CN105907815A - Preparation method of high-uniformity high-molecular-weight Levan fructan - Google Patents
Preparation method of high-uniformity high-molecular-weight Levan fructan Download PDFInfo
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- C12P19/00—Preparation of compounds containing saccharide radicals
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Abstract
The invention discloses a preparation method of high-uniformity high-molecular-weight Levan fructan. The method comprises the following steps: carrying out molecular modification on fructosyl sucrose transferase by using modern molecular enzyme engineering to obtain a fructosyl sucrose transferase mutant H280R, successfully carrying out prokaryotic expression in Escherichia coli, purifying the expressed protein, carrying out reaction on the purified enzyme and a substrate sucrose, and carrying out ethanol precipitation to obtain the Levan fructan sample. When the fructosyl sucrose transferase mutant H280R is used for preparing the Levan fructan, the concentration of the substrate sucrose can reach 40%. Gel chromatography is utilized to determine that the molecular weight is 2*10<6>Da, so the Levan fructan belongs to high-molecular polysaccharides. The Levan fructan has the advantages of high uniformity and higher dispersity (2.06) than that of the Levan fructan prepared by the existing biological method.
Description
Technical field
The invention belongs to biological technical field, particularly to the preparation method of a kind of high uniformity macromolecule levan type levan.
Background technology
Levan type levan is one of two kinds of major type of levan, the fructose homopolymer being naturally-occurring, and its main chain is connected by the β repeated-(2,6)-glycosidic bond, and side chain is connected by β-(2,1)-glycosidic bond.Levan type levan is all widely used in many fields, in the food industry, levan can be as fructose sources, emulsifying agent, encapsulation agent and flavor improving agent, owing to levan has the characteristic being difficult to digest, and have low in calories and cariogenicity, in addition being also used as prebiotics to stimulate probiotic bacteria in the propagation of intestinal microbial population, therefore levan is used as sugar replacement；In pharmaceuticals industry, levan can extend effect of drugs and as cholesterol adsorbent in intestinal as a kind of immunomodulator, the succedaneum of blood plasma；Levan also has moisture-keeping function, may apply in cosmetics；Additionally, the levan generated by zymomonas mobilis fructosyl sucrose transferring enzyme also has antitumor, antiviral activity.
Levan type levan can be produced by Transglycosylation from sucrose by fructosyl sucrose transferring enzyme (Levansucrase, EC2.4.1.10).Fructosyl sucrose transferring enzyme belongs to glycoside hydrolase (GH68) family, this family also includes saccharase and Inulosucrase, these three enzyme is all the substrate donor using sucrose as its prioritizing selection, its molecular structure is mainly made up of the signal peptide of N-terminal, catalytic center and three, C-terminal district part.The fructosyl sucrose transferring enzyme of the overwhelming majority all includes three conserved region, is made up of three different amino acid residues, and they are nucleophilic group respectively, and acid/base catalytic group and transition state stablize group, is the most important region of this enzyme enforcement catalysis.
The microorganism that can produce fructosyl sucrose transferring enzyme at present includes bacillus cereus (Bacillus), zymomonas mobilis (Zymomonas mobilis), sticks series bacillus (Paenibacillus polymyxa), Kingsoft lactobacillus (Lactobacillus sanfranciscensis), actinomyces viscosus (Actinomyces viscosus) and pseudomonas (Pseudomonas) etc. more.Zymomonas mobilis is gram negative bacteria, mainly bring as the bacterial strain producing ethanol, its production fructosyl sucrose transferring enzyme of Recent study also gets more and more, the zymomonas mobilis of wild type can produce the levan of macromolecule, but the molecular weight with the levan of substrate reactions generation of recombiant protein is the most relatively small, and have can only synthesize oligosaccharide.The chemosynthesis of levan and extract the high levan of uniformity all very difficulty from plant, fermentable produces the widest method that levan is current industrial application, but the molecular weight of its tunning often changes greatly, and catalytic efficiency is the highest.Therefore it provides the fructosyl sucrose transferring enzyme that can produce the high macromole levan of uniformity is of great significance for Levan type levan industrialized production and application tool.
Chinese invention patent 201310403373.9 discloses a kind of bacillus subtilis Gum levan saccharase mutant T305A at present, it is to utilize modern molecular enzyme engineering technology that the Gum levan saccharase aminoacid sequence deriving from bacillus subtilis (Bacillus subtilis) is carried out molecular modification, screened by fallibility PCR and vigor, obtain mutant T305A, its gene is inserted pSE380 and builds recombinant vector, import escherichia coli host and express.This mutant enzyme is compared with wild-type enzyme, and the concentration of substrate toleration in turn glucosides formation Gum levan reaction with sucrose for substrate significantly improves, and concentration of substrate when reaching maximum conversion is brought up to the 25% of mutant enzyme by the 10% of wild-type enzyme.The most do not mention this patent and can manufacture homogeneous, the Levan type levan of high molecular, and fructosyl sucrose transferring enzyme can only tolerate the substrate of 25% concentration to substrate tolerance.
Summary of the invention
It is an object of the invention to overcome the deficiencies in the prior art, the preparation method of a kind of high uniformity macromolecule Levan type levan is provided, to overcome existing method substrate tolerance the highest, the molecular weight of the levan produced is the most relatively small, the problem that can only synthesize oligosaccharide having, improves enzyme process and prepares the effect of Levan type levan.
In order to solve above-mentioned technical problem, the present invention is accomplished by:
The preparation method of a kind of high uniformity macromolecule Levan type levan, comprises the following steps:
(1) fructosyl sucrose transferring enzyme mutant H280R is built
It is 5'-CTCTTTACGATCAGCCGTGATTCGACTTATG-3' that design utilizes primers F: H280R, primer R:H280R is 5'-CATAAGTCGAATCACGGCTGATCGTAAAGAG-3', and with plasmid pET-32a-lev as template, reaction principle according to Dpn I method rite-directed mutagenesis, build fructosyl sucrose transferring enzyme mutant H280R, set PCR program；
Pcr amplification reaction system is: 10 μ L Primer STAR Max, 1 μ L primers F: H280R, 1 μ L primer R:H280R, 2 μ L templates pET-32a-lev, and reaction cumulative volume is 20 μ L；
PCR amplification condition is: 94 DEG C of denaturations 3min, 94 DEG C of degeneration 30s；54 DEG C take off fire 30s；72 DEG C extend 3min45s；Carry out 30 circular response altogether；Then continue to extend 10min in 72 DEG C；
After PCR primer checking, recovery, double digested through BamH I and Xho I, it is cloned into PGEX-4T-1 plasmid, proceeds to E.coli BL21 (DE3)；
(2) expression and purification of fructosyl sucrose transferring enzyme mutant H280R
By the wild type of the fructosyl sucrose transferring enzyme mutant H280R contained built in step (1) and the bacterium solution of saltant type recombinant bacterial strain, it is transferred to respectively in the 200ml LB liquid medium containing finite concentration ampicillin, cultivate 3～4h, work as OD for 37 DEG C600When reaching 0.6～0.8, add the derivant IPTG of final concentration of 0.2～0.5mmol/L, induction culturing about 4h at a temperature of 28 DEG C, and centrifugal 5min collects thalline under the rotating speed of 12000rpm, utilize in precipitation, add cell lysis buffer solution, and clarify at ultrasonication 2min on ice to bacterium solution, then centrifuge tube is put into rotating speed be 12000rpm, temperature be in the centrifuge of 4 DEG C, centrifugal 10min, takes supernatant and moves to another clean centrifuge tube；
Recombinant bacterial strain collects wet thallus after derivant IPTG overnight incubation, the broken collection crude enzyme liquid containing fructosyl sucrose transferring enzyme, then crude enzyme liquid is loaded in the purification column containing mesh His label, the imidazole buffer II of NaCl and 500mmol/L of the imidazole buffer I by NaCl and 20mmol/L of PB, 500mmol/L containing 20mmol/L and PB, the 500mmol/L containing 20mmol/L, the acidity-basicity ph of buffer I and buffer II is 7.4, carry out gradient elution, chromatograph the collection liquid to eluting after terminating, carry out SDS-PAGE checking；
(3) enzymatic clarification prepares Levan type levan
By the fructosyl sucrose transferring enzyme of purification in step (2) with 25～40% the sucrose of concentration react, adding pH is the citrate buffer solution of 6.0, the temperature keeping reaction is 40～50 DEG C, response time is 48h, sevag method is utilized to remove removing protein, add triploid and amass in the ethanol of fructosyl sucrose transferring enzyme reactant liquor, under 4 DEG C of cryogenic conditions, make polysaccharide precipitation and collect precipitation, recycling concentration be 60～80% ethanol solution wash twice after, with tertiary effluent, precipitation is dissolved collection again, and lyophilised machine freezing obtains the Levan type levan of powder.
Further, the concentration of the ampicillin in described step (2) is 30～60 μ g/mL.
In described step (2), the ultrasound wave demand power of ultrasonication on ice is 120W, and ultrasonication is every 2s time ultrasonication 1s.
Compared with prior art, the invention have the benefit that
When 1, using fructosyl sucrose transferring enzyme mutant H280R to prepare Levan type levan, the concentration of substrate sucrose can reach 40%；
2, present invention clone has obtained fructosyl sucrose transferring enzyme mutant H280R, and success carries out prokaryotic expression in BL21 (DE3) escherichia coli, by to the protein purification expressed, being reacted by the enzyme-to-substrate sucrose of purification and obtain polysaccharide sample through ethanol precipitation, the size being determined its molecular weight by gel chromatography is 2 × 106Da, belongs to macromolecular polysaccharide；
3. the Levan type levan homogeneity that prepared by the present invention is good, and dispersion is 2.06, is better than current bioanalysis and prepares Levan type levan.
Detailed description of the invention
Below in conjunction with specific embodiment, the detailed description of the invention of the present invention is described in further detail.
The preparation method of a kind of high uniformity macromolecule Levan type levan, comprises the following steps:
(1) fructosyl sucrose transferring enzyme mutant H280R is built
It is 5'-CTCTTTACGATCAGCCGTGATTCGACTTATG-3' that design utilizes primers F: H280R, primer R:H280R is 5'-CATAAGTCGAATCACGGCTGATCGTAAAGAG-3', and with plasmid pET-32a-lev as template, reaction principle according to Dpn I method rite-directed mutagenesis, build fructosyl sucrose transferring enzyme mutant H280R, set PCR program；
Pcr amplification reaction system is: 10 μ L Primer STAR Max, 1 μ L primers F: H280R, 1 μ L primer R:H280R, 2 μ L templates pET-32a-lev, and reaction cumulative volume is 20 μ L；
PCR amplification condition is: 94 DEG C of denaturations 3min, 94 DEG C of degeneration 30s；54 DEG C take off fire 30s；72 DEG C extend 3min45s；Carry out 30 circular response altogether；Then continue to extend 10min in 72 DEG C；
After PCR primer checking, recovery, double digested through BamH I and Xho I, it is cloned into PGEX-4T-1 plasmid, proceeds to E.coli BL21 (DE3)；
(2) expression and purification of fructosyl sucrose transferring enzyme mutant H280R
By the wild type of the fructosyl sucrose transferring enzyme mutant H280R contained built in step (1) and the bacterium solution of saltant type recombinant bacterial strain, it is transferred to containing in the 200ml LB liquid medium of the ampicillin that concentration is 30～60 μ g/mL respectively, cultivate 3～4h, work as OD for 37 DEG C600When reaching 0.6～0.8, add the derivant IPTG of final concentration of 0.2～0.5mmol/L, induction culturing about 4h at a temperature of 28 DEG C, and centrifugal 5min collects thalline under the rotating speed of 12000rpm, utilize in precipitation, add cell lysis buffer solution, and clarify at ultrasonication 2min on ice to bacterium solution, the ultrasound wave demand power of described ultrasonication on ice is 120W, and ultrasonication is every 2s time ultrasonication 1s, again centrifuge tube is put into rotating speed be 12000rpm, temperature be in the centrifuge of 4 DEG C, centrifugal 10min, takes supernatant and moves to another clean centrifuge tube；
Recombinant bacterial strain collects wet thallus after derivant IPTG overnight incubation, the broken collection crude enzyme liquid containing fructosyl sucrose transferring enzyme, then crude enzyme liquid is loaded in the purification column containing mesh His label, the imidazole buffer II of NaCl and 500mmol/L of the imidazole buffer I by NaCl and 20mmol/L of PB, 500mmol/L containing 20mmol/L and PB, the 500mmol/L containing 20mmol/L, the acidity-basicity ph of buffer I and buffer II is 7.4, carry out gradient elution, chromatograph the collection liquid to eluting after terminating, carry out SDS-PAGE checking；
(3) enzymatic clarification prepares Levan type levan
By the fructosyl sucrose transferring enzyme of purification in step (2) with 25～40% the sucrose of concentration react, adding pH is the citrate buffer solution of 6.0, the temperature keeping reaction is 40～50 DEG C, response time is 48h, sevag method is utilized to remove removing protein, add triploid and amass in the ethanol of fructosyl sucrose transferring enzyme reactant liquor, under 4 DEG C of cryogenic conditions, make polysaccharide precipitation and collect precipitation, recycling concentration be 60～80% ethanol solution wash twice after, with tertiary effluent, precipitation is dissolved collection again, and lyophilised machine freezing obtains the Levan type levan of powder.
Synthetically prepared Levan type levan is analyzed and identifies
The glucose taking the sucrose of 1%, the Levan type levan of 1% and 1% respectively is standard substance, utilize diphenylamines for developer, developing solvent is n-butyl alcohol: glacial acetic acid: water is 2～3:1～2:1～2, is placed on 10min in the baking oven that temperature is 80 DEG C, observes colour developing situation.
Choosing the Levan type levan powder of lyophilizing after purification, be configured to the solution that concentration is 5mg/ml, go the removal of impurity through 0.45um membrane filtration, use its relative molecular mass of gel chromatography, chromatographic condition is chromatographic column UltrahydrogelTMLinear 300mm × 7.8mm × 2, flowing is the NaNO of 0.1mol/L mutually3Solution, flow velocity is 0.9mL/min, and chromatogram column temperature is 45 DEG C, and the molecular weight measuring Levan type levan is 2 × 106Da。
The Levan type levan powder accurately weighing 20mg purification is placed in brown ampoule bottle, adds 1mol/L H2SO4Final vacuum tube sealing, sustained response 4h at a temperature of 90 DEG C, cool down after levan complete hydrolysis, use BaCO3Neutralize, centrifugal, repeatedly, until H2SO4Solution eliminates completely；Take its supernatant after the membrane filtration that aperture is 0.45um, be analyzed by high performance liquid chromatography, and with fructose, glucose is as standard substance, wherein chromatographic condition: chromatographic column is CarboPac PA20 (3mm × 150mm, 6 μm), and flow velocity is 0.5mL/min；The method using gradient elution, mixed flow is the NaOH solution of 250mmol/L, the NaAc solution of 1mol/L and ultra-pure water mutually, and wherein gradient elution process is: in 0～21min, NaOH solution 1.8%, NaAc solution 0%；In 21～30min, NaOH solution 1.8%, NaAc solution rise to 20% from 5%；In 30～50min, NaOH solution 80%, NaAc solution 0%.
The above is only embodiments of the present invention; again state, for those skilled in the art, under the premise without departing from the principles of the invention; the present invention can also be carried out some improvement, these improve in the protection domain also listing the claims in the present invention in.
Claims (3)
1. the preparation method of one kind high uniformity macromolecule Levan type levan, it is characterised in that: comprise the following steps:
(1) fructosyl sucrose transferring enzyme mutant H280R is built
It is 5'-CTCTTTACGATCAGCCGTGATTCGACTTATG-3' that design utilizes primers F: H280R, primer R:
H280R is 5'-CATAAGTCGAATCACGGCTGATCGTAAAGAG-3', and with plasmid pET-32a-lev as mould
Plate, according to the reaction principle of Dpn I method rite-directed mutagenesis, builds fructosyl sucrose transferring enzyme mutant H280R, sets PCR journey
Sequence；
Pcr amplification reaction system is: 10 μ L Primer STAR Max, 1 μ L primers F: H280R, 1 μ L primer R:
H280R, 2 μ L templates pET-32a-lev, reaction cumulative volume is 20 μ L；
PCR amplification condition is: 94 DEG C of denaturations 3min, 94 DEG C of degeneration 30s；54 DEG C take off fire 30s；72 DEG C extend 3min45s；
Carry out 30 circular response altogether；Then continue to extend 10min in 72 DEG C；
After PCR primer checking, recovery, double digested through BamH I and Xho I, it is cloned into PGEX-4T-1 plasmid, turns
Enter E.coli BL21 (DE3)；
(2) expression and purification of fructosyl sucrose transferring enzyme mutant H280R
Wild type and the saltant type of the fructosyl sucrose transferring enzyme mutant H280R contained built in step (1) are recombinated
The bacterium solution of bacterial strain, is transferred in the 200ml LB liquid medium containing finite concentration ampicillin respectively, 37 DEG C of cultivations
3～4h, work as OD600When reaching 0.6～0.8, add the derivant IPTG of final concentration of 0.2～0.5mmol/L, the temperature of 28 DEG C
Lower induction culturing about the 4h of degree, and centrifugal 5min collects thalline under the rotating speed of 12000rpm, utilization adds thin in precipitation
Cellular lysate buffer, and clarify at ultrasonication 2min on ice to bacterium solution, then centrifuge tube is put into rotating speed is 12000rpm, temperature
Degree is in the centrifuge of 4 DEG C, centrifugal 10min, takes supernatant and moves to another clean centrifuge tube；
Recombinant bacterial strain collects wet thallus after derivant IPTG overnight incubation, and broken collection contains the thick of fructosyl sucrose transferring enzyme
Enzyme liquid, is then loaded into crude enzyme liquid in the purification column containing mesh His label, by PB, 500mmol/L containing 20mmol/L
NaCl and 20mmol/L imidazole buffer I and containing 20mmol/L PB, 500mmol/L NaCl and
The imidazole buffer II of 500mmol/L, the acidity-basicity ph of buffer I and buffer II is 7.4, carries out gradient elution, chromatography
Collection liquid to eluting after end, carries out SDS-PAGE checking；
(3) enzymatic clarification prepares Levan type levan
By the fructosyl sucrose transferring enzyme of purification in step (2) with 25～40% the sucrose of concentration react, adding pH is the lemon of 6.0
Lemon acid buffer, the temperature keeping reaction is 40～50 DEG C, and the response time is 48h, utilizes sevag method to remove removing protein, adds three
Times volume, in the ethanol of fructosyl sucrose transferring enzyme reactant liquor, makes polysaccharide precipitation under 4 DEG C of cryogenic conditions and collects precipitation, then profit
With concentration be 60～80% ethanol solution wash twice after, will precipitation again with tertiary effluent dissolve collect, lyophilised machine freezing obtain powder
The Levan type levan of powder.
The preparation method of a kind of high uniformity macromolecule Levan type levan the most according to claim 1, it is characterised in that:
The concentration of the ampicillin in described step (2) is 30～60 μ g/mL.
The preparation method of a kind of high uniformity macromolecule Levan type levan the most according to claim 1, it is characterised in that:
In described step (2), the ultrasound wave demand power of ultrasonication on ice is 120W, and ultrasonication is ultrasonic broken every the 2s time
Broken 1s.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113444704A (en) * | 2021-07-27 | 2021-09-28 | 光明乳业股份有限公司 | Three levan sucrases and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113444704A (en) * | 2021-07-27 | 2021-09-28 | 光明乳业股份有限公司 | Three levan sucrases and application thereof |
| CN113444704B (en) * | 2021-07-27 | 2023-03-03 | 光明乳业股份有限公司 | Three levan sucrases and application thereof |
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