CN113201513A - Heat-resistant dextran sucrase mutant and preparation method and application thereof - Google Patents

Abstract

The heat-resistant dextran sucrase mutant is a new mutant obtained by intercepting amino acid peptide segments in the 37-1430 interval of Leuconostoc citreum (Leuconostoc citreum) dextran sucrase DsrV and performing structure optimization, and has the optimal temperature of 63 ℃ and good thermal stability at the high temperature of 58-68 ℃. The mutant can catalyze sucrose to generate dextran with micro molecular weight, and the catalysis process has the characteristics of low viscosity of conversion solution, no mixed bacteria growth, low energy consumption and simple operation. The mutant is suitable for industrial application, and is more favorable for reducing the production cost of the dextran with the micro molecular weight.

Description

Heat-resistant dextran sucrase mutant and preparation method and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering and molecular enzyme engineering, and particularly relates to a heat-resistant dextran sucrase mutant and a preparation method thereof, and application of the mutant in catalyzing sucrose to generate micro-molecular-weight dextran and dextran derivatives thereof by a one-step method.

Background

Dextran (Dextran), also known as Dextran, dextrane, Dextran, polydextrose, is a polymer of high molecular polysaccharides formed by linking several glucosyl groups, accompanied by the formation of fructose, by the catalytic action of Dextran sucrase (DSR, ec2.4.5.1) to transfer the D-glucosyl groups in sucrose molecules to receptor molecules. Dextran backbone is connected by alpha-D-1, 6 glycosidic bonds, and side chain branch points are connected by alpha-D-1, 3 and alpha-D-1, 2, which are the earliest microbial polysaccharides discovered and the microbial polysaccharides earlier used as plasma substitutes. Because the dextran has loose and soft texture, the dextran has the advantages of safety, no toxicity, no odor, high viscosity, good biocompatibility, good water solubility, strong water retention and the like, and is widely applied to a plurality of fields of pharmacy, food, chemical industry, agriculture and animal husbandry, chromatographic analysis and the like. Wherein, the dextran with low molecular weight (molecular weight is 10000-; the micro molecular weight dextran sulfate has antiviral properties. The dextran with micro molecular weight can also improve microcirculation, prevent or eliminate erythrocyte aggregation and thrombosis in blood vessels, has the functions of expanding blood volume and osmotic diuresis, can be used for treating acute hemorrhagic shock, myocardial infarction, cerebral thrombosis, peripheral vascular diseases, preventing disseminated intravascular coagulation and the like, and has wide application in clinical medicine. The low and micro molecular weight dextran can further form a series of derivatives with extremely wide application. Dextran derivative is usually prepared by using dextran with low and micro molecular weight as main carrier and performing complexation reaction with other metal compounds to generate dextran metal complex, which is important microelement supplement for human body and livestock, or has multiple pharmacological activities, has wide application prospects, and mainly comprises iron dextran, zinc dextran, copper dextran, calcium dextran, and the like. The preparation processes of different types of dextran metal complexes are different in magnitude, and dextran with low and micro molecular weight is activated under alkalescent conditions, then is subjected to complex reaction with different metal compounds respectively, and finished products with different purposes are obtained through product separation, purification and refining. Therefore, the efficient preparation of low-molecular, micro-molecular dextran has been of great interest to researchers of interest.

The dextran sucrase is a key enzyme for preparing dextran, and can be converted into sucrose to synthesize dextran or oligosaccharide with different molecular weights from 5 × 10 due to different enzyme sources and differences of functional characteristics and catalytic degrees of the enzyme³～3×10⁸Da are different. At present, the preparation of low and micro molecular dextran in China is generally a main means of fermentation and two-enzyme method, and from 1957, the research institute of blood transfusion and hematology of Chinese academy of medical sciences carries out systematic research on domestic dextran, selects a new dextran-producing strain-No. 1226 Leuconostoc mesenteroides (LM-1226), and has been popularized to the whole country in the last 60 th century. These methods all convert sucrose into dextran with larger molecular weight by dextransucrase, and then decompose macromolecular dextrose by acid hydrolysis (decomposition by HCl) or enzyme hydrolysis (by dextranase)And (4) obtaining a dextran product with low and micro molecular weight finally. Because the acid hydrolysis method introduces chloride ions, the enzyme hydrolysis method introduces new protein impurities, and the acidolysis or enzyme hydrolysis of macromolecular dextran makes the molecular weight of the product difficult to control, the molecular weight difference is large, the temperature in the production process is low (25-30 ℃), the viscosity of the fermentation liquor is high, the material transfer is slow, the conversion efficiency is low, the process control is complex, and the separation and purification of the product are difficult (Zhang Jiuhua, etc., in the sugarcane industry, 2018(2), 52-58.). Therefore, the preparation of dextran with low and micro molecular weight by directly catalyzing sucrose by one-step method through dextran sucrase becomes a research hotspot. In 2017, the French Marion Claverie et al (M.Claverie et al.2017.) developed a dextransucrase mutant DSR-M2 delta, which is a peptide segment consisting of amino acids in the region of 164-1433 of dextransucrase of Leuconostoc citreum NRRL B-1299 bacteria, the mutant enzyme can convert sucrose to directly generate dextran with low molecular weight, the reaction temperature of the mutant enzyme is 30 ℃, the thermal stability of the enzyme is yet to be improved, but the research results prove that the dextran sucrase is improved by the molecular enzyme engineering technology, and the mutant is an effective means for preparing dextran with low and micro molecular weight. The patent of Nanjing industry university (application No. 202010730704.X) discloses a dextran sucrase mutant, a preparation method and an application thereof, wherein the dextran sucrase mutant can catalyze sucrose to generate dextran with a micro molecular weight, and the technical achievement and the defects thereof are about the same as those of Marion Claverie of the French nation. In order to improve the thermal stability of enzyme, a patent of the university of fertilizer combination industry (application number 201710615998.X) discloses a genetic engineering bacterium for expressing heat-resistant dextran sucrase, a construction method and application thereof, wherein the activity and the thermal stability of double-mutant enzyme of the genetic engineering bacterium are improved, the double-mutant enzyme has the optimal enzyme activity at 25 ℃, but the enzyme activity of the mutant enzyme is sharply reduced after the temperature of the mutant enzyme is 40 ℃. Wang super et al (C.Wang et al.2017.) performed molecular splicing on dextran sucrase of Leuconostoc mesenteroides 0326, intercepted peptide segments in different intervals of a peptide chain were expressed or truncated, the mutant enzyme thereof catalyzes sucrose to generate dextran products or oligosaccharide products with low molecular weight or micro molecular weight, but the optimum temperature of the mutant is between 25-30 ℃,the activity was almost completely lost when the temperature reached 40 ℃. Therefore, by means of modern genetic engineering and molecular enzyme engineering technology, deep improvement and optimization design of dextran sucrase molecules are possible to obtain new dextran sucrase with enzymological characteristics and strong thermal stability.

The invention aims to develop dextran sucrase which has strong heat stability and can be industrially applied, and the dextran sucrase can be used for directly catalyzing sucrose to prepare low molecular weight and micro molecular weight dextran, so that the large-scale single enzyme method is realized to directly prepare the low molecular weight and micro molecular weight dextran and derivatives thereof, and the dextran sucrase has great academic value and practical application significance.

Disclosure of Invention

The invention overcomes the defects of the existing technology for preparing dextran with low molecular weight and small molecular weight, and discloses a heat-resistant dextran sucrase mutant and a preparation method and application thereof, wherein the inventor optimally designs a novel dextran sucrase, namely a dextran sucrase mutant DsrV delta, by comparing and analyzing amino acid sequences of a plurality of dextran sucrases from different sources and by means of homologous modeling, structure optimization, molecular design, molecular shearing and the like, the mutant has strong heat-resistant stability, and has good thermal stability at the temperature of 58-68 ℃, so that the dextran can catalyze sucrose to directly prepare the dextran with low molecular weight and small molecular weight, and can further prepare various dextran derivatives.

The preparation method of the dextransucrase mutant DsrV delta takes Leuconostoc citreum (Leuconostoc citreum) dextransucrase protein DsrV (Pid: AHC21928.1) as a base and a starting point, and particularly mainly intercepts and reserves a peptide segment consisting of amino acids in the interval of 37-1430 of the DsrV protein through sequence comparison, homologous modeling, molecular design and molecular shearing, and finally obtains the dextransucrase mutant DsrV delta through structural optimization, wherein the amino acid sequence of the mutant DsrV delta is shown in SEQ ID NO. 1. In order to efficiently express and prepare the dextran sucrase mutant, the nucleotide sequence of the coding gene of the dextran sucrase mutant is subjected to codon optimization according to the amino acid sequence of the mutant DsrV delta and the requirements of protein expression and purification of the dextran sucrase mutant by referring to the codon preference principle of escherichia coli to obtain the corresponding nucleotide sequence of the mutant gene dsrV delta, wherein the nucleotide sequence is shown as SEQ ID NO. 2. According to the nucleotide sequence of the mutant gene dsrV delta, a DNA fragment of the mutant gene dsrV delta is obtained by a whole-gene synthesis method, the DNA fragment is connected with an expression vector pET-22b (+) and then transformed into an escherichia coli host E.coli BL21(DE3), and then high-efficiency expression and enzymology property research of mutant enzyme are carried out, and relevant experiments of catalyzing sucrose to generate dextran are carried out.

The novel dextran sucrase mutant DsrV delta is obtained by optimization design through the method, the optimum catalytic pH of the dextran sucrase mutant is 5.7, the optimum temperature of the dextran sucrase mutant is 63 ℃, the dextran sucrase mutant has good thermal stability at 58-68 ℃, the dextran sucrase mutant can catalyze sucrose to generate dextran with low molecular weight and micro molecular weight through one-step method, and the processes of acid hydrolysis and alcohol fractional precipitation are not needed. The mutant can catalyze sucrose to directly generate dextran with low molecular weight and micro molecular weight under the condition of higher temperature, has the characteristics of high solubility of a substrate and a product, low viscosity of a conversion solution, no mixed bacteria growth, simple and convenient product separation and simple operation, and is a simple, convenient, economic and efficient method which is simple in process and suitable for industrial preparation of the dextran with low molecular weight and micro molecular weight and derivatives thereof.

The invention comprehensively applies the theory and means of modern bioinformatics and molecular biology to carry out sequence comparison, homologous modeling, structure comparison analysis and molecular design on the Leuconostoc citreum (Leuconostoc citreum) dextran sucrase protein DsrV, and based on the sequence comparison, the invention cuts out redundant structures on enzyme molecules, mainly intercepts and retains peptide segments formed by amino acids in the 37-1430 interval of the DsrV protein, and finally obtains dextran sucrase mutant DsrV delta with smaller enzyme and strong thermal stability through structure optimization, and the mutant can directly catalyze sucrose to directly generate dextran with low and differential molecular weight and derivatives thereof. The novel dextran sucrase mutant has good thermal stability, and is more favorable for reducing the production cost of the dextran with micro molecular weight and derivatives thereof and expanding the application field of the dextran sucrase mutant.

The heat-resistant dextran sucrase mutant provided by the invention has good thermal stability, the mutant enzyme does not need cold chain conditions in the processes of production, preparation, packaging, storage and transportation, the enzyme can keep good catalytic activity under a high-temperature condition, the mutant enzyme can adapt to unstable production conditions in production, the enzyme activity is not easy to inactivate under the high-temperature condition, immobilized enzymes (certain immobilized carriers such as PVA are solidified at about 50 ℃) can be prepared, the long-time catalytic ability is kept, and the utilization efficiency of the enzyme is favorably improved. In addition, the heat-resistant dextransucrase reacts at a higher temperature, the increase of the solubility of sucrose as a substrate at a high temperature is beneficial to improving the utilization efficiency of the substrate, the product, namely the dextran, has increased solubility at a high temperature, the viscosity of the conversion solution can be reduced, the material transfer and exchange are beneficial to accelerating the reaction speed, meanwhile, the energy consumption required by cooling in the production process can be reduced, the pollution of mixed bacteria can be prevented to a certain extent, and the advantages are beneficial to reducing the production cost. The invention is a successful example combining theory and experimental practice, the research strategy and the implementation technical scheme thereof also greatly reduce the blindness and invisibility in scientific research practice, effectively save scientific research resources and production cost, and embody the advancement of technical achievements and implementation means.

Drawings

FIG. 1 SDS-PAGE pattern of protein expression of dextran sucrase mutant DsrV Δ.

FIG. 2 pH analysis of dextran sucrase mutant DsrV Δ.

FIG. 3 graph of dextran sucrase mutant DsrV.DELTA.thermostability assay.

FIG. 4 is a high performance liquid chromatogram of dextran produced by catalyzing sucrose with dextran sucrase mutant DsrV delta.

Detailed Description

The technical solution of the present invention is further described with reference to the following examples, which belong to the main contents of the present invention, but are not limited to these contents. It should be noted that modifications and extensions which are obvious to those skilled in the art, although not shown in the present patent specification, are also included in the scope of the patent claims of the present invention.

Experimental materials, reagents and experimental methods show that the strains and vectors described in the invention and examples, such as host cell escherichia coli BL21(DE3) and expression vector pET-22b (+) are commercially available; enzymes, other biochemical reagents, culture media and other experimental consumables are purchased from biochemical reagent companies, and 1.5% (w/v) agar and 100 mu g/mL ampicillin are added into a solid LB culture medium; the molecular biological experimental methods not specifically described in the examples are carried out by referring to the specific methods and procedures listed in the molecular cloning experimental Manual (third edition) or according to the kit and product instructions; the methods of high-efficiency expression of mutant enzyme, enzyme activity detection, transformation experiment, product identification and detection and the like are carried out according to related reported conventional methods and documents.

Example 1 Synthesis of dextran sucrase mutant dsrV delta gene and construction of recombinant bacterium thereof

The inventor carries out homologous alignment analysis on dextran sucrase DSR from Leuconostoc citreum (Leuconostoc citreum NRRL B-1299), Leuconostoc mesenteroides (Leuconostoc mesenteroides 0326) and other thermophilic bacteria, and the like, and mainly intercepts and retains a peptide segment consisting of amino acids in the 37-1430 interval of the DsrV protein through sequence alignment, homologous modeling, structure alignment and optimization, molecular design and molecular shearing, and finally obtains the dextran sucrase mutant DsrV delta through structure optimization design, wherein the amino acid sequence of the dextran sucrase mutant DsrV delta is shown in SEQ ID No. 1. The inventor optimizes the codon of the nucleotide sequence of the coding gene according to the amino acid sequence of the mutant DsrV delta and the requirement of efficient expression and purification of the protein of the mutant DsrV delta and the principle of codon preference of escherichia coli to obtain the nucleotide sequence of the mutant gene dsrV delta, wherein the nucleotide sequence is shown as SEQ ID NO. 2.

A DNA fragment of a mutant gene dsrV delta of the mutant gene dsrV delta is obtained by a qualified biotechnology company according to a whole-gene synthesis method, the DNA fragment is connected with an expression vector pET-22b (+) to obtain a recombinant expression vector pET-22b-dsrV delta, then a strain of an escherichia coli host cell BL21(DE3) is introduced to obtain recombinant escherichia coli BL21(DE3)/pET-22b-dsrV delta, the recombinant strain is used for high-efficiency expression of a dextransucrase mutant DsrV delta and preparation of enzyme, and the prepared dextransucrase mutant enzyme is used for relevant experiments of catalytically converting sucrose to generate dextran and derivatives thereof.

Example 2 Studies of DsrV Delta Induction expression and enzymological Properties of dextran sucrase mutant

A single colony was picked on a plate coated with recombinant E.coli BL21(DE3)/pET-22 b-dsrV.DELTA.and cultured overnight at 37 ℃ and 220rpm in 5mL of LB liquid medium containing 100. mu.g/mL ampicillin, Amp. The inoculated amount of 2% was inoculated from 5mL of LB liquid medium into a 500mL flask containing 200mL of LB liquid medium (containing 100. mu.g/mL Amp), and the cell concentration was adjusted to OD when cultured at 37 ℃ and 220rpm₆₀₀When the concentration is 0.5-0.7, IPTG is added to the final concentration of 1mmol/L, 5mL of sterile Tris-HCI solution (pH value is 6.4, 100mmol/L) is added at the same time, and the shaking culture is carried out in a shaking table at 30 ℃ and 220rpm for 4-8 h. Collecting 200mL culture solution after inducing culture for 5h, centrifuging, collecting thallus, adding cell-breaking buffer (20mM sodium acetate buffer, pH 5.4, 20mM CaCl) 5mL per gram thallus₂0.2g/L of sodium amide), adding Triton X-100 to a final concentration of 0.5%, ultrasonically disrupting the cells (working time 10sec, interval time 10sec, 60 cycles), centrifuging at 12000rpm at 4 ℃ for 15min, and collecting the supernatant to obtain the crude enzyme solution. Meanwhile, SDS-PAGE electrophoresis is carried out on the recombinant bacteria for inducing expression to detect the expression condition of the mutant, as shown in figure 1, no target band (Lane 2) appears in blank control escherichia coli BL21(DE3)/pET-22b, and the molecular weight of the mutant enzyme (Lane 1) is about 155kDa, which indicates that the mutant enzyme is successfully expressed and the molecular weight is consistent with the theoretical value.

The enzyme activity of the dextran sucrase is determined by a 3, 5-dinitrosalicylic acid method (namely a DNS method): the principle is that glucose group in cane sugar is polymerized into glucan under the action of dextran sucrase catalysis, fructose is released at the same time, and the fructose is reducing sugar, so that the activity of the dextran sucrase can be detected by detecting the content of the fructose.

The method for determining the activity of the dextran sucrase comprises the following steps: 200. mu.L of reaction buffer (200mmol/L sucrose; 20 mmol/L)Sodium acetate buffer, pH 5.6; 20mmol/L CaCl₂) Pre-cooling for 5min in ice bath, loading into 96-well reaction plate, adding 20 μ L diluted enzyme solution with appropriate times, reacting at optimum temperature for 20min (with enzyme inactivated by boiling water for 10min as blank control), adding 200 μ L DNS to terminate reaction, boiling in water bath for 5min for full color development, cooling in cold water, diluting, and detecting OD with microplate reader₅₂₀Value, calculate enzyme activity.

Definition of enzyme activity unit: under the conditions of optimal reaction temperature and optimal pH, the enzyme amount required for generating 1 mu mol of reducing sugar per min is one enzyme activity unit (1U).

Sucrose is taken as a substrate, reaction buffers (pH value between 5.0 and 6.2) with the same substrate concentration and different pH values are prepared, the influence of different pH values on the activity of the dextransucrase is determined according to the method, the pH value with the highest relative activity is the optimal pH value of the dextransucrase, and experiments show that the optimal pH value of the dextransucrase mutant enzyme is 5.7 (figure 2).

Under the condition of optimum pH, using cane sugar as substrate to prepare reaction buffer solution according to the above-mentioned method, using thin-wall 96-hole reaction plate to make subpackage 100 microliter of reaction solution, adopting gradient PCR instrument to control the temp. of enzymatic reaction between 50 deg.C and 70 deg.C, under the different temp. conditions, using DNS method to measure the production quantity of fructose and define optimum reaction temp. of enzyme. As shown in figure 3, the optimum temperature of the mutant enzyme is 63 ℃, and the dextran sucrase mutant enzyme can keep more than 80% of activity in the temperature range of 58-68 ℃ and show good thermal stability.

Example 3 experiments on the conversion of dextran sucrase mutant DsrV Delta to sucrose

According to the method of the embodiment 2, the dextran sucrase mutant crude enzyme solution with enough quantity is prepared, and the experiment of converting sucrose to generate dextran is carried out. The inventors added 2000mL of sodium acetate buffer (20mmol/L sodium acetate, pH 5.7, 25mmol/L CaCl) in a 5.0L glass reactor₂) Stirring at the rotating speed of 100rpm, starting to raise the temperature of the kettle body, simultaneously adding sucrose, wherein the total amount of the added sucrose is 600g, stirring the sucrose to be fully dissolved, and then circulating the sucrose in a water bathControlling the temperature of the kettle to be about 63 ℃, adding 1000U of crude enzyme solution of the dextran sucrase mutant, carrying out catalytic reaction for 15 hours at the temperature of 63 ℃ and at 100rpm, and sampling every 3 hours during the reaction period to detect the molecular weight of the dextran (the sample is diluted by 200-300 times, and the molecular weight of the dextran in the solution and the content of the product are measured by using High Performance Liquid Chromatography (HPLC)). And when the reaction kettle is converted to 15 hours, stopping the reaction, raising the temperature of the reaction kettle to 85 ℃, preserving the heat for 1 hour to inactivate the dextran sucrase, filtering impurities such as denatured enzyme protein and the like by using gauze, putting the conversion solution into a boiling water bath for 30 minutes to inactivate the residual dextran sucrase, and filtering in vacuum to obtain clear and transparent filtrate, namely the mixed solution containing the dextran with low or micro molecular weight and the fructose. Removing fructose from the mixed solution by an alcohol precipitation separation method to obtain a dextran solution only containing low molecular weight. And (3) reheating and concentrating the dextran solution to half volume, cooling to room temperature until the dextran is crystallized and separated out, and further evaporating to dryness by a vacuum rotary evaporation concentration method (the vacuum degree is controlled to be 30-50 hPa, and the temperature is controlled to be 45-50 ℃), thus obtaining a crude dextran product.

When the catalytic reaction is carried out for 3, 6, 9, 12 and 15 hours through HPLC detection, the average molecular weight of dextran in the reaction system reaches about 4436, 8512, 13020, 14771 and 15135(Da), and the molecular weight growth rate of the product is obviously slowed down after the reaction is carried out for 9 hours, so that the mutant can convert sucrose to prepare dextran products with different molecular weight grades with low and micro molecular weights by properly controlling the time of the conversion reaction.

According to the method of the example 3, the reaction is terminated after 6 hours of the conversion reaction, the product is extracted and purified to obtain dextran product with micro molecular weight, the detection and analysis result of HPLC method is shown in (b) of figure 4, the molecular weight of the dextran product is mainly concentrated at about 8500Da (the retention time is 16.125min), and the product contains very little oligosaccharide. FIG. 4 (a) is an HPLC chromatogram of dextran standard, the molecular weights are 15000Da and 8000Da respectively, the retention times are 13.616min and 16.234min respectively, and the solvent peaks appear at 4-6 min in FIG. 4 (a) and (b).

Example 4 experiments on the conversion of dextran sucrase mutant DsrV Delta to sucrose to dextran derivatives

The dextran derivative prepared by the invention is mainly prepared from commonly used iron, copper, zinc and other metal complexes of dextran. According to the method of the embodiment 3 of the invention, when the dextran solution only containing low molecular weight is obtained (fructose component is removed by alcohol precipitation of the mixed solution), the dextran is not required to be purified and dried, but is directly concentrated or diluted in a reaction kettle to obtain the solution with the dextran content of 40 percent, sodium citrate with the final concentration of 5 percent (W/V) is added as a complexing catalyst, and the mixture is stirred for 1 hour at 70-80 ℃ for activation; adjusting with 20% sodium hydroxide solution to make the solution weakly alkaline, and stirring at 70-80 deg.C for 2-3 hr; cooling the reaction system to 60 ℃, adjusting the pH to 5.8-6.0 by using a hydrochloric acid solution, dropwise adding a ferric trichloride solution with the final concentration of 0.4-0.5mol/L, dropwise adding a 20% sodium hydroxide solution to stabilize the pH to 5.8-6.5, heating to 70-80 ℃, stirring at 100rpm for 3-5 hours to perform a complex reaction, and cooling to room temperature to obtain a low-molecular-weight iron dextran crude product solution.

The method comprises the steps of preparing a solution with a molecular weight of about 5000Da (fructose components are removed from a mixed solution by an alcohol precipitation method), directly concentrating or diluting the solution in a reaction kettle to obtain a solution with a dextran content of 50%, stirring the solution at a speed of 100rpm, adding a 20% sodium hydroxide solution to make the solution weakly alkaline, controlling the temperature to be 80-90 ℃, performing activation by a reaction for 90min, dropwise adding a copper chloride solution to make the final depth of the solution be 0.5mol/L, controlling the pH value of the solution to be 5.0-6.0 by using a 20% sodium hydroxide solution in the dropwise adding process, controlling the temperature to be 60-70 ℃ for reaction for 120min, and cooling the solution to room temperature after the reaction is finished to obtain a low-molecular-weight copper dextran crude product solution.

The method comprises the steps of preparing a solution with a molecular weight of about 5000Da (fructose components are removed from a mixed solution by an alcohol precipitation method), directly concentrating or diluting the solution in a reaction kettle to obtain a solution with a dextran content of 30%, stirring the solution at a speed of 100rpm, adding a 20% sodium hydroxide solution to make the solution alkalescent, controlling the temperature to be 90-100 ℃, performing activation by a 120min reaction, dropwise adding a zinc chloride solution to make the final depth of the zinc chloride solution be 0.4mol/L, controlling the pH of the zinc chloride solution to be 5.5-6.5 by using a 20% sodium hydroxide solution in the dropwise adding process, controlling the temperature to be 70-80 ℃, performing reaction for 150min, cooling the solution to room temperature after the reaction is finished, and obtaining a low-molecular-weight zinc dextran crude product solution.

And (2) adding 3 times of 85% ethanol into the crude iron, copper and zinc dextrans solution prepared by the method, washing, performing suction filtration, dissolving the precipitate with a proper amount of water, performing suction filtration to remove impurities, performing alcohol washing and water washing, finally washing with 95% ethanol to obtain a product with better purity, and drying to obtain the metal complex (namely the derivative) of the iron, copper and zinc dextrans.

It will be appreciated that modifications or alterations may be made by those skilled in the art in light of the above teachings, for example, the heat-resistant dextran sucrase mutants or functionally equivalent mutants thereof, or mutants with highly similar amino acid sequences, in addition to Leuconostoc citreum, may be derived from Leuconostoc mesenteroides, Streptococcus oralis, Leuconostoc lactis, Leuconostoc garlicum, Leuconostoc suis, Bacillus, Actinomyces, Pseudomona acidovorans, Bacillus stearothermophilus, Thermus thermophilus, etc. The vector is suitable for expression in hosts such as escherichia coli, bacillus licheniformis, bacillus subtilis, pichia pastoris and the like, and the dextran sucrase mutant can be transferred into prokaryotic or eukaryotic hosts by methods such as an electrical transformation method, a protoplast transformation method, homologous integration and the like so as to realize the expression and preparation of the dextran sucrase mutant. Or by the above-described methods of enzymatically engineering certain segments of the mutants of the present invention with amino acid residues, and all such modifications and alterations are intended to fall within the scope of the appended claims.

SEQUENCE LISTING

<110> Guangxi institute of Biomanufacturing technology, institute of research, Ltd

<120> heat-resistant dextran sucrase mutant and preparation method and application thereof

<130> heat-resistant dextran sucrase mutant and preparation method and application thereof

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 1395

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<213> Artificial sequence

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

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Asn Asn Val Gln Ser Thr Ser Asp Asn Ala Thr Asp Thr Gln Gln Asn

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

35 40 45

Ser Asp Asp Val Thr Thr Thr Ala Ala Asn Asp Lys Thr Gln Ser Ala

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Asp Thr Asn Val Thr Glu Lys Gln Ala Asp Asp His Thr Leu Asn Asn

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

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Asn Glu Asn Ser Glu Ser Val Ala Val Ser Thr Asn Asn Gly Ser Ala

100 105 110

Glu Lys Thr Thr Glu Glu Val Gln Gln Val Ser Gly Lys Tyr Val Glu

115 120 125

Lys Asp Gly Ser Trp Tyr Tyr Tyr Phe Asp Asp Gly Lys Asn Ala Lys

130 135 140

Gly Leu Ser Thr Ile Asp Asn Asn Ile Gln Tyr Phe Asp Glu Asp Gly

145 150 155 160

Lys Gln Val Lys Gly Gln Tyr Val Thr Ile Asp Asn Gln Thr Tyr Tyr

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Phe Asp Lys Asp Ser Gly Asp Glu Leu Ile Gly Leu Gln Ser Ile Asp

180 185 190

Gly Lys Ile Val Ala Phe Asn Asp Glu Gly Gln Gln Ile Phe Asn Gln

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Tyr Tyr Gln Ser Glu Asn Gly Thr Thr Tyr Tyr Phe Asp Asp Lys Gly

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His Ala Ala Thr Gly Ile Lys Asn Ile Glu Gly Lys Asn Tyr Tyr Phe

225 230 235 240

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

245 250 255

Gln Ile Met Thr Phe Asp Gln Asn Thr Gly Gln Glu Val Ser Asn Thr

260 265 270

Thr Ser Glu Ile Lys Glu Gly Leu Thr Thr Gln Asn Thr Asp Tyr Ser

275 280 285

Glu His Asn Ala Ala His Gly Thr Asp Ala Glu Asp Phe Glu Asn Ile

290 295 300

Asp Gly Tyr Leu Thr Ala Ser Ser Trp Tyr Arg Pro Thr Asp Ile Leu

305 310 315 320

Arg Asn Gly Thr Asp Trp Glu Pro Ser Thr Asp Thr Asp Phe Arg Pro

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Ile Leu Ser Val Trp Trp Pro Asp Lys Lys Thr Gln Val Asn Tyr Leu

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Asn Tyr Met Ala Asp Leu Gly Phe Ile Ser Asn Ala Asp Ser Phe Glu

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

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Lys Asp Ala Met Ala Ala Phe Ile Val Thr Gln Pro Gln Trp Asn Glu

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Thr Ser Glu Asp Met Ser Asn Asp His Leu Gln Asn Gly Ala Leu Thr

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Tyr Val Asn Ser Pro Leu Thr Pro Asp Ala Asn Ser Asn Phe Arg Leu

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Leu Asn Arg Thr Pro Thr Asn Gln Thr Gly Glu Gln Ala Tyr Asp Leu

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

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Asn Ser Asn Pro Val Val Gln Ala Glu Gln Leu Asn Trp Leu Tyr Tyr

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Gln Ile Ala Ala Asp Tyr Phe Lys Leu Ala Tyr Gly Val Asp Gln Asn

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Asp Ala Thr Ala Asn Gln His Leu Ser Ile Leu Glu Asp Trp Ser His

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Asn Asp Pro Leu Tyr Val Thr Asp Gln Gly Ser Asp Gln Leu Thr Met

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Asp Asp Tyr Met His Thr Gln Leu Ile Trp Ser Leu Thr Lys Ser Ser

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Asp Ile Arg Gly Thr Met Gln Arg Phe Val Asp Tyr Tyr Met Val Asp

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Arg Ser Asn Asp Ser Thr Glu Asn Glu Ala Ile Pro Asn Tyr Ser Phe

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Val Arg Ala His Asp Ser Glu Val Gln Thr Val Ile Ala Gln Ile Val

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

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Gln Leu Glu Ala Ala Phe Lys Ile Tyr Asn Glu Asp Glu Lys Leu Ala

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Asp Lys Lys Tyr Thr Gln Tyr Asn Met Ala Ser Ala Tyr Ala Met Leu

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Leu Thr Asn Lys Asp Thr Val Pro Arg Val Tyr Tyr Gly Asp Leu Tyr

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Thr Asp Asp Gly Gln Tyr Met Ala Thr Lys Ser Pro Tyr Tyr Asp Ala

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Ile Asn Thr Leu Leu Gln Ala Arg Ile Gln Tyr Val Ala Gly Gly Gln

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Ser Met Ser Val Asp Asn Asn Asp Val Leu Thr Ser Val Arg Tyr Gly

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Lys Asp Ala Met Thr Val Thr Asp Ala Gly Thr Ser Glu Thr Arg Thr

755 760 765

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

770 775 780

Glu Gly Asp Thr Val Thr Leu His Met Gly Ala Ala His Lys Asn Gln

785 790 795 800

Ala Tyr Arg Pro Leu Leu Ala Thr Thr Ser Asp Gly Leu Ser Tyr Tyr

805 810 815

Asp Thr Asp Asp Asn Ala Pro Val Glu Tyr Thr Asp Asp Asn Gly Asp

820 825 830

Leu Ile Phe Thr Ser Asn Ser Ile Tyr Gly Val Gln Asn Pro Gln Val

835 840 845

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

850 855 860

Asp Ala Arg Thr Ala Ser Asp Thr Thr Thr Asn Thr Ser Asp Lys Val

865 870 875 880

Phe His Ser Asn Ala Ala Leu Asp Ser Gln Val Ile Tyr Glu Gly Phe

885 890 895

Ser Asn Phe Gln Ala Phe Ala Thr Asp Ser Ser Glu Tyr Thr Asn Val

900 905 910

Val Ile Ala Gln Asn Ala Asp Gln Phe Lys Gln Trp Gly Val Thr Ser

915 920 925

Phe Gln Leu Ala Pro Gln Tyr Arg Ser Ser Thr Asp Thr Ser Phe Leu

930 935 940

Asp Ser Ile Ile Gln Asn Gly Tyr Ala Phe Thr Asp Arg Tyr Asp Leu

945 950 955 960

Gly Tyr Gly Thr Pro Thr Lys Tyr Gly Thr Ala Asp Gln Leu Arg Asp

965 970 975

Ala Ile Lys Ala Leu His Ala Ser Gly Ile Gln Ala Ile Ala Asp Trp

980 985 990

Val Pro Asp Gln Ile Tyr Asn Leu Pro Glu Gln Lys Leu Ala Thr Val

995 1000 1005

Thr Arg Thr Asn Ser Phe Gly Asp Asp Asp Thr Asp Ser Asp Ile

1010 1015 1020

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

1025 1030 1035

Gln Glu Met Tyr Gly Gly Ala Phe Leu Glu Glu Leu Gln Ala Leu

1040 1045 1050

Tyr Pro Ser Leu Phe Glu Val Asn Gln Ile Ser Thr Gly Val Pro

1055 1060 1065

Ile Asp Gly Ser Val Lys Ile Thr Glu Trp Ala Ala Lys Tyr Phe

1070 1075 1080

Asn Gly Ser Asn Ile Gln Gly Lys Gly Ala Gly Tyr Val Leu Lys

1085 1090 1095

Asp Met Gly Ser Asn Lys Tyr Phe Lys Val Val Ser Asn Thr Glu

1100 1105 1110

Asp Gly Asp Tyr Leu Pro Lys Gln Leu Thr Asn Asp Leu Ser Glu

1115 1120 1125

Thr Gly Phe Thr His Asp Asp Lys Gly Ile Ile Tyr Tyr Thr Leu

1130 1135 1140

Ser Gly Tyr Arg Ala Gln Asn Ala Phe Ile Gln Asp Asp Asp Asp

1145 1150 1155

Asn Tyr Tyr Tyr Phe Asp Lys Thr Gly His Leu Val Thr Gly Leu

1160 1165 1170

Gln Asn Ile Asn Asn His Thr Tyr Phe Phe Leu Pro Asn Gly Ile

1175 1180 1185

Glu Leu Val Asn Ser Phe Leu Gln Asn Glu Asp Gly Thr Thr Val

1190 1195 1200

Tyr Phe Asp Lys Lys Gly His Gln Val Phe Asp Gln Tyr Ile Thr

1205 1210 1215

Asp Gln Asn Gly Asn Ala Tyr Tyr Phe Asp Asp Ala Gly Val Met

1220 1225 1230

Leu Lys Ser Gly Phe Thr Met Ile Asp Gly His Gln Gln Tyr Phe

1235 1240 1245

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

1250 1255 1260

Asp Gly Tyr Lys Tyr Tyr Phe Glu Pro Gly Ser Gly Asn Leu Ala

1265 1270 1275

Ile Leu Arg Tyr Val Gln Asn Ser Lys Asn Gln Trp Phe Tyr Phe

1280 1285 1290

Asp Gly Ser Gly His Ala Val Thr Gly Phe Gln Thr Ile Asn Gly

1295 1300 1305

Lys Lys Gln Tyr Phe Tyr Asn Asp Gly His Gln Ser Lys Gly Glu

1310 1315 1320

Phe Ile Asp Ala Asp Gly Asp Thr Phe Tyr Thr Ser Ala Thr Asp

1325 1330 1335

Gly Arg Leu Val Thr Gly Val Gln Lys Ile Asn Gly Ile Thr Tyr

1340 1345 1350

Ala Phe Asp Asn Thr Gly Asn Leu Ile Thr Asn Gln Tyr Tyr Gln

1355 1360 1365

Leu Ala Asn Gly Lys Tyr Met Leu Leu Asp Asp Asn Gly Arg Ala

1370 1375 1380

Lys Thr Gly Phe Val Leu Gln Asp Gly Ile Ile Thr

1385 1390 1395

<210> 2

<211> 4188

<212> DNA

<213> Artificial sequence

<400> 2

atgaccccgt ctgttctggg tgactctagt gttccagacg taagcgctaa caacgttcag 60

tctacctctg acaacgctac cgacacccag cagaacacca ccaccgttac cgaagaaaac 120

gacaaagttc agccggctgc tacctctgac gacgttacca ccaccgctgc taacgacaaa 180

acccagtctg ctgacaccaa cgttaccgaa aaacaggctg acgaccacac cctgaacaac 240

gacaaagttg acaacaaaca gaacgaagtt gctccgacca acgacaccaa cgaaaactct 300

gaatctgttg ctgtttctac caacaacggt tctgctgaaa aaaccaccga agaagttcag 360

caggtttctg gtaaatacgt tgaaaaagac ggttcttggt actactactt cgacgacggt 420

aaaaacgcta aaggtctgtc taccatcgac aacaacatcc agtacttcga cgaagacggt 480

aaacaggtta aaggtcagta cgttaccatc gacaaccaga cctactactt cgacaaagac 540

tctggtgacg aactgatcgg tctgcagtct atcgacggta agatagttgc gttcaacgac 600

gaaggccagc agatcttcaa ccagtactac cagtctgaaa acggtaccac ctactacttc 660

gacgacaaag gtcacgctgc taccggtatc aaaaacatcg aaggtaaaaa ctactacttc 720

gacaacctgg gtcagctgaa aaaaggtttc tctggtgtta tcgacggtca gatcatgacc 780

ttcgaccaga acaccggtca ggaagtttct aacaccacct ctgaaatcaa agaaggtctg 840

accacccaga acaccgacta ctctgaacac aacgctgctc acggtaccga cgctgaagac 900

ttcgaaaaca tcgacggtta cctgaccgct tcttcttggt accgtccgac cgacatcctg 960

cgtaacggta ccgactggga accgtctacc gacaccgact tccgtccgat cctgtctgtt 1020

tggtggccgg acaaaaaaac ccaggttaac tacctgaact acatggctga cctcggcttc 1080

atctctaacg cggactcttt cgaaaccgaa gactctcagt ctctgctgaa cgaagctagt 1140

aactacgttc agaaatctat cgaaatgaaa atctctgctc agcagtctac cgaatggctg 1200

aaagacgcta tggctgcttt catcgttacc cagccgcagt ggaacgaaac ctctgaagac 1260

atgtctaacg accacctgca gaacggtgct ctgacctacg ttaactctcc gctgaccccg 1320

gacgctaact ctaacttccg tctgctgaac cgtaccccga ccaaccagac cggtgaacag 1380

gcttacgacc tggacaactc taaaggtggt ttcgaactgc tgctggctaa cgacgttgac 1440

aactctaacc cggttgttca ggctgaacag ctgaactggc tgtactacct gatgaacttc 1500

ggtaccatca ccgctaacga cgctgacgct aacttcgacg gtatccgtgt tgacgctgtt 1560

gacaacgttg acgctgacct gctgcagatc gctgctgact acttcaaact ggcttacggt 1620

gttgaccaga acgacgctac cgctaaccag cacctgtcta tcctggaaga ctggtctcac 1680

aacgacccgc tgtacgttac cgaccagggt tctgaccagc tgactatgga cgactacatg 1740

cacacccagc tgatctggtc tctgaccaaa tcttctgaca tccgtggtac catgcagcgt 1800

ttcgttgact actacatggt tgaccgttct aacgactcta ccgaaaacga agctatcccg 1860

aactactctt tcgttcgtgc tcacgactct gaagttcaga ccgttatcgc tcagatcgtt 1920

tctgacctgt acccggacgt tgaaaactct ctggctccga ccgctgaaca gttagaggct 1980

gcgttcaaaa tctacaatga agacgaaaaa ctggctgaca aaaaatacac ccagtacaac 2040

atggcttctg cttacgctat gctgctgacc aacaaagaca ccgttccacg tgtgtactac 2100

ggggacctgt acaccgacga cggtcagtac atggctacca aatctccgta ctacgacgct 2160

atcaacaccc tgctgcaggc tcgtatccag tacgttgctg gtggtcagtc tatgtctgtt 2220

gacaacaacg acgttctgac ctctgttcgt tacggtaaag acgctatgac cgttaccgac 2280

gctggtacct ctgaaacccg taccgaaggt atcggcgtta tcgtaagcaa caacgcttct 2340

ctgcagctgg acgaaggtga caccgttacc ctgcacatgg gtgctgctca caaaaaccag 2400

gcttaccgtc cgctgctggc taccacctct gacggtctgt cttactacga caccgacgac 2460

aacgctccgg ttgaatacac cgacgacaac ggtgacctga tcttcacctc taactctatc 2520

tacggtgttc agaacccgca ggtttcgggt tacctggctg tgtgggtgcc ggttggtgct 2580

cagcaggacc aggacgctcg taccgcttct gacaccacca ccaacacctc tgacaaagtt 2640

ttccactcta acgctgctct ggactctcag gttatctacg aaggtttctc taacttccag 2700

gctttcgcta ccgactcttc tgaatacacc aacgttgtta tcgctcagaa cgctgaccag 2760

ttcaaacagt ggggtgttac tagcttccag ctggctccac agtaccgttc ttctaccgac 2820

acctctttcc tggactctat catccagaac ggttacgctt tcaccgaccg ttacgacctg 2880

ggttacggta ccccgaccaa atacggtacc gctgaccagc tgcgtgacgc tatcaaagct 2940

ctgcacgctt ctggtatcca ggctatcgct gactgggttc cggaccagat ctacaacctg 3000

ccggaacagg aactggctac cgttacccgt accaactctt tcggtgacga cgacaccgac 3060

tctgacatcg acaacgctct gtacgttgtt cagtctcgtg gtggtggtca gtaccaggaa 3120

atgtacggtg gtgctttcct ggaagaactg caggctctgt acccgtctct gttcgaagtt 3180

aaccagatct ctaccggtgt tccgatcgac ggttctgtta aaatcaccga atgggctgct 3240

aaatacttca acggttctaa catccagggt aaaggtgctg gttacgttct gaaagacatg 3300

ggttctaaca aatacttcaa agttgtttct aacaccgaag acggtgacta cctgccgaaa 3360

cagctgacca acgacctgtc tgaaaccggt ttcacccacg acgacaaagg tatcatctac 3420

tacaccctgt ctggttacag ggctcagaac gcgttcatcc aggacgacga cgacaactac 3480

tactacttcg acaaaaccgg tcacctggtt accggtctgc agaacatcaa caaccacacc 3540

tacttcttcc tgccgaacgg tatcgaactg gttaactctt tcctgcagaa cgaagatggc 3600

accaccgtct acttcgacaa aaaaggtcac caggttttcg accagtacat caccgaccag 3660

aacggtaacg cttactactt cgacgacgct ggtgttatgc tgaaatctgg tttcaccatg 3720

atcgacggtc accagcagta cttcgaccag aacggtgttc aggttaaaga caaattcgtt 3780

gttggtaccg acggttacaa atactacttc gaaccgggtt ctggtaacct ggctatcctg 3840

cgttacgttc agaactctaa aaaccagtgg ttctacttcg acggttctgg tcacgctgtt 3900

actggcttcc agaccatcaa tggtaaaaaa cagtacttct acaacgacgg tcaccagtct 3960

aaaggtgaat ttatcgacgc tgacggtgac accttctaca cctctgctac cgacggtcgt 4020

ctggttaccg gtgttcagaa aatcaacggt atcacctacg ctttcgacaa caccggtaac 4080

ctgatcacca accagtacta ccagctggct aacggtaaat acatgctgct ggacgacaac 4140

ggtcgtgcta aaaccggttt cgttctgcag gacggtatca tcacctaa 4188

Claims (5)

1. A heat-resistant dextran sucrase mutant is characterized in that the preparation method is a protein obtained by intercepting an amino acid peptide segment in a 37-1430 interval of Leuconostoc citreum (Leuconostoc citreum) dextran sucrase DsrV and performing structure optimization, and the amino acid sequence of the protein is shown in SEQ ID NO. 1.

2. A gene encoding the mutant of claim 1, having the nucleotide sequence shown in SEQ ID No. 2.

3. An expression vector comprising the mutant gene of claim 2.

4. A host cell comprising a prokaryotic or eukaryotic cell transformed with the expression vector of claim 3.

5. The use of a mutant enzyme according to claim 1 for catalyzing the production of dextran from sucrose and from feedstocks containing sucrose components, and for the production of dextran derivatives.

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