably chemical transformation, heat shock method or electric robin.
Five of technical scheme provided by the invention is: a kind of recombinant lactic acid bacteria, is characterized in that, the foreign gene containing energy express amino acid sequence composition protein as shown in SEQ.IDNO:1 in sequence table in its genome.
In the present invention, the nucleotide sequence of described foreign gene is preferably as shown in SEQ.IDNO:2 in sequence table, and its express amino acid sequence forms the protein as shown in SEQ.IDNO:1 in sequence table.
Described recombinant lactic acid bacteria is a kind of recombinant lactic acid bacteria that can utilize oligofructose, obtain hydrolysis oligofructose, and it is the recombinant lactic acid bacteria of the protein of energy express amino acid sequence composition as shown in SEQ.IDNO:1 in sequence table.In the present invention, described milk-acid bacteria is the milk-acid bacteria that can not produce fructosidase, not have the routine utilizing oligofructose ability described in the routine of this area, preferred lactobacillus bulgaricus (Lactobacillusbulgaricus) ATCC11842 bacterial strain.
In the present invention, described recombinant lactic acid bacteria can be obtained by ordinary method, as long as in the milk-acid bacteria genome as described in being imported by the nucleic acid molecule (preferred nucleotide sequence is as shown in SEQ.IDNO:2 in sequence table) of the protein of energy express amino acid sequence composition as shown in SEQ.IDNO:1 in sequence table, make it stably express.Preferably, described recombinant lactic acid bacteria for by shuttle plasmid pSIP403 by can the nucleic acid molecule (preferred nucleotide sequence is as shown in SEQ.IDNO:2 in sequence table) of the protein of express amino acid sequence composition as shown in SEQ.IDNO:1 in sequence table import as described in milk-acid bacteria genome in, that is: by comprise the shuttle plasmid pSIP403 (recombinant expression vector pSIP403-sacA) that can express the nucleic acid molecule producing the protein of aminoacid sequence composition as shown in SEQ.IDNO:1 in sequence table import as described in milk-acid bacteria in, screening obtains recombinant lactic acid bacteria, described recombinant lactic acid bacteria can the protein of express amino acid sequence composition as shown in SEQ.IDNO:1 in sequence table.Those skilled in the art know, and other shuttle vectorss that can carry conventional nucleic acid molecule are also applicable to the present invention.
Six of technical scheme provided by the invention is: the protein of aforementioned amino acid sequences as shown in SEQ.IDNO:1 in sequence table is as the application of fructosidase, inulinase or levanase.
In the present invention, the protein of described aminoacid sequence as shown in SEQ.IDNO:1 in sequence table has fructosidase activity, and it can β (2-1) glycosidic link of hydrolysis substrate non-reducing end, generates fructose; The protein of described aminoacid sequence as shown in SEQ.IDNO:1 in sequence table also has inulinase activity, and the hydrolysis of inulin of long-chain can be the oligofructose of fructose or short chain by it; The protein of described aminoacid sequence as shown in SEQ.IDNO:1 in sequence table also has levanase activity, and it can be hydrolyzed β in levanase (2-6) glycosidic link, and the Polylevulosan of bacterial origin is also utilized.Due to the specificity of enzyme, the fructosidase in other sources mainly acts on β (2-1) glycosidic link of substrate non-reducing end, as the fructosidase of most of bifidus bacillus; And fructosidase of the present invention is with multiple enzyme activities such as fructosidase, inulinase and levanases, make it can act on the substrate of different connecting key and chain length, the carbohydrate having enriched its Host Strains utilizes diversity, promotes its propagation in enteron aisle.
The protein of described aminoacid sequence as shown in SEQ.IDNO:1 in sequence table has no at present as the fructosidase that plant lactobacillus is originated and reported, simultaneously because this enzyme has the active and levanase activity of inulinase concurrently, it also has no as a new fructoside enzyme viability and reported.
On the basis meeting this area general knowledge, above-mentioned each optimum condition, can arbitrary combination, obtains the preferred embodiments of the invention.
Agents useful for same of the present invention and raw material except special instruction, all commercially.
Positive progressive effect of the present invention is:
1, fructosidase of the present invention is intracellular enzyme, and fructosidases of other current reports have is extracellular enzyme, the enzyme for being connected with cell wall had.The position of fructosidase in cell determines the position that oligofructose hydrolysis occurs, in the gastrointestinal tract, oligofructose born of the same parents outer or cell wall hydrolysis hydrolysate can be made utilize by other entero-bacte; In born of the same parents, hydrolysis does not then have this inferior position, thus the beneficial function of oligofructose can be made to give full play to.
2, not all milk-acid bacteria can utilize oligofructose, and some milk-acid bacteria with prebiotic function can not utilize oligofructose to be exactly because it is not encoded and can be hydrolyzed the fructosidase of oligofructose.Due to the difference of compatibility etc., in these bacterium, the fructosidase in other sources of heterogenous expression also not necessarily can make it obtain the ability utilizing oligofructose.And fructosidase of the present invention heterogenous expression in lactobacillus bulgaricus ATCC11842, recombinant bacterium can be made to obtain the ability utilizing oligofructose, compensate for the defect that it can not utilize oligofructose.Meanwhile, the research that this application of the present invention also increases its metabolic function to other by gene engineering method transformation milk-acid bacteria provides helpful reference.
3, fructosidase of the present invention does not have and turns glycosyl ability, oligofructose can not be generated by sucrose, but this enzyme has best catalysis activity for the oligofructose of short chain, have exoinulinase concurrently simultaneously, the multiple enzyme activities such as circumscribed levanase and saccharase, the carbon sources such as oligose to be helped in its Host Strains better utilised enteron aisle, promotes the propagation of Host Strains in enteron aisle.
Accompanying drawing explanation
The swimming lane of numbering " 1 " in Fig. 1 is PCR primer; The swimming lane of numbering " 2 " is
the electrophorogram of Easy-sacA after NdeI and XhoI double digestion; The swimming lane of numbering " 3 " is the pET-28a (electrophorogram of+) – sacA after NdeI and XhoI double digestion; M
1for DL2000Marker; M
2for 1Kbladder.
Fig. 2 is the electrophoresis result figure after recombinant protein expression identification and purifying, and wherein, M is albumen Marker; 1 is the precellular broken supernatant of E.coliBL21-sacA induction; 2 is the cytoclasis supernatant after E.coliBL21-sacA induction; For the purpose of 3, albumen is through Ni
2+electrophoresis result after column purification.
Fig. 3 is the comparison diagram of MALDITOF/TOF mass spectrometric detection protein peptide spectrum and SacA protein sequence, and overstriking fragment is the peptide section that LC-MS obtains, and underscore fragment is the peptide section that expression vector is introduced.
Fig. 4 is pH (A), temperature (B, C), metal ion (D) affect result figure to restructuring SacA protease activity.
Fig. 5 is the HPLC analytical results figure that SacA is hydrolyzed oligofructose product, SacA acts on sucrose (A) respectively at 37 DEG C, kestose (B), GF3 (C), GF4 (D) 0 (I), 10 (II), after 30 (III) and 60 (IV) minutes, detect with the HPLC being furnished with nh 2 column, wherein, each abbreviation is: Glc, glucose; Fru, fructose; Suc, sucrose; 1-K, kestose 1-kestose; Nys, GF3; F-nys, GF4.
Fig. 6 be lactobacillus bulgaricus ATCC11842 wild type strain (left figure) and containing pSIP403-sacA recombinant bacterial strain containing 1% oligofructose modified MRS culture medium on the (IP-673 containing 25ng/mL, the erythromycin of 10 μ g/mL and the purpurum bromocresolis of 30mg/L, right figure) growing state.
Fig. 7 is the structure schematic diagram of expression vector pET-28a (+)-sacA.
Fig. 8 is the structure schematic diagram of expression vector pSIP403-sacA.
Embodiment
Mode below by embodiment further illustrates the present invention, but does not therefore limit the present invention among described scope of embodiments.The experimental technique of unreceipted actual conditions in the following example, conventionally and condition, or selects according to catalogue.
The determination of saccharase position and the extraction of thick enzyme in embodiment 1 plant lactobacillus
After plant lactobacillus (Lactobacillusplantarum) ST-III is cultivated 24h in 37 DEG C, centrifugal (4 DEG C, 12,000 × g, 10min) collect supernatant and thalline.After supernatant is degerming with the membrane filtration of 0.45 μm, through 25kDa ultra-filtration membrane ultrafiltration and concentration to 1/20 of original volume, be fermented liquid supernatant.Thalline, with after 50mMPBS (pH=6.0) centrifuge washing 2 times, adds N,O-Diacetylmuramidase to 2mg/mL, 37 DEG C of effect 1h.The ultrasonic wave broken 8min of interval (400W, ultrasonic 5s, interval 5s) in ice bath, rear centrifugal (4 DEG C, 12,000 × g, 10min) collect supernatant liquor and precipitation respectively.Precipitation is resuspended in isopyknic above-mentioned PBS, is smudge cells precipitation.Supernatant liquor molecular weight cut-off be 25kDa ultra-filtration membrane ultrafiltration and concentration to 1/5 of original volume, be cytoclasis supernatant.Measure fermented liquid supernatant respectively, in cytoclasis, the enzyme of cleer and peaceful precipitation is lived, and the results are shown in Table 1.As can be seen from the results, enzyme is lived and is almost all present in cytoclasis supernatant, shows that this enzyme is intracellular enzyme.
In table 1 plant lactobacillus, saccharase enzyme is lived and is distributed
Thus the cytoclasis supernatant that aforesaid method obtains is the thick enzyme of saccharase in plant lactobacillus, and enzyme is lived as 5.75U/mL.
The clonal expression of embodiment 2 fructosidase and property testing
1, the structure of recombinant expression plasmid
With SEQIDNo.3:5 '-GGAATTCCATATGGAGCTCATCGTCATGATATGGAAT-3 ' and SEQIDNo.4:5 '-CCGCTCGAGCCGCTAGCTTCGCAACATC-3 ' for primer, with plant lactobacillus ST-III genome for template, carry out PCR reaction.PCR reaction system is 20.0 μ L, and the final concentration of each reactant is: the Mg of 1 × PCRbuffer, 1mmol/L
2+, the dNTP of 0.2mmol/L, 0.4 μm of ol/L upstream and downstream primer (SEQIDNo.3, SEQIDNo.4), the Taq DNA polymerase of 0.1U/ μ L and the DNA profiling of 2.0ng/ μ L, use ddH
2o complements to 20.0 μ L.PCR reaction parameter is: 95 DEG C of denaturation 5min, 95 DEG C of sex change 30s, 56 DEG C of annealing 30s, and 72 DEG C extend 90s, 35 circulations; 72 DEG C extend 7min.Amplified production 1% agarose gel electrophoresis detects.The goal gene fragment of purifying is connected under the effect of T4 ligase enzyme
on Easy carrier (purchased from American Promega company), linked system, condition of contact are with reference to T4 ligase enzyme specification sheets.Connection product electricity is converted into bacillus coli DH 5 alpha, by blue hickie screening positive clone, performing PCR of going forward side by side qualification and order-checking qualification.Extracting contains the recombinant plasmid of correct Insert Fragment
easy-sacA, carry out double digestion (NdeI, XhoI) with expression plasmid pET-28a (+) (purchased from American Novagen company), enzyme cuts system with reference to restriction enzyme specification sheets, reclaim purifying digestion products, carry out 4 DEG C of connections of spending the night.Connect product conversion in BL21 (DE3) (purchased from American Novagen company), coat on the LB flat board containing sulphuric acid kanamycin, cultivate 16h for 37 DEG C, screening positive clone also carries out order-checking qualification, obtain correct target recombinant expression vector, by its called after pET-28a (+)-sacA, corresponding recombinant bacterium called after E.coliBL21-sacA.
2, the abduction delivering of target protein
The mono-bacterium colony of picking E.coliBL21-sacA contains in 20mL in the LB liquid medium of the sulphuric acid kanamycin of 100 μ g/mL, and 37 DEG C, 200rpm shake-flask culture spends the night.By incubated overnight liquid, be seeded to 200mL and contain in the LB liquid medium of kantlex, inoculum size is 1%, and culture temperature 37 DEG C, shaking table oscillation rate is 200rpm, at OD
600when reaching about 0.5-0.7, adding inductor IPTG to final concentration is 0.1mmol/L, 16 DEG C, induction 12h.After induction, in 4 DEG C, the centrifugal 10min of 10000g, collects somatic cells.
3, protein purification
Every gram of wet thallus adds 5mL cell lysis buffer solution (50mMTris-HCl, 300mMNaCl, 10mM imidazoles, pH8.0) resuspended somatic cells, adds N,O-Diacetylmuramidase to 1mg/mL, and phenylmethylsulfonyl fluoride (PMSF) is to final concentration 1mM, after ice bath 30min, carry out ultrasonication.Broken condition is as follows: ultrasonic 300W, ultrasonic 2s, interval 8s, and effect 10min, whole process is carried out in ice bath.Cytoclasis liquid is in 4 DEG C, and the centrifugal 20min of 16000g, collects supernatant.Supernatant adds and is chelated with NiSO
4sepharoseFastFlow gel (GE company of the U.S.) on, mixing, 4 DEG C, shake 1.5h.Latter 4 DEG C, the centrifugal 2min supernatant discarded of 3500g, all the other mix again, with washing soln (50mMTris-HCl, the 300mMNaCl of 10 times of volumes after loading affinity column, 50mM imidazoles, pH8.0) after washing away foreign protein, the elution buffer of different concns (imidazoles containing 50mMTris-HCl, 300mMNaCl and different concns) gradient elution target protein, get 50 μ L eluted protein liquid to mix with isopyknic 2 × sample-loading buffer, in order to carry out protein electrophoresis analysis.Merge the elutriant that purifying protein concentration is higher, with the 50mMTris-HCl (pH8.0, containing 20% glycerine) of 1000 times of volumes in 4 DEG C of 24h that dialyse, freeze-drying is for subsequent use.
4, SDS-PAGE electrophoresis
Protein sample is mixed with isopyknic 2 × sample-loading buffer, shakes in vortex oscillator even; After boiling water heating 5min, adopt the concentrated glue of 5% and 12% separation gel to carry out race glue, electrode buffer is Tris-glycine buffer.Voltage sets when sample is in concentrated glue is 90V, enters that to change voltage after separation gel be 120V until bromophenol blue indicator; After electrophoresis is complete, with coomassie brilliant blue staining liquid after room temperature dyeing 2h, clear to protein band with destainer decolouring.All complete on decolorization swinging table in dyeing and decolorization.By gel imaging instrument gray scale scanning electrophoresis offset plate, analyzing proteins distribution and concentration.
5, MALDITOF/TOF mass spectrometric detection protein peptide spectrum
Blob of viscose containing target protein is cut into 1mm
3the fritter of left and right, load in centrifuge tube, washing once.Decolour 15 minutes with 50% (v/v) acetonitrile/25mM bicarbonate of ammonia (100 μ L, pH8.0).3 times repeatedly, until color is to the greatest extent de-, distilled water wash 1 time, to be immersed by blob of viscose in 30 μ L100% acetonitriles 5 minutes, dehydration, blob of viscose bleaches, then room temperature is drained and is added 8 μ l trypsin Trypsin) enzyme liquid (0.005mg/ml), 37 DEG C of about 16h that spend the night.The sample of 0.3 μ L enzymolysis is added the matrix point of 0.3 μ L in sample panel, room temperature is dried, and upper 4700 protein ingredient analysis systems (purchased from American AppliedBiosystems company) select positive ion reflective-mode to carry out interpretation of mass spectra.
6, result
Using plant lactobacillus ST-III genome as template, be that primer carries out pcr amplification reaction by sequence shown in SEQIDNo.3 and SEQIDNo.4, obtain the gene fragment of about 1.5kb, size is consistent with expection, result such as Fig. 1 numbers shown in " 1 " swimming lane, namely contains goal gene sacA in target fragment.By amplification after reclaim purifying PCR primer fragment with
easy carrier connects, and by blue hickie screening positive clone, builds cloning vector
easy-sacA, and by order-checking and digestion verification.As numbered shown in " 2 " swimming lane in Fig. 1, its agarose gel electrophoresis shows two endonuclease bamhis of 1.5kb and 3kb, and illustration purpose fragment is inserted
easy carrier.By cloning vector
easy-sacA restriction enzyme NdeI and XhoI, digestion products connects with corresponding double digestion carrier pET-28b (a), construction of expression vector pET-28a (+)-sacA.Recombinant plasmid connector is converted into E.coliBL21 (DE3), 37 DEG C of slat chain conveyor 24h, select positive transformant and are seeded to overnight incubation in LB liquid nutrient medium, collected by centrifugation thalline, extract recombinant plasmid, with the restriction enzyme of correspondence, recombinant plasmid is identified.PET-28a-sacA is through NdeI and XhoI double digestion, obtain the pET28a linear fragment of 5kb and the goal gene fragment of 1.5kb, as Fig. 1 numbers shown in " 3 " swimming lane, prove that expression vector pET-28a (+)-sacA successfully proceeds in E.coliBL21 (DE3) Host Strains.Positive expression host called after E.coliBL21-sacA.
E.coliBL21-sacA is after IPTG induction, and in born of the same parents, supernatant has an obvious expression of recombinant proteins band at about 66kDa place, sees Fig. 2.Recombinant protein is because being adsorbed to affinity media (Ni specifically with 6 × His label
2+) go up thus be separated rapidly with foreign protein.The imidazoles of lower concentration can play removes foreign protein and the effect of purifying protein, and the recombinant protein strong with medium avidity can elute under the imidazoles effect of high density.The recombinant protein SacA expressed by E.coliBL21 (pET-28a (+)-sacA) passes through Ni
2+affinity chromatography carries out purifying, adds final concentration 50mmol/L imidazoles, namely can remove most foreign protein in adsorption-buffering liquid, and recombinant protein is adsorbed onto on filler to greatest extent; Adopt gradient concentration wash-out target protein afterwards, the target protein on the complete affine filler of wash-out of imidazoles energy of 300mmol/L concentration.Imidazoles is removed in the 50mmol/LTris-HCL dialysis of 1000 times of volumes of imidazoles eluted protein, and from 1L fermented liquid, about obtain the SacA albumen of 120mg purifying after freeze-drying, living than enzyme reaches 107.0U/mg.At N end, restructuring fructosidase adds that corresponding 6 × his tag size is about 58.1kDa, but the actual recombinant protein obtained about 6KDa bigger than normal than theoretical value.The electrophoresis result of purifying protein as shown in Figure 2.
In order to verify whether expressing protein is SacA, and recombinant protein, after cutting glue and being further purified, uses pancreatin enzymolysis, carries out mass spectrometric detection, see Fig. 3 to the peptide section of its enzymolysis.Find in enzymatic fragment that 10 fragments are consistent with SacA protein sequence, account for 21% of protein sequence total length.This albumen Mascotscore numerical value (KoenigT, MenzeBH, KirchnerM, etal.RobustpredictionoftheMASCOTscoreforanimprovedqualit yassessmentinmassspectrometricproteomics [J] .JProteomeRes, 2008,7 (9): 3708-3717.) reach 96 (>86 is significant difference), show SacA successful expression in E.coliBL21 (DE3).
The mensuration of embodiment 3 saccharase character
1, saccharase enzyme activity determination
It is substrate that saccharase measures with kestose, and system comprises: 50 μ L20mM kestoses, 50 μ L50mMPBS (pH6.0), and 15 μ L enzyme liquid (11 μ g/mL), supplying volume with water is 200 μ L.Mix rear about 37 DEG C 10 minutes, rear 100 DEG C of water-bath 5min.The fructose produced measures test kit (BioVision company of the U.S.) by fructose and detects.Saccharase enzyme is lived and is defined: at 37 DEG C, under pH6.0 condition, and per minute hydrolysis kestose produces 1 μm of ol fructose and is defined as a unit.And the ratio enzyme calculating enzyme by measuring protein content is lived.
2, the mensuration of saccharase optimal pH
Prepare damping fluid (the pH3.0 – 8.0,0.2MNa of different pH
2hPO
4/ 0.1M citrate buffer; PH8.5 – 9.0,50mMTris – HCl damping fluid) former pH6.0 buffered soln replaces to present different pH damping fluids, and enzyme-substrate reactions system is at 37 DEG C of reaction 10min, and the enzyme measured under each pH is lived, and studies its optimal pH.
3, the mensuration of saccharase optimal reactive temperature
Enzyme-substrate reactions system is placed in pH6.0 respectively, 30 DEG C, 37 DEG C, reacts 5min in the water-bath of 40 DEG C, 50 DEG C, 60 DEG C, 70 DEG C and 80 DEG C, the fructosidase enzyme measured at each temperature is lived, to study the impact that temperature is lived on enzyme.
5, the mensuration of saccharase temperature stability
For the thermostability of studying enzyme, by the enzyme liquid of same volume respectively at pH6.0,4 DEG C, 30 DEG C, 50 DEG C, 60 DEG C insulation 2h, every 30min sampling, cool rapidly, pH6.0,37 DEG C measure remnant enzyme activity, be that relative enzyme lives 100%, calculate enzyme activity with uninsulated enzyme activity.
6, metal ion is to the mensuration of fructosidase activity influence
First enzyme liquid with containing the 12h that dialyses at the phosphoric acid salt salt buffer (100mM, pH6.0) 4 DEG C of 10mMEDTA, then used not containing the 12h that dialyses at the phosphate buffered saline buffer (100mM, pH6.0) 4 DEG C of EDTA again.Enzyme liquid after process adds corresponding metal compound solution (KCl, AgNO
3, HgCl
2, MgCl
2, MnCl
2, CuCl
2, CaCl
2), make metal ion final concentration to 5mM, 37 DEG C of insulation 30min, ice bath about 5min, adds substrate, measures enzyme and lives.To be that relative enzyme lives 100% not containing target metal ions and without the fructosidase vigor of the enzyme liquid of insulation, calculate enzyme activity.
7, interpretation of result
The results are shown in Figure 4, as can be seen from Figure 4, the optimal pH value of this saccharase is 6.0, and the fructosidase of originating with other is similar; Its optimum temperuture is 37 DEG C, corresponding with plant lactobacillus optimum growth temperature; Thermostability experiment shows that this enzyme is more stable at low temperatures, and at high temperature (at 50 DEG C and 60 DEG C) its vigor is lost rapidly; Its enzyme is lived and is subject to Ag+, Cu
2+andHg
2+strongly inhibited.The conventional character of these character and other saccharases matches.
The substrate specificity of embodiment 4 saccharase
1, kinetic constant measures
The catalysis activity of fructosidase under the concentration of substrate of mensuration 0.2-200mmol/L, do figure method according to two inverse to map to 1/ [S] with 1/V, obtain fructosidase respectively to the K of sucrose, kestose, GF3, GF4, raffinose, inulin, bacterial levan, melizitose
mvalue and V
maxvalue, and according to the theoretical molecular of SacA and enzyme liquid concentration calculating K
catwith K
cat/ K
mvalue.
Table 2SacA is to the kinetic parameter of different substrate
2, the detection of hydrolysate
After SacA acts on 0,10,30,60 minute with sucrose, kestose, GF3, GF4 respectively at 37 DEG C, centrifugal (16,060 × gfor15min), adopt HPLC to detect after supernatant membrane filtration.Condition is as follows: Kromasil nh 2 column (200mmID × 4.6mm, 5 μm); Moving phase: acetonitrile: water=70:30; Elution flow rate: 1mL/min; Loading volume: 10 μ L; Detector: differential refraction detector.
3, interpretation of result
Measure the Michaelis-Menton constant (K of different substrate respectively
m), maximum reaction velocity (V
max), turn over number (K
cat) and K
cat/ K
mratio, the results are shown in Table 2.In table 2, each value is the mean value of three independent measured values, adopt respectively 5.5 and 2000.0kDa calculate accordingly as the molecular weight of inulin and bacterial levan.As can be seen from the results, compared with other substrates, restructuring SacA keeps high avidity (low km value) for hydrolysis oligofructose class, show that this enzyme has better Preference for the glycosidic link of the β (2-1) in the Polylevulosan of low polymerization degree between two residue of fructose, and the Bacterium lacticum that other have been reported is all have the highest avidity for bacterial levan.For Vmax, substrate be sucrose and kestose time, this value is maximum, be respectively GF3, GF4, raffinose, inulin, 1.3,1.5,3,15 and 24 times of bacterial levan.For all substrates, what the ratio of Kcat/Km was maximum is kestose, shows that kestose is the suitableeest substrate of this enzyme.It can also be seen that from table 2, this enzyme has substrate specificities widely, not only can be hydrolyzed the common substrate of fructosidase as oligofructose, sucrose etc., raffinose, inulin and bacterial levan can also be hydrolyzed, show that this enzyme also has and comprise exoinulinase, the multiple enzyme activities such as circumscribed levanase and saccharase.
SacA acts on 0,10,30,60 minute with sucrose, kestose, GF3, GF4 respectively at 37 DEG C.Hydrolysate is analyzed by HPLC, the results are shown in Figure 5.Result show, initial reaction stage when enzyme and sucrose effect time, product is fructose and glucose; During with kestose effect, product is sucrose and fructose; During with GF3 effect, product is kestose and fructose; During with GF4 effect, product is GF3 and fructose.Extend the reaction times, GF3 and pentasaccharides and enzyme effect can produce sugarcane really disaccharides and sucrose.Above result shows, SacA is by β (2-1) glycosidic link in circumscribed mode hydrolysis substrate, from the non-reducing end of substrate release residue of fructose.This mode of action and other fructosidases reported similar.
The expression of embodiment 5 fructosidase gene in lactobacillus bulgaricus ATCC11842
1, the structure of fructosidase gene expression vector in lactobacillus bulgaricus ATCC11842
With SEQIDNo.5:5 '-AGGAGCTCATCGTCATGATATGG-3 ' and SEQIDNo.4:5 '-CCGCTCGAGCCGCTAGCTTCGCAACATC-3 ' for primer, with ST-III genome for masterplate, carry out PCR reaction.PCR reaction system is 20.0 μ L, and each reactant final concentration is: the Mg of 1 × PCRbuffer, 1mmol/L
2+, the dNTP of 0.2mmol/L, 0.4 μm of ol/L upstream and downstream primer (SEQIDNo.3, SEQIDNo.4), the Taq DNA polymerase of 0.1U/ μ L and the DNA profiling of 2.0ng/ μ L, use ddH
2o complements to 20.0 μ L.PCR reaction parameter is: 95 DEG C of denaturation 5min, 95 DEG C of sex change 30s, 56 DEG C of annealing 30s, and 72 DEG C extend 90s, 35 circulations; 72 DEG C extend 7min.Amplified production 1% agarose gel electrophoresis detects.Double digestion (BsphI after purifying object fragment, XhoI), with double digestion (NcoI, XhoI) shuttle plasmid pSIP403 (SorvigE, MathiesenG, NaterstadK, etal.High-level, induciblegeneexpressioninLactobacillussakeiandLactobacil lusplantarumusingversatileexpressionvectors [J] .Microbiology, 2005,151 (Pt7): 2439-49.; This plasmid is given by Food Research Inst. of Norway) in 4 DEG C of connections of spending the night.Connect product conversion in bacillus coli DH 5 alpha (purchased from American Promega company), go forward side by side performing PCR and order-checking qualification.Extracting contains the recombinant plasmid pSIP403-sacA of correct Insert Fragment, electricity is transformed into lactobacillus bulgaricus ATCC11842 (purchased from American ATCC), dull and stereotyped upper 37 DEG C of the MRS coated containing erythromycin cultivates 48h, and screening positive clone also carries out order-checking qualification.
2, target protein abduction delivering and utilize the mensuration of oligofructose ability
The lactobacillus bulgaricus list bacterium colony that picking contains correct recombinant plasmid pSIP403-sacA contains in 20mL in the MRS medium liquid of the erythromycin of 10 μ g/mL, and 37 DEG C, quiescent culture spends the night.Incubated overnight liquid gradient dilution is coated containing 1% oligofructose modified MRS culture medium on, described modified MRS culture medium contains the IP-673 (EijsinkVG that concentration is 25ng/mL, BrurbergMB, MiddelhovenPH, etal.InductionofbacteriocinproductioninLactobacillussake byasecretedpeptide [J] .JBacteriol, 1996, 178 (8): 2232-7., it is synthesized by Invitrogen company, its aminoacid sequence is MAGNSSNFIHKIKQIFTHR), the erythromycin of 10 μ g/mL and the purpurum bromocresolis of 30mg/L), cultivate 48h for 37 DEG C, observe bacterium colony situation and substratum colour-change.
3, interpretation of result
The results are shown in Figure 6, as seen from Figure 6, lactobacillus bulgaricus ATCC11842 wild type strain can not utilize oligofructose as carbon source for growth, on the substratum containing purpurum bromocresolis taking oligofructose as sole carbon source, this bacterium can grow, but poor growth does not produce sour variable color; After sacA is recombinant expressed in lactobacillus bulgaricus by shuttle plasmid pSIP403, recombinant bacterium can grow rapidly, and produces the variable color circle being greater than 3mm, shows that this recombinant bacterium has the excellent ability utilizing oligofructose.
Claims (1)
1. the protein of aminoacid sequence as shown in SEQ.IDNO:1 in sequence table is as the application of fructosidase, inulinase or levanase.
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| CN115838710A (en) * | 2022-12-05 | 2023-03-24 | 江苏海洋大学 | Exo-levanase capable of degrading levan and application thereof |
| CN117074699A (en) * | 2023-10-17 | 2023-11-17 | 中国农业大学 | Method for screening lactobacillus casei starter for metabolizing sucrose |
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| WO2016090192A1 (en) * | 2014-12-05 | 2016-06-09 | Danisco Us Inc. | Compositions and methods comprising the use of a bacillus agaradhaerens inulosucrase (inuo) |
| AU2016228331A1 (en) * | 2015-03-11 | 2017-09-28 | B Food Science Co., Ltd. | Improved beta-fructofuranosidase |
| WO2019100676A1 (en) * | 2018-05-25 | 2019-05-31 | 邦泰生物工程(深圳)有限公司 | Preparation method for enzyme-modified stevioside, enzyme for said preparation method, and application thereof |
| CN109553663A (en) * | 2018-12-19 | 2019-04-02 | 上海应用技术大学 | A kind of local modulin and its regulation method of lactobacillus plantarum metabolism FOS |
| CN111235073A (en) * | 2020-03-30 | 2020-06-05 | 南宁中诺生物工程有限责任公司 | Lactobacillus plantarum producing inulinase and application thereof |
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| CN108570487B (en) * | 2017-03-08 | 2021-07-27 | 财团法人食品工业发展研究所 | Method for increasing content of fructosyl mangiferin in plant extract |
| CN115838710A (en) * | 2022-12-05 | 2023-03-24 | 江苏海洋大学 | Exo-levanase capable of degrading levan and application thereof |
| CN117074699A (en) * | 2023-10-17 | 2023-11-17 | 中国农业大学 | Method for screening lactobacillus casei starter for metabolizing sucrose |
| CN117074699B (en) * | 2023-10-17 | 2024-01-12 | 中国农业大学 | Method for screening lactobacillus casei starter for metabolizing sucrose |
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