Recombinant dextranase as well as preparation method and application thereof

Abstract

The invention discloses recombinant dextranase as well as a preparation method and application thereof. The amino acid sequence of the recombinant dextranase is shown as SEQ ID NO.6. The recombinant dextranase has different degrees of degradation activity on different types of dextran and soluble starch. In addition, the enzyme can tolerate various metal ions, low-concentration Ca < 2 + >, Ba < 2 + >, Fe < 3 + >, Ni < 2 + > and Co < 2 + > have an activation effect on the enzyme, and more than 88% of activity can be kept in an environment with high-concentration Ba < 2 + >, Ca < 2 + >, Fe < 3 + >, Ni < 2 + > and Mn < 2 + >. The activity of the enzyme in sodium fluoride is improved by 14.53%. Most of the hydrolysates acting on the dextran T20 and the dextran T500 are various isomaltooligosaccharides, and the contents of isomaltotetraose, isomaltopentaose and isomaltohexaose are the highest; the enzyme hydrolysate has excellent oxidation resistance, so that the enzyme hydrolysate has potential application value in the fields of food, medical treatment and the like.

Description

Technical Field

[0001] The invention relates to a recombinant dextranase and a preparation method and application thereof, belonging to the field of dextranase. Background Art

[0002] Dextranase is a carbohydrate-active enzyme that degrades high-molecular-weight dextran into medium- and low-molecular-weight polysaccharides. The enzyme and its hydrolyzate are widely used in many industries such as medicine, food, and chemical industry, and have huge market demand. In the medical field, low-molecular-weight dextranase hydrolyzate has the effects of anticoagulation, antiviral, blood volume supplementation, and microcirculation improvement, and is often made into a variety of drugs and injections for clinical treatment and research; in addition, dextranase can hydrolyze dental plaque on the surface of teeth, so it is often added to tooth cleaning products for the prevention and treatment of caries. In the food field, dextranase is often added to the production process of sugarcane sugar to hydrolyze dextran in sucrose to reduce the viscosity of sucrose. In the chemical industry, dextranase can be used to prepare chromatographic fillers such as cross-linked dextran gel.

[0003] At present, the research on dextranase at home and abroad is mostly focused on the screening and fermentation of producing bacteria, the separation and purification of enzymes, and the immobilization of enzymes. Most dextranases are derived from bacteria and fungi, and their physical and chemical properties such as temperature tolerance, pH, and product type vary greatly, but the types of dextranases that can meet the production requirements of different industrial fields are still limited. As the application field of dextranase becomes more and more extensive, the exploration of dextranases from different strains and different properties has guiding significance for exploring and realizing the wide application of enzymatic products, and the exploration of new dextranases will also lay a new theoretical foundation for industrial enzyme production.

[0004] Most of the dextranases obtained by the prior art are obtained by screening, fermenting and isolating enzyme-producing bacteria in natural environments, and the content and purity of the target fermentation products are low. In addition, metal ions have different degrees of strong inhibitory effects on some dextranases. The vast majority of dextranases known to date are inhibited by Fe 3+ The inhibitory effect of dextranase limits the development and utilization of dextranase. Summary of the invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide a recombinant dextranase and a preparation method and application thereof.

[0006] Technical solution: To solve the above technical problems, the present invention provides a recombinant dextranase, whose amino acid sequence is shown in SEQ ID NO.6.

[0007] The present invention also provides a gene encoding the recombinant dextranase, and the nucleotide sequence thereof is shown in SEQ ID NO.4.

[0008] The present invention also provides a method for constructing the recombinant dextranase, comprising the following steps:

[0009] (1) After amplifying the stdex gene, double-digesting the plasmid, ligating, transforming into competent cells, and culturing to obtain recombinant genetically engineered bacteria;

[0010] (2) culturing the recombinant genetically engineered strain described in step (1), ultrasonically disrupting the bacteria, collecting the supernatant, and purifying to obtain the SMT3-StDex fusion protein;

[0011] (3) Cutting and purifying the SMT3-StDex fusion protein described in step (2) to obtain recombinant dextranase.

[0012] Wherein, the primer pair for amplification in step (1) is shown as SEQ ID NO.1 and SEQ ID NO.2.

[0013] The specific preparation steps of the SMT3-StDex fusion protein in step (2) are as follows: kanamycin sulfate is added to the culture medium containing the recombinant genetically engineered strain, and when the bacteria grow to OD 600 When the pH is about 0.8, IPTG is added, and the shaking flask culture is continued. The bacterial cells are collected by centrifugation, and the bacterial cells are resuspended in equilibrium buffer A. The bacterial cells are ultrasonically disrupted, the supernatant is collected, and the supernatant is preliminarily purified. After purification, the eluate is collected to obtain the SMT3-StDex fusion protein; the equilibrium buffer A includes Tris, NaCl, imidazole and urea.

[0014] The present invention also provides a dextran iron drug, which contains the recombinant dextranase. The StDex of the present invention is 1-5 mmol / L Fe 3+ The activity was improved under 10mmol / L Fe 3+ This property has laid an important theoretical foundation for the development of drug preparations such as iron dextran.

[0015] The invention also provides a fluoride-containing oral cleaning agent, which contains the recombinant dextranase. 0.1% sodium fluoride can promote the improvement of the activity of dextranase StDex, which has guiding significance for the application of dextranase in fluoride toothpaste.

[0016] The present invention also provides the use of the recombinant dextranase in decomposing dextran or soluble starch with different molecular masses; the dextran with different molecular masses includes dextran T10, dextran T20, dextran T40, dextran T70 and dextran T500.

[0017] The relative activity of the dextranase StDex obtained in the present invention in degrading soluble starch can reach 46.74%, which is higher than the degradation activity of other dextranases.

[0018] Wherein, the concentration of the dextran with different molecular weights is 1% to 7%.

[0019] The present invention also provides the application of the recombinant dextranase in the preparation of oligosaccharides. The hydrolyzate of the dextranase StDex obtained by the present invention is different from other dextranases. The hydrolyzate of StDex is a variety of isomaltooligosaccharides, among which isomaltotetraose, isomaltopentaose and isomaltohexaose account for 64%. The dextranase hydrolyzate obtained by other technologies is mainly isomaltotriose, isomaltohexaose and isomaltoheptaose. It has been determined that the hydrolyzate of StDex has strong antioxidant properties and has potential application value in the fields of food and medical treatment.

[0020] The present invention also provides the use of the recombinant dextranase in clearing or inhibiting free radicals.

[0021] Wherein, the application temperature of the recombinant dextranase is 20-80°C.

[0022] Furthermore, the application temperature of the recombinant dextranase is 25-75°C.

[0023] Furthermore, the application temperature of the recombinant dextranase is 30-60°C.

[0024] Furthermore, the application temperature of the recombinant dextranase is 35-55°C.

[0025] Furthermore, the application temperature of the recombinant dextranase is 40-50°C.

[0026] Optimally, the application temperature of the recombinant dextranase is 50°C.

[0027] Wherein, the application pH of the recombinant dextranase is 3.0-8.0.

[0028] Furthermore, the application pH of the recombinant dextranase is 5.0-7.5.

[0029] Furthermore, the application pH of the recombinant dextranase is 5.5-7.5.

[0030] Furthermore, the application pH of the recombinant dextranase is 6.0-7.5.

[0031] Optimally, the recombinant dextranase is applied at a pH of 7.0.

[0032] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0033] The optimal temperature of the dextranase StDex of the present invention is 50°C, and the enzyme activity can be maintained at more than 70% when treated at 20-50°C for 1 hour; the optimal pH is 7.0, and the enzyme activity can be maintained at more than 90% when treated at pH 5.0-7.0 for 1 hour. Under the conditions of pH 7.0 and temperature 50°C, when dextran T20 is used as substrate, the specific activity of the enzyme is 174.48U / mg, and the kinetic parameter K m 、v max and k cat / K m The values ​​were 45.06 μmol·L -1 、849.56μmol·L -1 ·min -1 and 25.67 μmol -1 ·L·min -1 The enzyme has different degrees of degradation activity on dextran T10, T20, T40, T70, T500 and soluble starch, and its relative activity in degrading soluble starch is as high as 46.74%. The enzyme can tolerate a variety of metal ions, including low concentration (1mmol / L) of Ca 2+ , Ba 2+ , Fe 3+ 、Ni 2+ and Co 2+ It has an activating effect on the enzyme. The enzyme is activated in 5mmol / L Ba 2+ and 10mmol / L Ca 2+ , Fe 3+ 、Ni 2+ , Mn 2+ The activity of the enzyme can be maintained at more than 88% under the environment. In addition, the activity of the enzyme in 0.1% sodium fluoride increased by 14.53%. The hydrolysis products of the enzyme acting on dextran T20 and T500 are mostly various isomaltooligosaccharides, among which isomaltotetraose, isomalpentaose and isomaltohexaose have the highest content, accounting for 64.36% and 74.15% of the total hydrolysis products of T20 and T500, respectively; the scavenging ability and inhibiting ability of the hydrolysis products of the enzyme acting on dextran T20 for DPPH free radicals and hydroxyl free radicals are 84.66μg Trolox / mL and 444.76U / mg, respectively, and the scavenging ability and inhibiting ability of the hydrolysis products of the enzyme acting on dextran T500 for DPPH free radicals and hydroxyl free radicals are 90.00μg Trolox / mL and 463.11U / mg, respectively, showing the excellent antioxidant capacity of the hydrolysis products of the enzyme, and thus has potential application value in food, medical and other fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 It is the SDS-PAGE electrophoresis diagram of recombinant dextranase StDex;

[0035] Figure 2 Determination of the optimum temperature (A) and pH (B) of the recombinant dextranase StDex;

[0036] Figure 3 The temperature stability (A) and pH stability (B) of recombinant dextranase StDex were determined;

[0037] Figure 4 This is the TLC chart of the product of dextran T20 hydrolyzed by recombinant dextranase StDex. DETAILED DESCRIPTION

[0038] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0039] The gene of the dextranase StDex was derived from the NCBI database. Escherichia coli DH5α and BL21 (DE3) competent cells were purchased from Nanjing Novozyme Biotechnology Co., Ltd., and the gene manipulation plasmid pET28a-SMT3 was purchased from Wuhan Miaoling Biotechnology Co., Ltd.

[0040] Enzymes and other biochemical reagents: FastDigest restriction endonuclease and T4 DNA ligase were purchased from Thermo Fisher Scientific; Phanta Master Mix high-fidelity DNA polymerase was purchased from Nanjing Novozymes Biotech Co., Ltd.; SUMO protease was purchased from Yisheng Biotech Co., Ltd.; plasmid mini-extraction kit and agarose gel recovery kit were purchased from Tiangen Biochemical Technology Co., Ltd.; dextran T10, T20, T40, and T70 were purchased from Sangon Biotech Co., Ltd.; Bradford protein concentration assay kit was purchased from Beyotime Biotechnology Co., Ltd.; DPPH free radical scavenging ability kit and hydroxyl free radical assay kit were purchased from Nanjing Jiancheng Bioengineering Institute; others were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0041] LB medium: 5g yeast extract, 10g peptone, 10g sodium chloride, add distilled water to 1000mL. LB solid medium: add 1.5% (w / v) agar powder to the LB medium. The above medium is sterilized by high pressure steam at 121℃ for 20min before use.

[0042] The molecular biology experimental methods not specifically described in the following examples were all performed with reference to the specific methods listed in the book Molecular Cloning Experiment Guide (3rd edition) by J. Sambrook, or according to the kits and product instructions.

[0043] Example 1 Construction of recombinant dextranase StDex expression plasmid

[0044] The gene sequence stdex information of dextranase StDex in Streptococcus thermophiles LMG 18311 is derived from the NCBI database (GenBank accession number: NC_006448.1:162546-163043), and its sequence is synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. Using the synthesized stdex gene sequence as a template, the upstream primer FP-stdex (SEQID NO.1), the downstream primer RP-stdex (SEQ ID NO.2) and Phanta Master Mix high-fidelity DNA polymerase and the polymerase chain reaction (PCR) are used to amplify the target gene sequence stdex. Among them, the primer sequences used for amplification are shown in SEQID NO.1-2, the amplification system is shown in Table 1, and the amplification program is shown in Table 2.

[0045] SEQ ID NO.1: 5'-AAAGGATCCATGTTGCAGCTCTTAACGTC-3'

[0046] SEQ ID NO.2: 5'-AAACTCGAGTTAATCATTTTCATCTGTTTC-3'

[0047] Table 1 PCR reaction system for gene stdex amplification

[0048]

[0049] Table 2 PCR program for gene stdex amplification

[0050]

[0051] The amplified stdex fragment and pET28a-SMT3 plasmid were double-digested with FastDigest restriction endonucleases BamHI and XhoⅠ at 37°C, respectively. The enzyme digestion system is shown in Table 3.

[0052] Table 3 Double enzyme digestion reaction system

[0053]

[0054] The digested product was recovered using an agarose gel recovery box, and then the plasmid and fragment after restriction digestion were connected using T4 DNA ligase to form a connection product of the stdex fragment and the pET28a-SMT3 plasmid. The connection product was transformed into Escherichia coli DH5α competent cells and cultured in a 37°C constant temperature incubator. The transformation steps are as follows:

[0055] (1) Add 10 μL of the ligation product to 100 μL of E. coli DH5α competent cells, mix well on ice and incubate on ice for 30 min.

[0056] (2) Heat shock the suspension in a 42°C water bath for 45 seconds and then incubate on ice for 3 minutes.

[0057] (3) Add 1 mL of LB liquid culture medium and incubate in a shaker for 1 h (120 rpm, 37°C).

[0058] (4) The suspension was centrifuged, and 100 μL of the supernatant was used to resuspend the precipitated cells. The cells were then spread on a LB solid plate containing kanamycin sulfate at a final concentration of 50 μg / mL and cultured in a 37°C constant temperature incubator overnight.

[0059] After a single colony grows on the plate, pick out 2-3 single colonies and inoculate them into LB liquid medium (containing kanamycin sulfate with a final concentration of 50 μg / mL) for culture (200 rpm, 37°C). When the colony grows to turbidity, the bacteria are collected by centrifugation, and the recombinant plasmid pET28a-SMT3-stdex is extracted using a plasmid extraction kit for sequencing verification. The gene sequencing is completed by Suzhou Jinweizhi Biotechnology Co., Ltd. The sequencing results show that the recombinant plasmid pET28a-SMT3-stdex contains a fusion gene fragment of dextranase stdex and a lytic expression tag smt3. The DNA sequence of the smt3-stdex fusion gene fragment is shown in SEQ ID NO.3.

[0060] SEQ ID NO.3(5'-3'):

[0061] ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGCTAGCATGTCGGACTCAGAAGTCAATCAAGAAGCTAAGCCAGAGGTCAAGCCAGAAGTCAAGCCTGAGACTCACATCAATTTAAAGGTGTCCGATGGATCTTCAGAGATCTTCTTCAAGATCAAAAAGACCACTCCTTTAAGAAGGCTGATGGAAGCGTTCGCTAAAAGACAGGGTAAGGAAATGGACTCCTTAAGATTCTTGTACGACGGTATTAGAATTCAAGCTGATCAGACCCCTGAAGATTTGGACATGGAGGATAACGATATTATTGAGGCTCACAGAGAACAGATTGGTGGATCCATGCTGCAGCTGCTGACGAGCTTTAAAGTGGCGTATTATCAGACCCCGTATCTGAGCGTGGCGAAATATACCATTCGCAAACTGTATAACTATCAGCAGTTTATTACCGCGTATGAAAACCTGCTGCGCAGCGAAGGCGCGACCAACAGCAACCGCAGCGTGAGCACCAAAAACGTGACGGGCGAAATTCTGAGCAAAGATGCGCTGGGCGTTACCGGCGATAAGGTGTGGATTTTTGTGAAAAGCGGCAAAGGCCTGAGCACCGTGCAGATGATTAACATGATTGGCATTAACGCGAGCTGGCATAACGATGAAGGCGATGTGGATAACAAAACCCCGTATGCGCAAGAAAACCTGACCGTGCGCCTGAGCCTGAGCGGCAAAATTGCGCAAGAAGCGGTGAAAATTGCGGATCAGCTGTATGTGACGAGCCCGGATGATTGGGCGACGAGCATTATGCGCAAAGCGCAAGCGCGCCTGGAAACCGATGAAAACGAT

[0062] Result analysis: According to the sequencing results, the full length of the smt3-stdex fusion gene fragment is 864bp, which is formed by connecting the smt3 and stdex gene fragments, of which the length of the stdex gene is 501bp and the length of the smt3 gene is 363bp, indicating that the expression plasmid of the recombinant dextranase StDex has been successfully constructed. The gene fragment of stdex is shown in SEQ ID NO.4, and the gene fragment of smt3 is shown in SEQ ID NO.5.

[0063] SEQ ID NO.4(5'-3'):

[0064] TCCATGCTGCAGCTGCTGACGAGCTTTAAAGTGGCGTATTATCAGACCCCGTATCTGAGCGTGGCGAAATATACCATTCGCAAACTGTATAACTATCAGCAGTTTTATTACCGCGTATGAAAACCTGCTGCGCAGCGAAGGCGCGACCAACAGCAACCGCAGCGTGAGCACCAAAAACGTGACGGGCGAAATTCTGAGCAAAGATGCGCTGGGCGTTACCGGCGATAAGGTGTGGATTTTTGTGAAAAGCG GCAAAGGCCTGAGCACCGTGCAGATGATTAACATGATTGGCATTAACGCGAGCTGGCATAACGATGAAGGCGATGTGGATAACAAAACCCCGTATGCGCAAGAAAACCTGACCGTGCGCCTGAGCCTGAGCGGCAAAATTGCGCAAGAAGCGGTGAAAATTGCGGATCAGCTGTATGTGACGAGCCCGGATGATTGGGCGACGAGCATTATGCGCAAAGCGCAAGCGCGCCTGGAAAACCGATGAAAACGAT

[0065] SEQ ID NO.5(5'-3'):

[0066] ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGGCAGCCATATGGCTAGCATGTCGGACTCAGAAGTCAATCAAGAAGCTAAGCCAGAGGTCAAGCCAGAAGTCAAGCCTGAGACTCACATCAATTTAAAGGTGTCCGATGGATCTTCAGAGATCTTCTTCA AGATCAAAAAGACCACTCCTTTAAGAAGGCTGATGGAAGCGTTCGCTAAAAGACAGGGTAAGGAAATGGACTCCTTAAGATTCTTGTACGACGGTATTAGAATTCAAGCTGATCAGAACCCTGAAGATTTGGACATGGAGGATAACGATATTATTGAGGCTCACAGAGAACAGATTGGTGGA

[0067] Example 2 Construction of genetically engineered bacteria for recombinant dextranase StDex

[0068] The recombinant plasmid pET28a-SMT3-stdex constructed in Example 1 above was transformed into Escherichia coli BL21 (DE3), and the transformation steps were as follows:

[0069] (1) Add 1 μL of recombinant plasmid pET28a-SMT3-stdex to 100 μL of E. coli BL21 (DE3) competent cells, mix well on ice and incubate on ice for 30 min.

[0070] (2) Heat shock the suspension in a 42°C water bath for 45 seconds and then incubate on ice for 3 minutes.

[0071] (3) Add 1 mL of LB liquid culture medium and incubate in a shaker for 1 h (120 rpm, 37°C).

[0072] (4) The suspension was centrifuged, and 100 μL of the supernatant was used to resuspend the precipitated cells. The cells were then spread on a LB solid plate containing kanamycin sulfate at a final concentration of 50 μg / mL and cultured in a 37°C constant temperature incubator overnight.

[0073] After a single colony grew on the LB plate, a single colony was picked up with a toothpick and inoculated into LB liquid culture medium (containing kanamycin sulfate with a final concentration of 50 μg / mL) to obtain the recombinant genetically engineered bacteria StDex-SMT3-BL21.

[0074] Example 3 Preparation of recombinant dextranase StDex

[0075] The genetically engineered strain StDex-SMT3-BL21 constructed in Example 2 was inoculated into LB liquid culture medium, and kanamycin sulfate with a final concentration of 50 μg / mL was added to culture the strain (200 rpm, 37°C). 600 When the pH is about 0.8, IPTG with a final concentration of 0.2 mmol / L is added, and the shake flask culture is continued for 3-4 hours (150 rpm, 37 ° C). After the culture is completed, the bacteria are collected by centrifugation (8000 rpm, 5 min), the bacteria are resuspended in equilibrium buffer A, and the bacteria are ultrasonically broken in an ice bath. After breaking, the supernatant is collected by centrifugation, and the supernatant is preliminarily purified in the AKTA protein purification system using a nickel column affinity chromatography method. After purification, the eluate is collected to obtain the SMT3-StDex fusion protein. The fusion protein buffer is replaced with equilibrium buffer B using an ultrafiltration centrifuge tube, and SUMO protease is added to cut the fusion protein (4 ° C, 12h) to obtain a mixture of SMT3 and StDex proteins. Subsequently, the protein mixture is purified again in the AKTA protein purification system using a nickel column affinity chromatography method, and the filtrate is collected to obtain the target enzyme StDex. The purified protein is detected by SDS-PAGE. Protein quantitative analysis is completed using a Bradford detection kit. The reagents and methods used for nickel ion affinity chromatography are as follows:

[0076] (1) Reagents

[0077] Equilibrium buffer A: 20mmol / L Tris, 500mmol / L NaCl, 20mmol / L imidazole, 8mol / L urea, pH 8.0;

[0078] Equilibration buffer B: 20 ​​mmol / L Tris, 500 mmol / L NaCl, pH 8.0;

[0079] Elution buffer C: 20mmol / L Tris, 500mmol / L NaCl, 500mmol / L imidazole, pH 8.0.

[0080] Storage buffer D: 20mmol / L Tris, 50mmol / L NaCl, pH 7.0.

[0081] All the above buffers were prepared with ultrapure water.

[0082] (2) Operation steps

[0083] ① Filter the protein supernatant, buffer AD and ultrapure water with a 0.22 μm microporous filter membrane;

[0084] ② Rinse the AKTA system, pump head A, and pump head B with ultrapure water;

[0085] ③ Column loading: Place pump head A in ultrapure water, adjust the flow rate to 1 mL / min, and load the nickel ion affinity column on the AKTA protein purifier;

[0086] ④ Column flushing: Place pump head A in equilibration buffer A / B, and pump head B in elution buffer C. Set the flow rate to 2 mL / min, first flush 5 column volumes with elution buffer C, and then flush the column with equilibration buffer A / B until the baseline is balanced;

[0087] ⑤ Loading: Place pump head A in the protein supernatant / mixed solution for loading, and collect the components such as the flow-through and eluate. After loading, place pump head A back into the equilibrium buffer for flushing until the baseline is balanced.

[0088] ⑥ Elution: Use elution buffer C to perform gradient elution of the target protein, and set the elution time to 15 minutes. During this period, when the UV absorption peak rises, collect the eluate at a volume of 2 mL / tube.

[0089] ⑦ Use an ultrafiltration centrifuge tube with a molecular weight cutoff of 3kDa to concentrate the collected target protein and replace it with enzyme storage buffer D.

[0090] Result analysis: Sequence analysis showed that the fusion protein SMT3-StDex expressed by smt3-stdex contained 288 amino acids, of which dextranase StDex contained 167 amino acids, with a theoretical molecular weight of 18.69 kDa and a theoretical isoelectric point of 6.56. Its amino acid sequence is shown in SEQ ID NO.6. The purification results showed that Figure 1 As shown in the figure, the purified recombinant dextranase StDex is a single band, and the actual molecular weight is consistent with the theoretical molecular weight. Wherein, M: standard protein marker; 1: purified dextranase StDex.

[0091] SEQ ID NO.6:

[0092] SMLQLLTSFKVAYYQTPYLSVAKYTIRKLYNYQQFITAYENLLRSEGATNSNRSVSTKNVTGEILSKDALGVTGDKVWIFVKSGKGLSTVQMINMIGINASWHNDEGDVDNKTPYAQENLTVRLSLSGKIAQEAVKIADQLYVTSPDDWATSIMRKAQARLETDEND Example 4 Enzymatic Characteristics of Recombinant Dextranase StDex

[0093] 1. Activity determination of recombinant dextranase StDex

[0094] (1) Prepare a standard curve: Dry 0.5g of glucose standard to constant weight, accurately weigh 0.1g of glucose and dissolve it in distilled water, dilute to 100mL with a volumetric flask, and prepare a 1mg / mL glucose standard solution. Pipette 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, and 0.7mL of the glucose standard solution into a 10mL EP tube, respectively, and fill to 2mL with the preservation buffer D in Example 3. Add 3mL of DNS solution to each of the above centrifuge tubes, and then boil for 5 minutes. After each tube has cooled to room temperature, pipette 200μL of sample into a 96-well plate, and detect the absorbance value A of each tube at 540nm. 540 The mass of glucose in each tube is used as the horizontal axis, A 540 Draw the standard curve with y as the vertical axis.

[0095] After measurement, the standard curve of glucose is y=2.324x-0.2127, where x is the mass of glucose (mg) and y is the absorbance value A 540 .

[0096] (2) Determination of the activity of dextranase StDex: Experimental group: 20 μL of the enzyme solution to be tested was taken, 180 μL of a 3% dextran T20 solution (prepared with the storage buffer D in Example 3) was added, mixed and placed in a 50°C water bath for reaction for 10 min, then 200 μL of DNS was added and boiled for 5 min to inactivate the enzyme solution; after adding 3 mL of distilled water, 200 μL of the sample was taken and the absorbance value A of the sample was detected at a wavelength of 540 nm. 540 Control group: The enzyme solution to be tested was replaced with inactivated enzyme solution (boiled at 100℃ for 5 minutes for standby use), and the rest of the methods and reagent dosages were the same as those of the experimental group. The enzyme activity unit (1U) is defined as the amount of enzyme required to hydrolyze 1μmol of glucose per minute.

[0097] The total activity of the dextranase StDex prepared in Example 3 was calculated based on the standard curve to be 39.08 U, and the specific activity was 174.48 U / mg.

[0098] 2. Determination of the Optimal Temperature and pH of Recombinant Dextranase StDex

[0099] The method for determining the optimum temperature is the same as the method for determining the dextranase activity in Example 4, except that the reaction temperature is set to 20-80°C (20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80°C). The group with the highest enzyme activity is set as 100%, and the relative activities at the other groups of temperatures are calculated as the percentage of the maximum activity.

[0100] After determination, Figure 2 As shown in A, the optimum temperature of dextranase StDex is 50℃.

[0101] The method for determining the optimum pH is the same as the method for determining the activity of dextranase in Example 4, except that 3% dextran T20 is dissolved in different pH buffers, and the pH is set to 3.0-8.0 (3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0). The buffers of dextran T20 at different pH are: 20mmol / L acetic acid-sodium acetate buffer (pH3.0-5.5), 20mmol / L Bis-Tris buffer (pH 6.0-7.0), 20mmol / LTris-HCl buffer (pH 7.5-8.0). The group with the highest enzyme activity is set to 100%, and the relative activities at the other groups of pH are calculated as the percentage of the maximum activity.

[0102] After determination, Figure 2 As shown in B, the optimum pH of dextranase StDex is 7.0.

[0103] 3. Temperature and pH stability determination of recombinant dextranase StDex

[0104] The method for determining temperature stability is the same as the method for determining dextranase activity in Example 4, except that the enzyme samples are placed in a water bath at 20-80°C (20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80°C) for 1 hour before the determination. The activity of the original enzyme solution that has not been treated for 1 hour is set as 100%, and the relative activities of the enzyme solutions of the other groups treated at different temperatures are calculated as the percentage of the activity.

[0105] The results of the test are as follows Figure 3 As shown in A, with the increase of temperature, the activity of dextranase StDex showed an overall downward trend, but after being treated at 20-50°C for 1 hour, it was still able to maintain more than 70% of the enzyme activity.

[0106] The method for determining pH stability is the same as the method for determining the optimal pH in Example 4, except that before the determination, the enzyme samples are placed in buffers of different pH (3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0) and incubated at 50°C for 1 hour. 20mmol / L acetic acid-sodium acetate buffer (pH 3.0-5.5), 20mmol / L Bis-Tris buffer (pH 6.0-7.0), 20mmol / L Tris-HCl buffer (pH 7.5-8.0). The activity of the original enzyme solution that has not been treated for 1 hour is set as 100%, and the relative activities of the enzyme solutions of the other groups treated with different pH are calculated as the percentage of the activity.

[0107] The results of the test are as follows Figure 3 As shown in B, the dextranase StDex activity can maintain more than 90% of the enzyme activity when treated at pH 5.0-7.0 for 1 hour.

[0108] 4. Effects of different metal ions on the activity of recombinant dextranase StDex

[0109] The metal ion Na with a final concentration of 1 mmol / L, 5 mmol / L and 10 mmol / L was added to the storage buffer D in Example 3. + (NaCl), K + (KCl), Ca 2+ (CaCl2), Mg 2+ (MgCl2), Al 3+ (AlCl3), Ba 2+ (BaCl2), Cu 2+ (CuSO4), Fe 3+ (FeCl3), Zn 2+ (ZnSO4), Co 2+ (CoCl2), Ni 2+ (NiCl2) and Mn 2+ (MnSO4). Under the optimal conditions (50°C, pH 7.0), buffer solutions containing different metal ion components were prepared to determine the effects of different metal ions on enzyme activity. The determination method was the same as the dextranase activity determination method in Example 4, except that 3% dextran T20 was dissolved in the above-mentioned different metal ion solutions. The control group was set to not add any metal ions, and the activity of dextranase StDex was calculated as 100%, and the relative activities of the enzymes in the other groups were calculated as the percentage of the activity.

[0110] The results are shown in Table 4. The enzyme can tolerate a variety of metal ions, including 1-5 mmol / L Ca 2+ , Ba 2+ , Fe 3 + 、Ni 2+ and Co 2+ etc. all have an activating effect on StDex. The enzyme is activated in 10mmol / L Ca 2+ , Fe 3+ 、Ni 2+ , Mn 2+ Under the same environment, the activity of K + Mg 2+ 、Al 3+ 、Zn 2+ etc. have an inhibitory effect on the enzyme. 3+The inhibitory effect of StDex was 1-5mmol / L Fe 3+ The activity is enhanced in the environment, and the Fe 3+ This property has laid an important theoretical foundation for the development of drug preparations such as iron dextran.

[0111] Table 4 Effect of metal ions on the activity of dextranase StDex

[0112]

[0113] in, a ND means that the value was not detected in the corresponding metal ion environment.

[0114] 5. Effects of chemical reagents on the activity of recombinant dextranase StDex

[0115] In the storage buffer D in Example 3, ethanol, sodium benzoate, sorbic acid, sodium fluoride and 0.5% sodium dodecyl sulfate were added to a final concentration of 0.1%, and the effects of different chemical reagents on the activity of StDex enzyme were determined under optimal conditions (50°C, pH 7.0). The determination method is the same as the dextranase activity determination method in Example 4, except that 3% dextran T20 is dissolved in the above-mentioned different chemical reagent solutions. The control group was set to not add any additional chemical reagents, and the activity of dextranase StDex was calculated as 100%, and the relative activities of the enzymes in the other groups were calculated as the percentage of the activity.

[0116] The test results are shown in Table 5. Ethanol, sodium benzoate, sorbic acid, sodium dodecyl sulfate and other reagents all have different degrees of inhibitory effects on the enzyme, while the activity of the enzyme in 0.1% sodium fluoride is increased by 14.53%. This property has laid an important theoretical foundation for the development of fluoride-containing oral cleaners.

[0117] Table 5 Effects of chemical reagents on the activity of dextranase StDex

[0118]

[0119] 6. Determination of substrate specificity of recombinant dextranase StDex

[0120] The method for determining substrate specificity is the same as the method for determining dextranase activity in Example 4, except that the 3% dextran T20 substrate is replaced with dextran T20 of different concentrations (1%, 1.5%, 2%, 3%, 5%, 7%), dextran of different molecular weights (T10, T40, T70, T500), soluble starch, β-cyclodextrin, maltose, sucrose, chitosan and agarose, etc., and the effects of different substrates on StDex enzyme activity are determined under optimal conditions (50°C, pH 7.0). The enzyme activity of StDex with 3% dextran T20 as the substrate is set as 100%, and the relative activities of the enzymes in the other groups are calculated as the percentage of the activity.

[0121] The results are shown in Table 6. Dextranase StDex can hydrolyze dextran T20 of different concentrations, and compared with 3% dextran T20, the relative activity of hydrolyzing other concentrations of dextran T20 is maintained at more than 95%. Dextranase StDex can hydrolyze dextran of different molecular weights, and the hydrolysis efficiency gradually decreases with the increase of dextran molecular weight. Dextranase StDex has no hydrolysis activity on β-cyclodextrin, maltose, sucrose, chitosan and agarose, but can hydrolyze soluble starch, and the hydrolysis activity reaches 46.74%, which is much greater than the hydrolysis activity of other known dextranases.

[0122] Table 6 Effects of different substrates on the activity of dextranase StDex

[0123]

[0124] in, a ND means that the value was not detected in the corresponding substrate environment.

[0125] 7. Determination of Enzyme Kinetic Parameters of Recombinant Dextranase StDex

[0126] The method for determining the enzyme kinetic parameters is the same as the method for determining the dextran activity in Example 4, except that the concentration of the 3% dextran T20 substrate is adjusted to 5-40 mg / mL, and the substrate types are respectively changed to 3% dextran of different molecular weights (T10, T40, T70, T500) and 3% soluble starch, and the effects of various substrates of different concentrations on the StDex enzyme activity are determined under the optimal conditions (50°C, pH 7.0). m 、v max and k cat / K m The value of is calculated from the Lineweaver-Burk plot.

[0127] The results are shown in Table 7. As the molecular weight of the dextran substrate increases, K mGradually decreases, k cat / K m The results show that the affinity of dextranase StDex to low molecular weight dextran substrate is greater and the catalytic efficiency is higher. Although the catalytic activity of dextranase StDex to soluble starch is not as good as that to dextran substrate, its affinity and catalytic efficiency are stronger than those to dextran substrate.

[0128] Table 7 Kinetic parameters of dextranase StDex under different substrates

[0129]

[0130]

[0131] Example 5 Application of recombinant dextranase StDex

[0132] 1. Preparation of Oligosaccharides with Dextranase StDex and Dextran T20

[0133] The dextranase StDex purified in Example 3 was added to 1% dextran T20, with the ratio of enzyme to substrate being 1:1, and hydrolyzed for 30 min and 60 min respectively under optimal conditions (50° C., pH 7.0) to obtain oligosaccharide products of dextran T20 hydrolyzed by dextranase StDex.

[0134] 2. Identification of oligosaccharide products from the hydrolysis of dextran T20 by dextranase StDex

[0135] (1) Oligosaccharide products were identified by thin layer chromatography (TLC). The thin layer chromatography method is as follows:

[0136] ① Prepare the developing agent: prepare the developing agent in a volume fraction of n-butanol: isopropanol: glacial acetic acid: water = 7:5:4:2.

[0137] ②Prepare the color developer: Mix 50 mL of acetone, 1 g of diphenylamine, 1 mL of aniline, and 1 mL of phosphoric acid to obtain the color developer.

[0138] ③ Loading samples: draw lines on the silica gel plate at 1 cm above and below, with the lower line as the loading point. Pipette 1 μL of glucose standard, 1 μL of isomaltooligosaccharide standard (1 mg / mL), 1 μL of StDex inactivated enzyme solution (treated at 100°C for 5 min), and 1 μL of oligosaccharide product sample (reaction for 30 min and reaction for 60 min) on the silica gel plate, leaving 1 cm between each sample and let it dry.

[0139] ④ Color development: Place the silica gel plate in a chromatography tank containing a developing agent, and take out the plate to dry when the solvent front reaches the end line. Spray the color developer evenly on the silica gel plate, and then place it in an 80℃ oven for 20 minutes.

[0140] The TLC results of oligosaccharide products of dextran T20 hydrolyzed by dextranase StDex are shown in Figure 4 , it can be seen that the oligosaccharide products of StDex hydrolysis of dextran T20 for 30min and 60min are similar, all of which are various isomaltooligosaccharides, while glucose products are not detected. Therefore, oligosaccharide products were successfully prepared using dextranase StDex and dextran T20, and the products were identified as isomaltooligosaccharides.

[0141] (2) Identification of oligosaccharide products by LC-MS

[0142] After the sample was freeze-dried, 10 mg of the oligosaccharide product sample was weighed and placed in a 2 mL EP tube. 1 mL of 50% methanol / water was added, vortexed for 30 seconds, left for 30 seconds, and vortexed for 30 seconds again, and repeated several times until the sample was dissolved. Then, it was placed in a centrifuge (13000 rpm, 10 min), and 500 μL of the supernatant was placed in an injection vial for liquid chromatography-mass spectrometry analysis. LC-MS was completed by Yangzhou Boruitang Biotechnology Co., Ltd.

[0143] The oligosaccharide product components of dextran T20 hydrolyzed by dextranase StDex are shown in Table 8. Various isomaltooligosaccharides such as isomaltose, isomaltotriose, isomaltotetraose, isomalpentaose, isomaltohexaose, isomaltoheptaose, isomaltooctaose, and arabinooligosaccharides (or xylooligosaccharides) were detected in the StDex hydrolyzate components. Among them, isomaltotetraose, isomaltopentaose and isomaltohexaose have the highest content, accounting for 64.36% of the dextran T20 hydrolyzate, while the content of arabinooligosaccharides (or xylooligosaccharides) is less.

[0144] Table 8 LC-MS identification of product components of dextranase StDex acting on dextran T20

[0145]

[0146] The components of the oligosaccharide products of dextran T500 hydrolyzed by dextranase StDex are shown in Table 9. A variety of isomaltooligosaccharides and arabinooligosaccharides (or xylooligosaccharides) were also detected in the StDex hydrolysis product components, among which isomaltotetraose, isomaltopentaose and isomaltohexaose were the most abundant, accounting for 74.15% of the total hydrolysis products of dextran T500.

[0147] Table 9 LC-MS identification of product components of dextranase StDex acting on dextran T500

[0148]

[0149]

[0150] By comparison, it was found that the types of products produced by the hydrolysis of dextran T20 and T500 by dextranase StDex were very similar, but the contents of various oligosaccharides were different. Isomaltotetraose accounted for the largest proportion of dextran T20 and T500 hydrolysates, 30.51% and 39.62% respectively, followed by isomaltotetraose and isomaltohexaose. In addition, isomaltododecanose was also detected in the T500 hydrolysate, accounting for 3.11% of the total content of the product.

[0151] 3. Determination of the antioxidant activity of dextranase StDex hydrolysis products

[0152] The DPPH free radical scavenging ability and hydroxyl free radical inhibition ability of the enzymatic hydrolysis products of dextran T20 and T500 were determined by dextranase StDex to evaluate the antioxidant activity of the oligosaccharide products. The determination method was carried out according to the DPPH free radical scavenging ability kit and hydroxyl free radical determination kit of Nanjing Jiancheng Bioengineering Institute.

[0153] Table 10 Determination of the antioxidant capacity of dextran products acted by dextranase StDex

[0154]

[0155] The measurement results are shown in Table 10. The scavenging ability of the hydrolyzate of dextran T20 by the enzyme to scavenging DPPH free radicals and inhibiting hydroxyl free radicals are 84.66 μg Trolox / mL and 444.76 U / mg, respectively. The scavenging ability of the hydrolyzate of dextran T500 by the enzyme to scavenging DPPH free radicals and inhibiting hydroxyl free radicals are 90.00 μg Trolox / mL and 463.11 U / mg, respectively, showing the excellent antioxidant capacity of the hydrolyzate of the enzyme, and thus has potential application value in the fields of food and medicine.

Claims

1. A recombinant dextranase, characterized in that Its amino acid sequence is shown in SEQ ID NO.

6.

2. A gene encoding the recombinant dextranase according to claim 1.

3. The gene according to claim 2, characterized in that Its nucleotide sequence is shown in SEQ ID NO.

4.

4. A method for constructing the recombinant dextranase according to claim 1, characterized in that: The following steps are involved: (1) After amplifying the stdex gene, double-digesting the plasmid, ligating, transforming into competent cells, and culturing to obtain recombinant genetically engineered bacteria; (2) culturing the recombinant genetically engineered strain described in step (1), ultrasonically disrupting the bacteria, collecting the supernatant, and purifying to obtain the SMT3-StDex fusion protein; (3) Cutting and purifying the SMT3-StDex fusion protein described in step (2) to obtain recombinant dextranase.

5. The method according to claim 3, characterized in that: The specific preparation steps of the SMT3-StDex fusion protein in step (2) are as follows: adding kanamycin sulfate to the culture medium containing the recombinant genetically engineered strain, adding IPTG, continuing the shake flask culture, collecting the bacteria by centrifugation, resuspending the bacteria in equilibrium buffer A, ultrasonically disrupting the bacteria, collecting the supernatant, and preliminarily purifying the supernatant. After purification, collecting the eluate to obtain the SMT3-StDex fusion protein; the equilibrium buffer A includes Tris, NaCl, imidazole and urea.

6. A dextran iron drug, characterized in that: It contains the recombinant dextranase according to claim 1.

7. A fluoride-containing oral cleaning agent, characterized in that: It contains the recombinant dextranase according to claim 1.

8. Use of the recombinant dextranase according to claim 1 in decomposing dextran or soluble starch of different molecular masses; the dextran of different molecular masses includes dextran T10, dextran T20, dextran T40, dextran T70, and dextran T500.

9. The use according to claim 8, characterized in that: The concentration of the dextran with different molecular weights is 1% to 7%.

10. Use of the recombinant dextranase according to claim 1 in the preparation of oligosaccharides.

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