Dextran sucrase truncated mutant, preparation and application in dextran synthesis

Abstract

The invention discloses a dextran sucrase truncated mutant and application in preparation and synthesis of dextran, the mutant retains 45-1370 site amino acid residues of dextran sucrase DarM, through truncated mutation, the heterologous expression of the mutant in an escherichia coli host is remarkably improved compared with that before truncated, and the enzymatic property is stable. The dextransucrase truncated mutant can be used for generating dextran with the average molecular weight of about 40kDa through a single-enzyme one-step method under specific reaction conditions, and commercial strains of dextran can be industrially prepared based on the dextransucrase truncated mutant.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme engineering and bioengineering, and specifically relates to a mutant and an application thereof, in particular to a dextran sucrase truncated mutant and a preparation method thereof, and an application of the mutant to catalyze sucrose to generate medium molecular weight dextran with an average molecular weight of about 40 kDa under specific conditions in a one-step process. Background Art

[0002] Dextran is the most widely used type of a-glucan with extremely high commercial value. Dextran with different molecular weights has different application values ​​in the food industry, pharmaceutical and medical fields. Among them, dextran T40 and dextran T70 have been included in the list of essential drugs for rural and pastoral cooperation. Clinical-grade dextran of 40, 60 and 70 kDa is dissolved in 6% or 10% aqueous solution and can be used as a plasma substitute by restoring plasma lost due to severe bleeding. Dextran T40 can enhance microcirculation, improve blood flow by reducing viscosity, inhibit red blood cell and platelet aggregation, prevent thrombosis, and is beneficial to the prevention and treatment of cardiovascular diseases. The traditional preparation method of dextran is mainly microbial fermentation, but this method is usually complicated, costly and produces ethanol waste liquid that has a certain impact on the environment. The single enzyme one-step method for synthesizing dextran with a specific molecular weight is green and fast, and can convert low-cost sucrose into high-value-added dextran and oligosaccharides. Dextransucrase (Dextransucrase, EC2.4.5.1) is a member of the glycoside hydrolase 70 family. It hydrolyzes sucrose into D-glucose and fructose, and then extends the D-glucose to the non-reducing end of the sugar chain through transglycosylation to synthesize dextran of different molecular weights. Usually, the dextran synthesized by sucrose catalyzed by dextransucrase has a large molecular weight and high viscosity, poor fluidity, or the product is unevenly distributed and difficult to separate. In addition, dextransucrase is easily inactivated and difficult to separate and purify, so it is of great significance to prepare an engineered strain of dextransucrase with good stability and high product specificity. Summary of the invention

[0003] Technical problems to be solved: Currently, there are few industrial strains of dextran sucrase, and they are easily inactivated. The molecular weight of the product dextran is large or unevenly distributed, and separation and purification are difficult. In view of the deficiencies described in the prior art, the present invention provides a truncated mutant of dextran sucrase and its application, including a vector of the gene and a recombinant truncated mutant; the truncated mutant of dextran sucrase can be used to convert sucrose into medium molecular weight dextran under specific reaction conditions, and the product is evenly distributed, expanding the scope and field of application.

[0004] In order to achieve the above-mentioned invention object, the present invention provides the following technical solutions:

[0005] The present invention provides a truncated mutant of dextransucrase, wherein the truncated mutant deletes amino acid residues 1-44 and 1371-1935 at the C-terminus and N-terminus of dextransucrase DarM respectively, and retains amino acid residues 45-1370. The amino acid sequence of the mutant is shown in SEQ ID NO.3;

[0006] The present invention also provides a gene encoding a truncated mutant of dextransucrase, the nucleotide sequence of the gene is shown in SEQ ID NO.2;

[0007] The present invention also provides a genetically engineered strain comprising a gene encoding a dextransucrase mutant;

[0008] The plasmid vector of the above-mentioned engineering bacteria is pET-28a(+);

[0009] The host bacteria of the above-mentioned engineering bacteria is Escherichia coli.

[0010] The mutant is used in preparing medium molecular weight dextran by a single enzyme one-step method.

[0011] The application of the above-mentioned engineered bacteria in the preparation of medium molecular weight dextran.

[0012] The specific application steps are: the reaction is carried out in 50mM acetic acid-calcium acetate buffer with a pH of 5.4, the reaction temperature is 45°C, 200mM sucrose is used as the substrate, the enzyme addition amount is 2-4mg pure enzyme per milliliter of reaction solution, and the reaction time is 12h.

[0013] The beneficial technical effect of the present invention is that compared with the dextran sucrase before truncation, the dextran sucrase mutant has a higher heterologous expression amount in Escherichia coli, stable enzymatic properties, and uniform product distribution. In particular, compared with other reported dextran sucrases, the dextran sucrase mutant of the present invention can generate dextran with an average molecular weight of about 40 kDa in a single enzyme one-step method, and the molecular weight distribution of the generated dextran is relatively concentrated, and the solubility is good. Based on the present invention, a commercial strain for industrial preparation of dextran can be developed. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the embodiments of the present application document or the technical solutions in the prior art, the drawings required for use in the description of the embodiments or the prior art are briefly introduced below. Obviously, the drawings described below are only references to some embodiments in the present application document. For those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0015] Figure 1Schematic diagram of the truncation mutation strategy of dextransucrase DarM and its truncation mutant DarM-ΔV through sequence alignment and structure prediction; where: gray: signal peptide; red: domain V; yellow: domain IV; green: domain B; purple: domain C.

[0016] Figure 2 Schematic diagram of the prediction and visualization of the three-dimensional structure of a truncated mutant according to one embodiment of the present invention, wherein the color marking of the structural domains is Figure 1 correspond.

[0017] Figure 3 The SDS-PAGE image of soluble expression of dextransucrase DarM and its truncated mutant DarM-ΔV protein.

[0018] Figure 4 is an analysis of the enzymatic properties of the dextran sucrase mutant DarM-ΔV (A is a schematic diagram of the optimal reaction temperature. B is a schematic diagram of the optimal reaction pH. C is a schematic diagram of temperature stability. D is a schematic diagram of pH stability.)

[0019] Figure 5 Schematic diagram of the HPLC analysis of sucrose production products catalyzed by the dextransucrase mutant DarM-ΔV under specific conditions. DETAILED DESCRIPTION

[0020] The present invention is further described in detail below in conjunction with embodiment, it should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall equally within the scope limited by the appended claims of the application.

[0021] Example 1 Design of truncation mutation of dextransucrase gene, gene cloning and construction of recombinant genetic engineering strain

[0022] 1) The darm sequence of dextran sucrase (nucleotide sequence as SEQ NO.1) was derived from Leuconostoc citreum CBA3623, and was homologously compared with other identified α-glucan sucrases and analyzed for genetic evolution. It was also compared with dextran sucrase DSR-M and alternan sucrase ASR, which have similar homology and are more studied. The DarM domain was divided according to the secondary structural characteristics of the two dextranases ( Figure 1 ), predicted the structure of DarM based on the three-dimensional structures of two glucansucrases and performed homology modeling ( Figure 2), rationally designed truncation based on structural features. The amino acids 45-1370 of DarM protein were retained, and the amino acids 1-44 and 1371-1935 were deleted to obtain the dextran sucrase truncation mutant DarM-ΔV.

[0023] 2) Using the dextran sucrase darm gene (nucleotide sequence such as SEQ NO.1) as a template, primers were designed to amplify the truncated sequence darm-ΔV (nucleotide sequence such as SEQ NO.2) by PCR. The primer sequences are as follows:

[0024] Primer 1 sequence: gtgccgcgcggcagccatatgATGACAACACAGAGCCAACAGGA;

[0025] Primer 2 sequence: gtggtggtggtggtgctcgagTTACACACCTAAAGTTGACGAAGTATCA;

[0026] 3) After the PCR product was detected by 1% agarose gel electrophoresis, the target fragment was recovered and connected to the pET-28a(+) E. coli expression vector using homologous recombination reagents. The ligation product was transformed into RIPL(DE3) E. coli expression competent cells. The specific method is as follows: RIPL(DE3) competent cells were thawed on ice, the ligation product was added to the competent cells, and the mixture was mixed gently and placed on ice for 30 minutes. After the ice bath, it was heat-shocked at 42°C for 90 seconds, and then ice-bathed for 3-5 minutes. Subsequently, 900 μL of anti-LB medium without antibiotics was added, and it was incubated at 37°C and 200 rpm for 1 hour; finally, after centrifugation at 6000 rpm for 2 minutes at room temperature, the supernatant was discarded, 100 μL of resuspended bacteria was left and coated on a resistance plate with 50 μg / mL kanamycin, and inverted in a 37°C constant temperature incubator for overnight incubation. The next day, the clones on the plate were picked for colony PCR identification, and the positive clones were sent to the sequencing company for verification, and the positive strains were preserved.

[0027] Example 2 Preparation of the truncated mutant DarM-ΔV of dextransucrase

[0028] 1) The positive clones and the full-length engineered bacteria before truncation were inoculated into LB medium containing 50 μg / mL kanamycin, and cultured and activated in a shaker at 37°C and 220 rpm. 600 When the concentration reached 0.6-0.8, 0.25 mM IPTG was added and the mixture was placed in a shaker at 16°C and 220 rpm to induce expression for 20 h.

[0029] 2) After induction, the cells were collected by centrifugation and an appropriate amount of acetic acid-calcium acetate solution with a pH of 5.4 was added for ultrasonic cell disruption. After cell disruption, the cells were centrifuged at 4°C and 12,000 rpm for 20 min, and the supernatant was collected for SDS-PAGE detection and enzyme activity determination. The SDS-PAGE results are shown in Figure 3 As shown: before the truncation mutation, the heterologous expression level of dextran sucrase DarM in the E. coli expression system was extremely low, and it was difficult to observe obvious specific bands with the naked eye. After the truncation mutation, the expression yield of DarM-ΔV increased significantly, and the apparent molecular weight was about 146 kDa, which was consistent with the theoretical molecular weight.

[0030] 3) In order to purify DarM-ΔV, the strain was inoculated into 500 mL of LB medium containing 50 μg / mL kanamycin for large-scale induction expression. The culture and induction conditions were the same as those in step 1 of Example 2). An appropriate amount of cell lysis buffer was added to the collected bacteria, and the supernatant obtained after ultrasonic cell lysis and centrifugation was purified by nickel column affinity chromatography. The purity of the purified product was identified by SDS-PAGE detection, and the protein concentration was determined by NanoDrop by measuring the absorbance value at 280 nm.

[0031] Example 3 Characterization of the enzymatic properties of the dextransucrase truncation mutant DarM-ΔV

[0032] The enzyme activity was determined by 3,5-dinitrosalicylic acid method (DNS) to determine the yield of reducing sugar (fructose) in the reaction product. Definition of enzyme activity unit: 1 enzyme activity unit (U) is the amount of enzyme required to produce 1 μmol of fructose per minute under the assay conditions.

[0033] 1) Determination of the optimal reaction temperature: 10 μL of enzyme solution was added to 990 μL of 200 mM sucrose substrate (dissolved in acetic acid-calcium acetate buffer at pH = 5.4), and the reaction was carried out at 20°C to 60°C (increments of 5°C) for 10 minutes. After the reaction was completed, 500 μL of DNS was added to terminate the reaction, and the reaction was placed in a boiling water bath for 5 minutes to fully develop the color. After cooling with cold water, 200 μL was taken on an ELISA plate and the absorbance at OD540nm was measured. The group with the highest enzyme activity was set as 100%. The results are as follows: Figure 4A As shown, DarM-ΔV showed the highest enzyme activity at 45°C.

[0034] 2) Determination of the optimal reaction pH: 10 μL of enzyme solution was added to 990 μL of 200 mM sucrose substrate at different pH values ​​(sucrose dissolved in acetic acid-calcium acetate buffer at pH 4.0-7.0). The reaction conditions and enzyme activity determination were carried out as described above. The pH value with the highest activity was set as 100%. The results are as follows: Figure 4B As shown, the enzyme activity of DarM-ΔV was highest at pH = 4.5.

[0035] 3) Temperature stability determination: The enzyme solution was stored at 20°C-60°C (increments of 5°C) for 1 hour, reacted under the optimal temperature and pH conditions, and the temperature stability of the recombinant dextran sucrase truncated mutant DarM-ΔV was determined. The results are shown in Figure 4C. The enzyme activity can be maintained at more than 60% when stored at 20°C-45°C for one hour.

[0036] 4) pH stability determination: The enzyme solution was diluted with acetic acid-calcium acetate buffer at pH 4.0-7.0 and stored for 1 hour. Then, an appropriate amount of the enzyme solution was reacted under the optimal temperature and pH conditions to determine the pH stability of the recombinant dextran sucrase truncated mutant DarM-ΔV. The results are as follows: Figure 4D As shown, the enzyme activity can be maintained at more than 85% when stored in the pH range of 4-7 for one hour.

[0037] The above results confirm that the dextransucrase truncated mutant DarM-ΔV has good temperature stability and pH stability.

[0038] Example 4 Synthesis of medium molecular weight dextran using dextran sucrase engineered bacteria DarM-ΔV

[0039] 1) DarM-ΔV was purified and concentrated according to the experimental method described in Example 2, and the concentration was measured for later use. The enzymatic reaction was carried out under the optimal reaction conditions determined in Example 3. After adjusting the reaction time, substrate concentration and enzyme dosage, the optimized optimal dextran production conditions were as follows: the reaction was carried out in 50 mM acetic acid-calcium acetate buffer with a pH of 5.4, the reaction temperature was 45°C, 200 mM sucrose was used as the substrate, the enzyme addition amount was 2-4 mg pure enzyme per ml of reaction solution, and the reaction time was 12 h.

[0040] 2) Product analysis was performed using high performance liquid chromatography (HPLC) (TOSOH, TSK G3000, 7.8×300 mm) with parallax detection. The column temperature was set at 60°C, the mobile phase was dd H2O, the flow rate was 0.6 mL / min, and the injection volume was 10 μL. The standard products were commercial dextran with molecular weights of 5000 Da (T5), 20000 Da (T20), 50000 Da (T50), 100000 Da (T100), and 400000 Da (T400). The results are as follows: Figure 5 As shown, the black line is the characteristic peak of the T20 standard, and the red line is the characteristic peak of the T50 standard; the products are mainly fructose and some oligosaccharides with low polymerization degree, and only one characteristic peak of polysaccharide (calculated molecular weight is about 40kDa).

[0041] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0042] References to "one embodiment", "another embodiment", "embodiment", etc. in this specification refer to specific features, structures or characteristics described in conjunction with the embodiment as included in at least one embodiment generally described in this application. The appearance of the same expression in multiple places in the specification does not necessarily refer to the same embodiment. Further, when a specific feature, structure or characteristic is described in conjunction with any embodiment, it is claimed that the realization of such feature, structure or characteristic in conjunction with other embodiments also falls within the scope of the present invention.

[0043] Although the present invention is described herein with reference to a plurality of illustrative embodiments of the present invention, it should be understood that those skilled in the art may devise many other modifications and implementations that fall within the scope and spirit of the principles disclosed herein. More specifically, within the scope of the present disclosure and claims, a variety of variations and improvements may be made to the components and / or layout of the subject combination layout. In addition to variations and improvements made to the components and / or layout, other uses will also be apparent to those skilled in the art.

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Claims

1. A truncated mutant of dextransucrase, characterized in that: The amino acid residues 1-44 and 1371-1935 at the C-terminus and N-terminus of dextransucrase DarM are truncated, and the amino acid residues 45-1370 are retained. The amino acid sequence of the dextransucrase mutant is shown in SEQ ID NO.

3.

2. A gene encoding the truncated mutant of dextransucrase according to claim 1, characterized in that The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

3. A genetically engineered strain, characterized in that A gene encoding the dextransucrase mutant according to claim 2.

4. A method for preparing the dextransucrase mutant according to claim 1, characterized in that Follow these steps: 1) Design primers to amplify the truncated sequence darm-ΔV; Primer 1 sequence: gtgccgcgcggcagccatatgATGACAACACAGAGCCAACAGGA; Primer 2 sequence: ggtggtggtggtgctcgagTTACACACCTAAAGTTGACGAAGTATCA. 2) amplifying the truncated gene of claim 2 using primers 1 and 2, ligating the truncated gene to the pET-28a(+) prokaryotic expression vector, and transforming the ligated product into RIPL(DE3) Escherichia coli expression competent cells to obtain a genetically engineered strain containing the encoding gene; 3) Cultivate the genetically engineered strain in a shaker at 37°C and 220 rpm until OD 600 When the pH reaches 0.6-0.8, add IPTG with a final concentration of 0.25 mM, induce at 16°C for 20 h, and collect the cells by centrifugation; 4) adding acetic acid-calcium acetate buffer with a pH of 5.4 to the bacterial cells to resuspend the bacterial cells, performing ultrasonic disruption using an ultrasonic disruptor, and then collecting the supernatant by centrifugation to obtain a crude enzyme solution containing a dextransucrase mutant heterologously expressed in Escherichia coli.

5. An application of a dextransucrase mutant, characterized in that: The dextran sucrase truncation mutant is used to catalyze sucrose to synthesize medium molecular weight dextran.

6. The use of the dextransucrase mutant according to claim 5, characterized in that: Sucrose was catalyzed under specific conditions to prepare medium molecular weight dextran with an average molecular weight of about 40 kDa. The reaction conditions were as follows: the reaction was carried out in 50 mM acetic acid-calcium acetate buffer at a pH of 5.4, the reaction temperature was 45°C, 200 mM sucrose was used as the substrate, 2-4 mg of pure enzyme was added per ml of reaction solution, and the reaction time was 12 h.

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