Levosan sucrase capable of preparing levosan with different molecular weights as well as preparation method and application of levosan sucrase

Abstract

The invention provides levan sucrase capable of preparing levan with different molecular weights as well as a preparation method and application of the levan sucrase. A levan sucrase gene is amplified by using a primer containing a restriction enzyme cutting site, and the levan sucrase gene is cloned to a pET32a-Acma plasmid to obtain a recombinant plasmid pET32a-lsc-BS-Acma, and the recombinant plasmid pET32a-lsc-BS-Acma is converted into a recombinant plasmid pET32a-lsc-BS-Acma. The method comprises the following steps: converting a recombinant plasmid pET32a-lsc-BS-Acma into a host E.coli BL21 through heat shock, so as to obtain a BS0506-lsc recombinant strain; the method comprises the following steps: inoculating an activated BS0506-lsc recombinant strain into a culture medium containing ampicillin resistance, inducing, treating to obtain a crude enzyme solution, and purifying the crude enzyme solution by adopting a bacterial enhancer matrix method to obtain the levan sucrase capable of preparing levan with different molecular weights after purification. The levan sucrase is efficiently cloned and expressed, and is quickly and efficiently purified by using a BEM method, so that a method for quickly synthesizing uniform, high-purity and high-yield levan by using an enzymic method is provided, and controllable production of the levan with different molecular weights is realized.

Description

Technical Field

[0001] The invention belongs to the technical field of gene engineering and enzyme engineering, and in particular relates to a levansucrase capable of preparing levans with different molecular weights, and a preparation method and application thereof. Background Art

[0002] Levan is a non-reducing polymer composed of fructosyl units connected by β-(2,6) bonds, occasionally with β-(2,1) branches, and may contain a glucosyl unit at the end. Plant-based levan is mainly found in grasses, such as Jerusalem artichoke, Duckgrass, and Poa macrophylla, with a molecular weight of 10-33kDa; levan has a wide range of microbial sources, such as Bacillus, Acetobacter, Erwinia herbicola, Pseudomonas, Streptococcus, and yeast, and is produced by levansucrase.

[0003] The molecular weight of plant-derived levan is between 10 and 33 kDa, while the molecular weight of bacterial-derived levan has a wide range, and some microorganisms can even synthesize high-molecular-weight levan with a molecular weight of up to 104 kDa. Levan with different molecular weight distributions not only plays a role in preventing drying and nutrient storage of bacterial biofilms, but also has different functional properties. As soluble dietary fiber, oligosaccharides often have higher prebiotic effects and antioxidant capacity; high-molecular-weight levan has more possibilities in emulsification, water retention, intrinsic viscosity, and plasticity. Therefore, it is of great practical significance to prepare levan with different molecular weights and realize molecular weight controllable production.

[0004] At present, the synthesis of levan is divided into plant extraction, microbial fermentation and enzymatic synthesis. Among them, plant synthesis has low yield and different characteristics; the separation steps of levan from microbial sources are cumbersome, low purity and high cost, resulting in its limited yield; the enzymatic synthesis of levan is simple, easy to purify and has high yield, which is conducive to the large-scale industrial production of levan. Therefore, it is crucial to find a method that can secrete a large amount of levansucrase.

[0005] Levansucrase (EC 2.4.1.10) belongs to the glycoside hydrolase family GH68. The enzyme has substrate specificity. When sucrose is the only carbon source, it is first hydrolyzed into fructose and glucose, and the fructosyl group is transferred to a suitable acceptor, which can be water, another sucrose or a growing levan chain, that is, hydrolysis and transfructosylation are carried out through a double displacement reaction. The synthesis of levan can be divided into three identifiable stages: FOS synthesis, levan elongation and levan degradation.

[0006] Traditional methods for purifying soluble proteases include gel filtration, ion exchange, affinity chromatography, etc. Recombinantly expressed proteins can be selectively separated by adding affinity tags and then using affinity chromatography or specific chromatographic adsorption columns. There are many purification tags, such as 6×His, CAT, GST, etc. The most commonly used is the 6×His tag. The recombinant protein solution with the tag is concentrated, purified by nickel column, eluted, and de-tagged in turn to obtain a purified recombinant protein with biological activity. The protein obtained by this method has high purity and strong specificity, but it has high requirements for the chelating environment, is greatly affected by other affinity media, and the filler is expensive, time-consuming, and not suitable for high-throughput proteins.

[0007] At present, the patents invented in China are focused on the production of levan, focusing on direct sugar production by wild fungi (publication number: CN113684157A), extracting and purifying wild-type levansucrase in wild fungi to catalyze the production of various sugar compounds (publication number: CN104073456A), expressing and purifying mutants of levansucrase to catalyze sugar production (publication number: CN105907815A), cloning and expressing levansucrase in Bacillus pumilus or Pichia pastoris for heterologous expression, and then catalyzing sugar production (publication number: CN106434504A; Publication number: CN113293153A). Therefore, there is currently a lack of technical reports on the efficient secretory expression of levansucrase in Escherichia coli BL21. In addition, the direct production of levan by wild strains has uncontrollable product structure, low yield, and cumbersome purification process; the mutant strains have high technical requirements and high costs; the expression systems of Bacillus and Pichia pastoris have high technical requirements, relatively low yield, and complex enzyme purification. Therefore, constructing a recombinant strain that can efficiently secrete levansucrase, seeking a simplified recombinant enzyme purification method, and using it to produce levan of different molecular weights are of great significance for increasing the yield of levan and promoting the industrial application of levan in food, cosmetics, functional materials, etc. Invention content

[0008] In view of this, the present invention aims to provide a levansucrase capable of preparing levans of different molecular weights and a preparation method and application thereof, so as to solve at least one technical problem raised in the background technology.

[0009] To achieve the above object, the technical solution created by the present invention is implemented as follows:

[0010] A preparation method of levansucrase capable of preparing levans with different molecular weights comprises the following steps: amplifying a levansucrase gene using a primer containing a restriction site, cloning the levansucrase gene into a pET32a-Acma plasmid to obtain a recombinant plasmid pET32a-lsc-BS-Acma, and transforming the recombinant plasmid pET32a-lsc-BS-Acma into a host E.coliBL21 through heat shock to obtain a BS0506-lsc recombinant strain; inoculating the activated BS0506-lsc recombinant strain into a culture medium containing ampicillin resistance, inducing and treating the activated BS0506-lsc recombinant strain to obtain a crude enzyme solution, and purifying the crude enzyme solution using a bacterial enhancer matrix method to obtain the levansucrase capable of preparing levans with different molecular weights after purification;

[0011] The sequence of the levansucrase gene is: SEQ NO 1.

[0012] Furthermore, the upstream primer comprises a Sac I restriction site, and the downstream primer comprises an EcoR I restriction site.

[0013] Furthermore, the preparation of the pET32a-Acma plasmid includes inoculating Escherichia coli containing the pET-32a-Acma plasmid into a culture medium containing Amp antibiotics, centrifuging after culturing, discarding the supernatant, and extracting to obtain the pET32a-Acma plasmid.

[0014] The integration plasmid is the pET32a-Acma integration plasmid with Acma purification tag, which has ampicillin resistance, contains seven restriction sites including NcoⅠ, BamHⅠ, SacⅠ, KpnⅠ, PstⅠ, EcoRⅠ, HindⅢ, and a 6×glycine linker.

[0015] Furthermore, the preparation of the recombinant plasmid pET32a-lsc-BS-Acma includes: performing double enzyme digestion of SEQ NO 1 and plasmid pET32a-Acma with Sac I and EcoR I at the same time, connecting the enzyme digestion products, transforming into E. coli DH5α competent medium, coating with ampicillin-resistant culture medium, and obtaining the recombinant plasmid pET32-lsc-BS-Acma.

[0016] Furthermore, the induced expression includes inoculating the activated BS0506-lsc recombinant strain into a culture medium containing ampicillin resistance and shaking and culturing at 36.5-37.5°C and 190-210rpm for 6-8h, adding IPTG and inducing expression at 15.5-16.5°C, centrifuging the obtained fermentation broth to collect the bacteria, and taking the supernatant after ultrasonic disruption as the crude enzyme solution.

[0017] Furthermore, the fermentation conditions of the culture medium are: 10 g / L tryptone, 5 g / L yeast extract powder, 10 g / L sodium chloride, prepared with pure water.

[0018] Furthermore, the crude enzyme solution is purified by the bacterial enhancer matrix method, which comprises the following steps:

[0019] 1) Select a Bacillus strain or a lactic acid bacteria strain for expansion culture, collect the cells by centrifugation, resuspend them in HCl solution, place them in a boiling water bath, cool them to room temperature, and replace the HCl with Tris-HCl buffer to obtain a bacterial enhancer matrix BEM;

[0020] 2) Take an equal amount of BS0506-lsc crude enzyme solution and mix it with BEM matrix at room temperature to obtain levansucrase capable of preparing levan with different molecular weights.

[0021] The levansucrase prepared by the above-mentioned method for preparing levansucrase capable of preparing levan with different molecular weights.

[0022] The levansucrase prepared by the preparation method of the levansucrase capable of preparing levan with different molecular weights is used to prepare levan with different molecular weights. The prepared levansucrase is used to transform and produce levan with sucrose as substrate, the sucrose concentration is 100-300 g / L, the enzyme addition amount is 0.5-5 U / g substrate, the buffer solution is acetic acid-sodium acetate solution with pH 5.8, the reaction temperature is 25-50°C, the rotation speed is 160 rpm, alcohol precipitation is performed, and freeze-drying is performed.

[0023] The production conditions of high molecular weight levan are: enzyme addition amount 3-5U / g substrate, sucrose concentration 100-120g / L, reaction time 12-18h;

[0024] The production conditions of low molecular weight levan are: enzyme addition amount 0.5-2U / g substrate, sucrose concentration 280-300g / L, reaction time 36-48h.

[0025] A method for characterizing levan with different molecular weights, characterized in that it comprises the following steps:

[0026] 1) Performing gel permeation chromatography on the freeze-dried levan to determine the molecular weight;

[0027] 2) Performing Fourier transform infrared spectroscopy on the freeze-dried levan and comparing it with commercially available levan to identify the functional groups;

[0028] 3) Perform scanning electron microscopic analysis on lyophilized levan with different molecular weights to observe the microscopic morphology;

[0029] 4) Test the water holding capacity and emulsification properties of freeze-dried levan with different molecular weights to evaluate its application potential in food, medicine and cosmetics.

[0030] Compared with the prior art, the levansucrase capable of preparing levan with different molecular weights and its preparation method and application have the following advantages:

[0031] The present invention can efficiently secrete and express levansucrase (BS-lsc), collect the supernatant after ultrasonic crushing, use the bacterial enhancer matrix method (BEM) to perform one-step immobilization-purification, obtain high-purity BS-lsc enzyme, and evaluate the activity of the recombinantly expressed BS-lsc enzyme. The produced BS-lsc enzyme preparation is used to rapidly produce levan of different molecular weights in vitro, that is, sucrose is added as a substrate to an acetic acid-sodium acetate buffer solution of pH 5.6, and high-purity levan can be produced in a short time. The present invention is simple in method for producing levan, mild in conditions, easy to obtain, high in product purity, high in yield, short in production cycle, and low in cost, and solves the problem of levan yield, and has great application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The drawings constituting part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments and descriptions of the present invention are used to explain the present invention and do not constitute an improper limitation on the present invention. In the drawings:

[0033] Figure 1 It is the PCR amplification product of the BS0506 levansucrase gene identified by agarose gel electrophoresis as described in the embodiment of the present invention;

[0034] Figure 2 It is the agarose gel electrophoresis to identify the colony PCR product described in the embodiment of the present invention;

[0035] Figure 3 The recombinant levansucrase analyzed by SDS-PAGE in the embodiment of the present invention;

[0036] Figure 4 The synthesized lyophilized levan sample described in the embodiment of the present invention;

[0037] Figure 5 It is the permeabilization gel chromatogram of the molecular weight determination of different levans described in the examples of the present invention (a is a high molecular weight levan sample, b is a low molecular weight levan sample);

[0038] Figure 6 : are infrared spectra of levan with different molecular weights described in the examples of the present invention (a is a high molecular weight levan sample, b is a low molecular weight levan sample);

[0039] Figure 7 It is a scanning electron microscope image of levan with different molecular weights described in the examples of the present invention (a is a high molecular weight levan sample, b is a low molecular weight levan sample). DETAILED DESCRIPTION

[0040] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0041] The present invention will be described in detail below with reference to the accompanying drawings and in combination with embodiments.

[0042] Example 1

[0043] 1. Amplification of SEQ NO 1

[0044] The test strain Bacillus siamese BS0506 was screened from Northeastern soybean paste and cultured in LB liquid medium for 2 days. The bacteria were collected by centrifugation at 10,000 rpm, and the total bacterial DNA of BS0506 was extracted using Tiangen's bacterial genomic DNA extraction kit.

[0045] Primer F1: 5′-TGAGTGCCTCTGAAACC-3′ was designed based on other Bacillus Levansucrase genes available in the NCBI database;

[0046] R2:5'-ACAAACAGGAACGCACCC-3'.

[0047] SEQ NO 1 can be synthesized artificially;

[0048] The preparation of clone SEQ NO 1 includes:

[0049] The PCR system is: 10×Buffer 5μL, dNTP 1μL, primers F1 and R1 2μL each, Pfu DNA polymerase 1μL, total genomic DNA 1-3μL, ddH2O to make up 50μL system. The total amount of DNA is 10-100ng. PCR program: 95℃ pre-denaturation 5min; 95℃ denaturation 30s, 55℃ annealing 30s, 72℃ extension 4min, 30 cycles; 72℃ final extension 10min; 4℃ storage forever. The obtained PCR product was subjected to agarose gel electrophoresis. Figure 1 The figure is the electrophoresis result. The PCR product was recovered by gel excision using Tiangen DNA purification kit and sent to Genewise for sequencing.

[0050] 2. Construction of expression vector

[0051] Primers containing restriction sites were designed based on the sequencing results, wherein the upstream primer contained a restriction site for Sac I and the downstream primer contained a restriction site for EcoR I.

[0052] F2:5'-CGC GAGCTC AAAGAAAATACCCAAAAACC-3'

[0053] R2:5'-CGC GAATTC GTTGTTAACCGTAAGCTGTC-3'.

[0054] SEQ NO 1 was amplified using primers containing restriction sites, and the PCR product was recovered and purified.

[0055] Extract the plasmid pET32a-Acma, that is, inoculate the E. coli containing the pET-32a-Acma plasmid (the construction method of the course refers to "Research on the purification and immobilization method of recombinant protein based on BEM and AcmA expression system", "Tianjin University" 2020 paper, author Zhao Fangkun) into 5mL LB liquid culture medium containing Amp antibiotics (final concentration 100μg / mL), and culture in a shaking incubator at 37℃ 180rpm until the logarithmic growth phase. Centrifuge at 8000×g for 5min, discard the supernatant, extract the plasmid using a plasmid mini-extraction kit, and store at -20℃ for later use.

[0056] The PCR product and vector pET32a-Acma were double-digested, ligated with T4 DNA ligase at 25°C for 40 min, transformed into E.coil DH5α, spread on LB plates containing Amp antibiotics (final concentration 100 μg / mL), cultured at 37°C overnight, and some transformants were selected for PCR ( Figure 2 ), select universal primers T7 and T7 Term, extract recombinant plasmid from the successfully transformed transformants (Tian Gen Plasmid Extraction Kit), determine the DNA sequence, and the successful plasmid was named pET32a-lsc-BS-Acma.

[0057] The successfully transformed plasmid pET-32a-lsc-Acma was introduced into the expression vector E.coli BL21 (DE3), and the colony PCR verification was performed again. The successfully transformed transformants were selected to obtain the Levansucrase recombinant strain of BS0506. The recombinant expression strain was added to the LB liquid medium containing Amp antibiotics, cultured to the logarithmic growth phase, and the glycerol was preserved in a -80℃ refrigerator.

[0058] 3. Inducible Expression of Recombinant Levansucrase

[0059] The Levansucrase recombinant strain of BS0506 was inoculated into LB liquid culture medium containing Amp, and cultured at 37°C with shaking until OD600nm was 0.6, then IPTG inducer with a final concentration of 0.25mM was added, and cultured at 16-25°C with shaking for 18-24h, and then the bacteria were collected by centrifugation at 4°C for 15min, and the precipitate was repeatedly washed 2-3 times with appropriate 50mM Tris-HCl buffer (pH6.8), and finally Tris-HCl buffer was added at a ratio of 10 (50mM Tris-HCl buffer): 100 (initial LB liquid culture medium) to resuspend the washed bacteria.

[0060] Ultrasonic disruption to obtain crude enzyme solution: The resuspended bacterial solution was subjected to ultrasonic disruption for 24 minutes to obtain the recombinant enzyme Levansucrase expressed in the cell. The ultrasonic working power was 270Hz, the working temperature was 35℃, the ultrasonic was performed for 2s, and the pause was 4s. An ice water bath was added during the whole process. The obtained cell fragments were centrifuged at 8000×g for 10 minutes at 4℃. The supernatant was the crude enzyme solution of Levansucrase. The recombinant enzyme was named BS0506-lsc. The samples were analyzed by SDS-PAGE electrophoresis. The results are shown in Figure 3 .

[0061] 4. Purification of Recombinant Levansucrase

[0062] Preparation of the purified material bacterial enhancer matrix BEM: Select any Gram-positive bacillus or lactic acid bacteria strain (non-pathogenic) to prepare BEM. Here, Lactobacillus casei ATCC 393 is taken as an example. L. casei is cultured to an OD600nm of 1-1.2, and the precipitate is collected by centrifugation. After the bacteria are resuspended in 0.1M HCl, the mixture is boiled in water for 30 minutes. After cooling, the precipitate is repeatedly washed 3 times with 50mM Tris-HCl buffer (pH6.8), and finally, Tris-HCl buffer is added to resuspend the washed bacteria in a ratio of 20 (50mM Tris-HCl buffer): 100 (initial liquid culture medium volume), and BEM particles are prepared and stored at 4°C for use, and stored at -20°C for several years for use.

[0063] Use BEM to purify recombinant Levansucrase enzyme: add BEM resuspension and recombinant crude enzyme solution in a volume ratio of 1:1, mix well and let stand at room temperature for 30 minutes to allow BS0506-lsc with Acma purification tag to specifically bind to the peptidoglycan of bacterial cell wall. Centrifuge at 8000×g for 5 minutes, resuspend the precipitate with appropriate 50mM Tris-HCl buffer (pH6.8) to obtain the purified BS0506-lsc enzyme solution for the synthesis of levans. Store at 4℃ for future use, and store at -20℃ for several years for future use. Collect the supernatant and precipitate before and after purification, and BEM particles, and perform SDS-PAGE protein electrophoresis analysis ( Figure 3 ).

[0064] 5. Determination of the Enzyme Activity of Recombinant Levansucrase

[0065] The steps of enzyme activity determination are as follows: take an appropriate amount of purified levansucrase solution, react with 100g / L sucrose solution in pH 5.6 acetic acid-sodium acetate buffer system for a certain time. After the reaction is completed, add 3 times pre-cooled 95% ethanol solution to the reaction system, and let it stand overnight at 4°C to fully precipitate the polysaccharide. Then centrifuge at 10000×g for 30 minutes at 4°C, discard the supernatant, re-dissolve in pure water and freeze-dry in vacuum, weigh the levan product, and calculate its transfructose enzyme activity (U1). The content of monosaccharides in the reaction system is determined by DNS method, and its hydrolase activity (U2) is calculated.

[0066] The fermentation conditions of the above culture medium are: 10 g / L tryptone, 5 g / L yeast extract powder, 10 g / L sodium chloride, prepared with pure water.

[0067] 6. In vitro enzymatic synthesis of levan

[0068] Sucrose was used as substrate to transform and produce levan. The sucrose concentration was 100 g / L, the enzyme dosage was 0.5-5 U / g substrate, the buffer solution was acetic acid-sodium acetate solution with pH 5.6, the reaction temperature was 25-50°C, the rotation speed was 160 rpm, 3 times 95% pre-cooled ethanol was used for precipitation, vacuum freeze-drying ( Figure 4 ).

[0069] 7. Synthesis of levan with different molecular weights

[0070] The conversion and production of levan using sucrose as substrate, sucrose as substrate, acetic acid-sodium acetate solution with a buffer pH of 5.6;

[0071] The production conditions of high molecular weight levan are: enzyme addition amount 3-5U / g substrate, sucrose concentration 100g / L, reaction at 45℃ for 12-18h;

[0072] The production conditions of low molecular weight levan are: enzyme addition amount 0.5-2U / g substrate, sucrose concentration 300g / L, reaction at 45°C for 36-48h.

[0073] 8. Characterization of Levan

[0074] Determination of relative molecular weight (Mw): Evaluation was performed using a multi-detection gel permeation chromatography system. 5 mg / mL of levan was filtered through a 0.22 μm membrane and equipped with an e2695 liquid phase system, a differential detector and an Ultrahydrogel TM Linear column, 35°C, 0.5 mL / min flow rate, 0.1 M sodium nitrate as mobile phase, injection volume 40 μL ( Figure 5 ).

[0075] Functional group determination: Fourier transform infrared spectroscopy (FTIR) was used for evaluation. 2 mg of levan sample and 200 mg of potassium bromide powder were pressed into tablets, and the potassium bromide powder alone was used as a blank control ( Figure 6 ).

[0076] Microscopic morphology observation: Scanning electron microscope (SEM) was used to observe the microscopic morphology of levan samples. Hitachi S-4800 scanning electron microscope was used to observe the microscopic morphology of levan samples. A small amount of freeze-dried levan sample was placed on the sample stage, sprayed with gold, and observed on the microscope ( Figure 7 ).

[0077] Water holding capacity test (WHC): Take samples of levofloxacin with different molecular weights and dissolve them into 10 mg / mL solutions. After they are fully dissolved, centrifuge the solutions at high speed (10000×g, 20 minutes) and calculate the amount of precipitated water. The water holding capacity of levofloxacin with different molecular weights is calculated by the formula: WHC (%) = (total volume - volume of supernatant water / total volume) / total volume.

[0078] Emulsification and emulsion stability test: Determination of the emulsification activity and emulsion stability of levan samples with different molecular weights. That is, add 5mL of peony seed oil to 15mL of sugar solution with a concentration of 10.0mg / mL, vortex and oscillate for 5 minutes, take 100μL of the emulsified mixture and add it to 10mL of 0.1% SDS solution, homogenize for 5s, and measure the absorbance at 500nm. By formula:

[0079]

[0080]

[0081] Where A0 is the absorbance at 500 nm, N is the dilution factor of the emulsion in 0.1% SDS solution, and ρ is the mass concentration of polysaccharide in the sample solution (10,000 g / m 3 ), is the proportion of the oil phase in the emulsion (0.25), L is the optical diameter of the cuvette (1 cm), and t is the standing time (min).

[0082] Table 1 Water holding capacity, emulsification and emulsion stability of levan with different molecular weights

[0083] High molecular weight levan Low molecular weight levan <![CDATA[ WHC (%)]]> 84.9±0.78 89.16±1.21 <![CDATA[ E A I (g / m 2 )]]> 9.27±0.48 6.66±0.74 <![CDATA[ E SI (min)]]> 1246.08±213 887.73±36

[0084] At present, there is no related technical invention on water holding property, emulsification property and emulsion stability of levan. In the present invention, high molecular weight levan (Mw = 3.42 × 10 6 Da) were all multilayered flake structures under scanning electron microscopy, among which the low molecular weight levan (Mw = 6.93 × 10 3 Da) shows more finely divided serrated flakes with pores, while the large molecular weight presents a larger irregular flake structure. Both large and small molecular weight levan are completely soluble in water. The water holding capacity of large molecular weight levan is 84.9±0.78%, and the water holding capacity of small molecular weight levan is 89.16±1.21%. That is, when applied to food or cosmetics that need to hold water, choosing low molecular weight levan has better water retention and stability. As shown in Table 1, the emulsification of large molecular weight levan is 9.27±0.48g / m 2 The emulsification stability was 1246.08±213min, while the emulsification stability of low molecular weight levan was 6.66±0.74g / m 2 , and the emulsification stability is 888±36min. High molecular weight polysaccharides tend to have higher emulsification and emulsification stability, thus giving polysaccharides higher thickening and gelling abilities, and can be used as stable flocculants in beverages, emulsion thickening and emulsification in cosmetics, and for encapsulating and protecting hydrophobic active substances in oil droplets in medicine.

[0085] The present application has important application prospects. The purification method of the recombinant enzyme is simple and rapid. The enzymatic synthesis of levan has high yield, high purity, simple process and low cost. In addition, the environmental requirements are very low, and the experiment can be completed at room temperature, which greatly promotes its industrial production. Levans of different molecular weights have different application scenarios. The present invention provides a method for synthesizing levans of different molecular weights, which has universality in the enzymatic synthesis of other polysaccharides. The present invention provides a practical solution for industrial production, which is conducive to reducing production costs and is expected to produce significant economic benefits.

[0086] The present invention can efficiently secrete and express levansucrase (BS-lsc), collect the supernatant after ultrasonic crushing, use the bacterial enhancer matrix method (BEM) to perform one-step immobilization-purification, obtain high-purity BS-lsc enzyme, and evaluate the activity of the recombinantly expressed BS-lsc enzyme. The produced BS-lsc enzyme preparation is used to rapidly produce levan of different molecular weights in vitro, that is, sucrose is added as a substrate to an acetic acid-sodium acetate buffer solution of pH 5.6, and high-purity levan can be produced in a short time. The present invention is simple in method for producing levan, mild in conditions, easy to obtain, high in product purity, high in yield, short in production cycle, and low in cost, and solves the problem of levan yield, and has great application value.

[0087] The gene sequence of SEQ NO 1 is:

[0088]

[0089] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing levansucrase capable of preparing levan with different molecular weights, characterized in that: A levansucrase gene is amplified using primers containing restriction sites, and the levansucrase gene is cloned into a pET32a-Acma plasmid to obtain a recombinant plasmid pET32a-lsc-BS-Acma, and the recombinant plasmid pET32a-lsc-BS-Acma is transformed into a host E. coli BL21 by heat shock to obtain a BS0506-lsc recombinant strain; the activated BS0506-lsc recombinant strain is inoculated into a culture medium containing ampicillin resistance, and a crude enzyme solution is obtained after induction and treatment, and the crude enzyme solution is purified by a bacterial enhancer matrix method, and the purified levansucrase capable of preparing levans of different molecular weights is obtained; The sequence of the levansucrase gene is: SEQ NO 1.

2. The method for preparing levansucrase capable of preparing levan with different molecular weights according to claim 1, characterized in that: The upstream primer contains a Sac I restriction site, and the downstream primer contains an EcoRI restriction site.

3. The method for preparing levansucrase capable of preparing levan with different molecular weights according to claim 1, characterized in that: The preparation of pET32a-Acma plasmid includes inoculating Escherichia coli containing pET-32a-Acma plasmid into a culture medium containing Amp antibiotic, centrifuging after culturing, discarding supernatant, and obtaining pET32a-Acma plasmid after extraction.

4. The method for preparing levansucrase capable of preparing levan with different molecular weights according to claim 1, characterized in that: The preparation of the recombinant plasmid pET32a-lsc-BS-Acma includes: performing double enzyme digestion of SEQ NO 1 and plasmid pET32a-Acma with Sac I and EcoR I at the same time, connecting the enzyme digestion products, transforming into E. coli DH5α competent medium, coating with ampicillin-resistant culture medium, and obtaining the recombinant plasmid pET32-lsc-BS-Acma.

5. The method for preparing levansucrase capable of preparing levan with different molecular weights according to claim 1, characterized in that: The induced expression includes inoculating the activated BS0506-lsc recombinant strain into an ampicillin-resistant culture medium and shaking and culturing at 36.5-37.5°C and 190-210rpm for 6-8h, adding IPTG and inducing expression at 15.5-16.5°C, centrifuging the obtained fermentation liquid to collect the bacteria, and taking the supernatant after ultrasonic disruption as the crude enzyme solution.

6. A method for preparing levansucrase capable of preparing levan with different molecular weights according to any one of claims 1 to 5, characterized in that: The fermentation conditions of the culture medium are: 10 g / L tryptone, 5 g / L yeast extract powder, 10 g / L sodium chloride, prepared with pure water.

7. The method for preparing levansucrase capable of preparing levan with different molecular weights according to claim 1, characterized in that: Purification of crude enzyme solution using bacterial enhancer matrix method includes the following steps: 1) Select a Bacillus strain or a lactic acid bacteria strain for expansion culture, collect the cells by centrifugation, resuspend them in HCl solution, place them in a boiling water bath, cool them to room temperature, and replace the HCl with Tris-HCl buffer to obtain a bacterial enhancer matrix BEM; 2) Take an equal amount of BS0506-lsc crude enzyme solution and mix it with BEM matrix at room temperature to obtain levansucrase capable of preparing levan with different molecular weights.

8. Levansucrase prepared by the method for preparing levansucrase capable of preparing levan with different molecular weights according to any one of claims 1 to 7.

9. The method for preparing a levansucrase capable of preparing levans of different molecular weights according to any one of claims 1 to 7, wherein the levansucrase prepared is used for preparing levans of different molecular weights, characterized in that: The prepared levansucrase is used to convert sucrose as a substrate to produce levan, the sucrose concentration is 100-300 g / L, the enzyme addition amount is 0.5-5 U / g substrate, the buffer solution is acetic acid-sodium acetate solution with pH 5.8, the reaction temperature is 25-50°C, the rotation speed is 160 rpm, alcohol precipitation is performed, and freeze-drying is performed.

10. The use according to claim 9, characterized in that: The production conditions of high molecular weight levan are: enzyme addition amount 3-5U / g substrate, sucrose concentration 100-120g / L, reaction time 12-18h; The production conditions of low molecular weight levan are: enzyme addition amount 0.5-2U / g substrate, sucrose concentration 280-300g / L, reaction time 36-48h.

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