A branched-type glucan sucrose and its application to preparation of a medicinal gel dressing loaded with rhubarb

Abstract

The application discloses a branched dextran sucrose enzyme and application of the branched dextran sucrose enzyme in preparation of a rhubarb drug gel dressing. The branched dextran sucrose enzyme is obtained by mutating a known dextran sucrose enzyme gene sequence, and a gene engineering bacterium is constructed. The branched dextran sucrose enzyme has a high degree of branching, and the proportion of bond types is changed, so that the gel product has excellent mechanical properties. The dextran has good performance in promoting skin microcirculation, so the product has a good application prospect in the field of drug-loaded gel dressings. The enzyme can synthesize dextran oligosaccharides by using maltose, and the enzyme also has a good application in the field of skin prebiotics. The application lays a foundation for rational design and practical application and enzyme engineering production of dextran, and provides a broad market for development and application of a new drug dosage form.

Description

TECHNICAL FIELD

[0001] The present application relates to a preparation method of a branched dextran sucrose and its application in preparing a rhubarb drug gel dressing, belonging to the field of enzyme engineering. BACKGROUND

[0002] Dextran refers to a homopolysaccharide composed of glucose as monosaccharide, and the glucose units are connected by glycosidic bonds. Common dextran in life includes starch, glycogen, cellulose, etc. According to the type of glycosidic bond in dextran, dextran can be divided into two types: alpha-dextran and beta-dextran. When glucose exists in the form of pyranose six-membered ring, the hydroxyl groups on C1, C2, C3, C4 and C6 can form glycosidic bonds. Common glycosidic bonds in dextran include alpha-1, 4 glycosidic bond, alpha-1, 6 glycosidic bond and beta-1, 4 glycosidic bond, etc. In addition, dextran can also form other types of glycosidic bonds, such as alpha-1, 3 glycosidic bond, beta-1, 2 glycosidic bond, alpha-1, 2 glycosidic bond, etc. Alpha-dextran is a high molecular glucose polymer generated after sucrose is fermented by Leuconostoc mesenteroides, mainly connected by alpha-1, 6 glycosidic bonds, accompanied by a small amount of alpha-1, 3 and alpha-1, 4 glycosidic bonds, and can also be prepared by dextran sucrose produced by engineering bacteria. Due to its safety, non-toxicity, biocompatibility, structural specificity and other advantages, it has been widely used in food, medicine, chromatographic analysis, materials and other fields. The physical and chemical properties of dextran with different structures are quite different, and it has specific uses according to its functions. For example, 95% alpha-1, 3 glycosidic bond dextran 70 fermented by Leuconostoc mesenteroides is currently recognized as an excellent plasma substitute, and dextran fermented by baker's yeast can be used as a food additive to improve the taste of food.

[0003] Gel dressing is a colloidal substance with three-dimensional network structure and insoluble in water formed by special processing of water-soluble polymer materials. In hydrogel matrix, polysaccharides, a kind of biological macromolecules, play an important role, and the good biocompatibility and degradability of polysaccharides make their application more extensive. Natural polysaccharides extracted from food and plants have great potential for wound healing. Functional composite hydrogel not only can fully play the slow release effect of hydrogel in the application process, but also can load various cells, growth factors and cytokines, effectively improving the quality of wound healing. Dextran hydrogel has significant water absorption capacity, providing a biocompatible and hydrated environment conducive to cell growth and tissue regeneration, and can continuously absorb exudates from the wound without causing adhesion during replacement. Due to the high elasticity of hydrogel dressing, it can tightly adhere to uneven wounds, reducing bacterial growth, and providing a scaffold for fibroblast growth to accelerate the formation of new blood vessels. Dextran hydrogel has become a promising candidate for drug delivery applications due to its high water absorption, non-toxicity and biocompatibility.

[0004] Rhubarb is bitter and cold in nature, and has the effects of clearing heat and resolving toxins, drying dampness and astringency. It has good therapeutic effect on ulcers and abscesses caused by furuncles, burns, external injuries, etc., chronic ulcers caused by pressure sores, ischemic peripheral vascular diseases, and suppurative wounds such as surgical incision infections.

[0005] Dextran has the characteristics of high safety, good biocompatibility, antioxidant and gelation, and low molecular weight dextran is the only skin prebiotic that can promote the stability of skin microecology. Based on the gelation, antioxidant and prebiotic properties of dextran, it is applied to the secondary dosage form development of drugs, and a dextran / rhubarb drug composite hydrogel is designed and constructed to solve the problems of poor stability and short duration of action of existing active ingredients of drugs, and further maintain the balance of wound microecology, promote wound healing and shorten healing time, which has good market prospect. SUMMARY

[0006] The purpose of the present application is to provide a branched dextran sucrase, the amino acid sequence of which is shown in SEQ ID NO. 1.

[0007] The second purpose of the present application is to provide a coding gene of branched dextran sucrase, the nucleotide sequence of which is shown in SEQ ID NO. 2.

[0008] The third purpose of the present application is to provide a recombinant expression vector containing the coding gene of branched dextran sucrase.

[0009] The fourth purpose of the present application is to provide a genetically engineered strain containing the coding gene of branched dextran sucrase.

[0010] The fifth purpose of the present application is to provide a construction method of genetically engineered bacteria of branched dextran sucrase, which comprises the processes of primer design, recombinant plasmid construction and host bacteria transformation.

[0011] The primer design is to design mutant primers according to the sequence of dextran sucrase gene and the sequence of vector pET28a(+) by means of SnapGene software as follows:

[0012] The upstream mutant primer is as follows:

[0013] 5'-GTTGATGCTGTTGACAACGTGGATGCTGATTTGTTGCAAATTG-3'

[0014] The downstream mutant primer is as follows:

[0015] 5'-CAATTTGCAACAAATCAGCATCCACGTTGTCAACAGCATCAAC-3'

[0016] The recombinant plasmid construction is to use reverse PCR amplification technology to obtain the long gene cloning fragment of the mutant branched glucan sucrose enzyme and the vector plasmid by taking the glucan succharase expression plasmid as a template; the gene fragments are connected by ligase to obtain the recombinant expression plasmid pET28a(+)-DsrI-ND.

[0017] The host bacteria transformation is to transform the recombinant expression plasmid pET28a(+)-DsrI-ND into the E. coli competent cell BL21(DE3), and after kanamycin resistance screening, enzyme cutting, bacterial liquid PCR and DNA sequencing verification, the branched glucan sucrose enzyme engineering strain BL21(DE3) / DsrI-ND is obtained.

[0018] The sixth purpose of the application is to provide a branched glucan sucrose enzyme gene engineering bacteria expression application,

[0019] The genetic engineering bacteria BL21(DE3) / DsrI-ND is inoculated into the LB culture medium containing 40-60 ug / ml kanamycin at an inoculation amount of 0.5%, the rotation speed is 180 r / min, and the culture is carried out at 35-40℃ for 16-18h; 2mL of the culture liquid is taken from the above culture liquid and added into 200mL of culture medium, and the culture is carried out at 37℃ in a shaking bed; when the OD 600 When the 0.60-0.80 is reached, 500ul of IPTG is added to start inducing enzyme production, the fermentation is induced at 18℃ for 20-26h, the bacterial suspension after induction is centrifuged at 8000r / min for 10min at 0℃, one centrifuge tube corresponds to one bottle of bacterial suspension, then distilled water is added for washing, and centrifugation is carried out again; 20mL of PBS buffer with a pH value of 5.5 is added into each centrifuge tube, and the mixture is shaken and shaken uniformly, an ice water bath is added, ultrasonic crushing is carried out for 20min, and centrifugal separation is carried out; the supernatant is the crude enzyme liquid, and the enzyme activity is 20-30U / mL.

[0020] The seventh purpose of the application is to provide a catalytic process of the branched glucan sucrose enzyme,

[0021] The reaction conditions of the catalytic process are as follows: the pH value is 5.0-6.5, the reaction temperature is 30-35℃, the enzyme addition amount is 1-10U, the sucrose addition amount is 200g / L, and the reaction time is 8-12h.

[0022] The eighth purpose of the application is to provide an application of the branched glucan sucrose enzyme, 1-10ml of rhubarb medicine leaching liquid is added into the catalytic system of the branched glucan sucrose enzyme, a rhubarb medicine gel dressing is synthesized by one-step enzyme method, a new dosage form of medicine is prepared, and the application of improving the performance of the medicine is provided.

[0023] Further, in the catalytic process reaction system, maltose is added, and the branched glucan sucrose enzyme uses maltose as a receptor substrate to catalyze sucrose to prepare glucan oligosaccharide.

[0024] Further, in the preparation process of the rhubarb medicinal gel dressing, glucan oligosaccharide from the receptor reaction of the branched glucan sucrose enzyme is added to improve the effect of the rhubarb medicinal gel dressing on promoting skin microcirculation and further improve the performance application of the new dosage form of the medicine.

[0025] The branched glucan sucrose enzyme has excellent catalytic performance, overcomes the dependence of the existing branched glucan sucrose enzyme on the synthesis of linear glucan, can use sucrose as the only substrate to prepare branched glucan, greatly reduces the production process, and further reduces the production cost. The branched glucan has a high branching degree, which further improves the crosslinking degree of the product, is applied to the production of medicinal gel dressings, and is free from the dependence on crosslinking agents, thereby reducing the allergic reactions in the use of medicines, greatly improving the safety of the medicines as dressings, and the crosslinking structure has a strong drug release control ability, which greatly improves the drug efficacy of the loaded drugs. The branched glucan sucrose enzyme uses maltose as a receptor substrate to prepare glucan oligosaccharide, which reduces the complexity of glucan hydrolysis and the discharge amount of high-salt wastewater in the process; the product has uniform molecular weight, narrow distribution, and few impurities, greatly reduces the allergic reactions and other discomfort reactions as a skin prebiotic, further improves the safety of the medicine, and can be used as an excellent adjuvant to act on skin wounds. Therefore, the branched glucan sucrose enzyme can be applied to the medical field, has excellent safety, drug release control ability, and the advantages of improving the drug efficacy; in the industrial production process, biomass resources are fully utilized, the process is green and pollution-free, and the production cost is greatly reduced; the related products have excellent biodegradability, further reducing the pollution to the environment, and greatly improving the economic and ecological value of the branched glucan sucrose enzyme in the related application fields. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 It is a schematic diagram of the catalytic product of branched glucan sucrose enzyme Dsr-I-ND.

[0027] Figure 2 It is a nuclear magnetic detection diagram of the catalytic product of branched glucan sucrose enzyme Dsr-I-ND.

[0028] Figure 3 It is a GPC detection diagram of the catalytic product of branched glucan sucrose enzyme Dsr-I-ND.

[0029] Figure 4 It is a schematic diagram of the branched glucan loaded rhubarb medicinal gel dressing synthesized by branched glucan sucrose enzyme Dsr-I-ND. Detailed implementation method

[0030] The technical solutions in the embodiments of the present application will be described in detail below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application. Based on the embodiments of the present application, all other embodiments obtained by a person of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0031] Construction of recombinant expression plasmid pET28a(+)-DsrI-ND of Example 1

[0032] According to the sequence of the glucansucrase gene and the sequence of the vector pET28a(+), the mutant primers are designed by means of SnapGene software as follows:

[0033] Upstream mutant primer:

[0034] 5'-GTTGATGCTGTTGACAACGTGGATGCTGATTTGTTGCAAATTG-3'

[0035] Downstream mutant primer:

[0036] 5'-CAATTTGCAACAAATCAGCATCCACGTTGTCAACAGCATCAAC-3'; the recombinant plasmid construction is to take the glucansucrase expression plasmid as a template, use reverse PCR amplification technology to obtain the long gene cloning fragment of the mutant branched glucansucrase and the vector plasmid, and connect the above gene fragments by ligase to obtain the recombinant expression plasmid pET28a(+)-DsrI-ND.

[0037] Construction of genetically engineered bacterium BL21(DE3) / DsrI-ND of branched glucansucrase of Example 2

[0038] Preparation of E. coli BL21(DE3) competent cells: 50 μl of E. coli BL21(DE3) bacterial solution was taken into a 5 mL LB test tube, cultured at 37℃, 180 rpm, and a shaking bed for 12 h, 1 mL of bacterial solution in the test tube was taken and added into a shaking flask containing 100 mL of LB culture medium, and cultured at 37℃, 180 rpm, and a shaking bed, when the OD 600 of the bacterial solution was 0.5-0.6, the shaking flask was taken out, placed on ice for 10 min, centrifuged at 4100 rpm, 4℃ for 10 min, the supernatant was discarded, resuspended with 2 mL of 0.05M CaCl2 solution containing 15% glycerol, stored in a refrigerator at-80℃ after dispensing, and used as needed.

[0039] The recombinant expression plasmid pET28a(+)-DsrI-ND was transformed into E. coli BL21(DE3) by one-step cloning, and the specific method was as follows: 100 μL of prepared E. coli BL21(DE3) competent cells were placed in an ice box to thaw for 5-10 min; 10 μL of cooled one-step cloning reaction system was added to the competent cells, which were flicked with fingers, mixed, and placed on ice for 30 min; then, the reaction system was heated at 42°C for 90 s and quickly placed in an ice bath for 3 min. 900 μL of LB medium was added to the above reaction system, which was cultured at 37°C and 180 rpm for 1 h for cell recovery. 100 μL was inoculated on a resistant plate and cultured in a 37°C incubator for 12 h, and single colonies were picked and subjected to colony PCR verification using the primers in step 2. The colonies that could amplify the target gene fragment were the strains in which the recombinant expression plasmid pET28a(+)-DsrI-ND was successfully expressed.

[0040] Example 3 Fermentation of branched glucosucrase by branched glucosucrase genetically engineered bacteria BL21(DE3) / DsrI-ND

[0041] Seed medium: 10 g / L of proteose peptone, 5 g / L of yeast powder, 10 g / L of NaCl, sterilized at 121°C for 15 min.

[0042] Fermentation medium: 12 g / L of proteose peptone, 24 g / L of yeast powder, 2.31 g / L of KH2PO4, 16.43 g / L of K2HPO4, 10 g / L of glucose, sterilized at 121°C for 15 min.

[0043] Preparation method: 1 mL of the bacterial solution was inoculated into a 500 mL flask containing 100 mL of medium, and the flask was cultured at 37°C and 180 rpm for 0-4 h. Then, 0.1-0.5 mM of IPTG was added to induce the expression of BL21(DE3) / DsrI-ND protein, and the flask was further cultured at 20-24°C for 20-26 h. After the fermentation was stopped, the fermentation broth was centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and the resuspended cells were washed with PBS buffer solution with a pH of 5.5. The cells were broken by high-pressure homogenization to obtain a crude enzyme solution.

[0044] Example 4 Preparation of catalytic product of branched glucosucrase Dsr-I-ND

[0045] A certain concentration of sucrose is added to the branched glucan sucrose enzyme Dsr-I-ND crude enzyme solution, so that the final concentration of sucrose in the reaction system is 100 g / L; the reaction time is 4-6 h, preferably 4 h, the pH of the reaction system is 5.0, 5.5, 6.0, 6.5, and 7.0; the reaction temperature is 30-40°C, preferably 30°C; the enzyme addition amount is 1-10 U, preferably 5 U; cold ethanol is added to the enzyme reaction solution, the volume of cold ethanol is controlled to be 2-3 times the volume of the reaction solution, and the mixture is placed on ice for 30-60 min, then centrifuged at 8000 rpm for 10 min to obtain a precipitate, the supernatant is discarded, and the branched glucan is dried by a vacuum freeze dryer. The nuclear magnetic detection result of the product is shown in Figure 2 , the proportion of α-1,3 bond type is about 15%, and the proportion of α-1,6 bond type is about 80%.

[0046] Example 5: Preparation of glucan oligosaccharide by branched glucan sucrose enzyme Dsr-I-ND acceptor reaction

[0047] A certain concentration of sucrose is added to the branched glucan sucrose enzyme Dsr-I-ND crude enzyme solution, so that the final concentration of sucrose in the reaction system is 100 g / L, and an equal proportion of maltose is added; the reaction time is 4-6 h, preferably 4 h, the pH of the reaction system is 5.0, 5.5, 6.0, 6.5, and 7.0; the reaction temperature is 30-40°C, preferably 30°C; the enzyme addition amount is 1-10 U, preferably 5 U; the detection result of the preparation of glucan oligosaccharide by branched glucan sucrose enzyme Dsr-I-ND is shown in Figure 3 , and the molecular weight is about 1000 Da.

[0048] The catalyzed reaction solution is filtered, alcohol precipitated and washed, and vacuum dried to obtain the glucan oligosaccharide.

[0049] Example 6: Preparation of rhubarb medicinal gel dressing by branched glucan sucrose enzyme Dsr-I-ND catalysis

[0050] To the branched glucan sucrose enzyme Dsr-I-ND catalytic system, 2-10 mL / L of rhubarb medicinal extract is added, and 2 g / L of glucan oligosaccharide is added; the reaction time is 4-6 h, and the catalytic product is vacuum dried at 30°C for 20 min to obtain the branched glucan rhubarb medicinal gel dressing product, as shown in Figure 4 .

Claims

1. A branching-type glucansucrase characterized in that, The amino acid sequence of the branched glucan sucrose enzyme is shown as SEQ ID NO. 1, and the nucleotide sequence of the coding gene of the branched glucan sucrose enzyme is shown as SEQ ID NO.

2.

2. A recombinant expression vector comprising the coding gene of the branched glucan sucrose enzyme according to claim 1.

3. A genetically engineered strain comprising the coding gene of the branched glucan sucrose enzyme according to claim 1.

4. The genetically engineered strain comprising a branching glucosaccharase-encoding gene according to claim 3, characterized in that, The construction method of the genetically engineered bacteria comprises the following unit processes: primer design, recombinant plasmid construction, and host bacteria transformation: The primer design is to design mutant primers according to the sequence of the glucan sucrose enzyme gene and the sequence of the vector pET28a(+) by means of SnapGene software as follows: Upstream mutant primer: 5'-GTTGATGCTGTTGACAACGTGGATGCTGATTTGTTGCAAATTG-3' Downstream mutant primer: 5'-CAATTTGCAACAAATCAGCATCCACGTTGTCAACAGCATCAAC-3'; The recombinant plasmid construction is to use the dextran sucrose enzyme expression plasmid as a template, uses reverse PCR amplification technology to obtain the long gene cloning fragment of the mutated branched dextran sucrose enzyme and the carrier plasmid; the gene piece end is connected by ligase to obtain the recombinant expression plasmid pET28a(+) DsrI -ND; The transformation of the host bacterium involves transferring the recombinant expression cytokine pET28a(+)- DsrI -ND was transformed into E. coli competent cells BL21(DE3), and after kanamycin resistance selection, enzyme digestion, bacterial PCR, and DNA sequencing verification, a branched dextran sucrase engineered strain BL21(DE3) / was obtained. DsrI -ND.

5. Use of a branched glucansucrase according to claim 1, characterized in that: Under the reaction conditions of the catalytic process system of the branched glucan sucrose enzyme, 1-10 ml of a rhubarb drug extract is added, and a rhubarb drug gel dressing is synthesized by one-step enzyme method to prepare a new dosage form of the drug.

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