High-activity and high-stability starch branching enzyme as well as preparation and application thereof

Abstract

The invention discloses a high-activity and high-stability starch branching enzyme as well as preparation and application thereof, and belongs to the technical field of biology. A wild type starch branching enzyme truncated body PtGBE is expressed in E.coli BL21 (DE3), the amino acid sequence of the wild type starch branching enzyme truncated body PtGBE is shown in SEQ ID NO.1, gene site-directed modification is carried out on the PtGBE, the mutant type PtGBE with high activity and high stability is obtained, and the amino acid sequence of the mutant type PtGBE is shown in SEQ ID NO.3. The specific enzyme activity of the mutant type PtGBE is 7119.35 U / mg and is improved by 119% compared with that of PtGBE, and the thermal stability and the pH stability are improved. After in-vitro digestion experiment analysis, the content of resistant starch is increased, the content of cassava starch RS is increased to 73.81%, and the content of pyrodextrin RS is increased by 5.83%.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly relates to a highly active and highly stable starch branching enzyme and its preparation and application. Background Art

[0002] Starch branching enzyme (1,4-α-glucan branching enzyme; EC 2.4.1.18) is a class of glycosyltransferases belonging to the α-amylase family, which can catalyze the hydrolysis of α-1,4-glycosidic bonds in starch molecules to produce free short chains with non-reducing ends, and then connect them to the acceptor chain in the form of α-1,6-glycosidic bonds through transglycosylation, thereby forming new branch points, increasing the degree of starch branching, and increasing the content of resistant starch.

[0003] Resistant starch refers to a class of starches and their degradation products that cannot be digested and absorbed in the small intestine of healthy humans but can be utilized by microorganisms in the large intestine, and it has good effects in regulating blood sugar, reducing blood lipids, and improving intestinal function. The traditional production methods of resistant starch mainly include physical methods and chemical methods, which have the advantages of high conversion rate of resistant starch, relatively short production cycle, and relatively simple operation, etc. However, low safety, serious environmental pollution, and high energy consumption are also undeniable disadvantages. The enzymatic production of resistant starch has many advantages, such as high reaction specificity, environmental friendliness, and high safety, etc.

[0004] Starch branching enzyme is the main enzyme preparation for the enzymatic production of resistant starch. It can not only act on starch raw materials but also act on dextrin. The former is used to produce resistant starch, and the latter is used to produce resistant dextrin. If resistant dextrin is prepared based on resistant starch, the quality of resistant dextrin can be improved and the production cost can be reduced. In recent years, scientific researchers have been committed to using starch branching enzyme to produce resistant starch. For example, Li Lingling et al. used RoBE (derived from Rhodothemus obamensis) and VvGBE (derived from Vibrio vulnificus) to modify corn starch for 12 h and 10 h respectively, and the resistant starch content increased from 35% to 41% and 51% respectively. However, due to reasons such as low activity and poor stability of the starch branching enzyme used for starch modification, it will lead to situations such as long modification time and poor modification effect in its application. For example, Li Lingling et al. determined that the optimal temperature of VvGBE is 35°C, and the activity will be lost above 45°C; Jiang Haimin et al. determined that the enzyme activity of RoBE is 6003 U / mg; Ban Xiaofeng et al. determined that the enzyme activity of GBE Gt (derived from Geobacillus thermoglucosidans STB02) is 282 U / mg, and the remaining enzyme activity is 50% after incubation at 65°C for 6.9 min.

[0005] Therefore, the development of a starch branching enzyme with high activity and stability to improve the efficiency of starch modification to meet the needs of industrial production has become an urgent problem to be solved. Summary of the Invention

[0006] To overcome the disadvantages and deficiencies of the prior art, the primary object of the present invention is to provide a starch branching enzyme with high activity and high stability. The truncated form of the starch branching enzyme PtGBE derived from Parageobacillus thermantarcticus was recombinantly expressed in BL21(DE3). By site-directed modification of the PtGBE gene to improve its enzyme activity, a mutant PtGBE with high activity was obtained, and its thermal stability and pH stability were both improved compared with PtGBE. The results of in vitro digestibility analysis showed that the starch samples modified by the mutant PtGBE for 2 h had improved anti-digestibility, indicating that the mutant PtGBE has good application prospects in the preparation of resistant dextrin.

[0007] Another object of the present invention is to provide a method for preparing the above-mentioned starch branching enzyme with high activity and high stability.

[0008] Another object of the present invention is to provide the application of the above-mentioned starch branching enzyme with high activity and high stability.

[0009] The object of the present invention is achieved by the following technical solutions:

[0010] The starch branching enzyme is the starch branching enzyme PtGBE or the mutant starch branching enzyme PtGBE. Among them, the amino acid sequence of the starch branching enzyme PtGBE is shown in SEQ ID NO.1, and the amino acid sequence of the mutant starch branching enzyme PtGBE is shown in SEQ ID NO.3.

[0011] Specifically, the mutant starch branching enzyme PtGBE is obtained by site-directed modification of the starch branching enzyme PtGBE with the amino acid sequence shown in SEQ ID NO.1, and the 416th position is mutated to glutamate and the 349th position is mutated to lysine.

[0012] The related biological materials of the above-mentioned starch branching enzyme are any one of the following biological materials:

[0013] (1) A DNA molecule encoding the starch branching enzyme PtGBE or the mutant starch branching enzyme PtGBE;

[0014] (2) An expression cassette containing the DNA molecule described in (1);

[0015] (3) A recombinant vector containing the expression cassette described in (2);

[0016] (4) A recombinant bacterium containing the recombinant vector described in (3).

[0017] Preferably according to the present invention, the nucleotide sequence of the DNA molecule encoding the starch branching enzyme PtGBE is as shown in SEQ ID NO.2.

[0018] Preferably according to the present invention, the nucleotide sequence of the DNA molecule encoding the mutant starch branching enzyme PtGBE is as shown in SEQ ID NO.4.

[0019] Specifically, the nucleotide sequence of the DNA molecule encoding the mutant starch branching enzyme PtGBE is obtained by mutating five bases of the starch branching enzyme PtGBE with the nucleotide sequence shown in SEQ ID NO.2.

[0020] Preferably according to the present invention, the expression vector used for the recombinant vector is pET-28a(+).

[0021] Preferably according to the present invention, the expression bacterium used for the recombinant bacterium is Escherichia coli BL21(DE3).

[0022] The preparation method of the above-mentioned starch branching enzyme comprises the following steps: introducing the DNA molecule encoding the starch branching enzyme PtGBE or the mutant starch branching enzyme PtGBE into the pET-28a(+) expression vector, then transforming it into Escherichia coli BL21(DE3), inducing the expression of the mutant PtGBE, and purifying and preparing the starch branching enzyme PtGBE or the mutant starch branching enzyme PtGBE by metal chelate chromatography.

[0023] The application of the above-mentioned starch branching enzyme in modified starch.

[0024] Preferably according to the present invention, the above-mentioned application comprises the following steps: dissolving starch in a buffer solution, heating and gelatinizing it under stirring, incubating the gelatinized starch, then adding the above-mentioned starch branching enzyme, reacting, heating and boiling to inactivate and terminate the reaction after the reaction is completed, and freeze-drying the reacted sample.

[0025] The conditions for the above-mentioned heating and gelatinizing are preferably: temperature 90±5°C, time 30±5 min.

[0026] The conditions for the above-mentioned incubation are preferably: water bath shaker, temperature 50±5°C, time 10±2 min.

[0027] The conditions for the above-mentioned reaction are preferably: time 2±0.5 h.

[0028] The conditions for the above-mentioned inactivation are preferably: time 20±5 min.

[0029] The addition amount of the above-mentioned starch branching enzyme is 400±100 U / g.

[0030] The starch described above is at least one of corn starch, cassava starch, and potato starch.

[0031] The application of the above starch branching enzyme in modified dextrin.

[0032] Preferably according to the present invention, the application includes the following steps: dissolving dextrin in a buffer solution, incubating, then adding the above starch branching enzyme, reacting, heating to boiling for inactivation to terminate the reaction after the reaction is completed, and freeze-drying the reacted sample.

[0033] The conditions of the incubation are preferably: water bath shaker, temperature 50 ± 5 °C, time 10 ± 2 min.

[0034] The conditions of the reaction are preferably: time 2 ± 0.5 h.

[0035] The conditions of the inactivation are preferably: time 20 ± 5 min.

[0036] The addition amount of the starch branching enzyme is 400 ± 100 U / g.

[0037] The present invention has the following advantages and effects compared with the prior art:

[0038] 1. The specific activity of the mutant PtGBE described in the present invention is 7119.35 U / mg, which is higher than that of the reported starch branching enzyme with relatively high activity. The optimal temperature is 50 °C and the optimal pH is 7.0, providing a better enzyme preparation for subsequent applications.

[0039] 2. The gene site-directed modified starch branching enzyme obtained in the present invention can increase the content of resistant components in starch, can provide a better starting material for the preparation of resistant dextrin, thereby improving the yield and quality of subsequent resistant dextrin, and has good application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 It is the PCR amplification diagram of the DNA molecule of mutant PtGBE; where lane M is the DNA Marker and lane 1 is the PCR amplification product of the DNA molecule of mutant PtGBE.

[0041] Figure 2 It is the purification result diagram of PtGBE and mutant PtGBE; where M is the protein loading Marker; lane 1 is the loading solution after disrupting and centrifuging the adjusted component; lane 2 is the permeate; lane 3 is the eluate without the target protein; lane 4 is the eluate containing the target protein.

[0042] Figure 3 It is the optimal reaction pH diagram of PtGBE and mutant PtGBE.

[0043] Figure 4Optimal reaction temperature graph of PtGBE and mutant PtGBE.

[0044] Figure 5 Thermal stability graph of PtGBE and mutant PtGBE.

[0045] Figure 6 pH stability graph of PtGBE and mutant PtGBE. Specific implementation mode

[0046] The present invention will be further described below in conjunction with specific embodiments, but the cited embodiments are only used to illustrate the present invention and not to limit the scope of the present invention.

[0047] Unless otherwise specified, various reagents and raw materials used in the present invention are all commercially available products or products that can be prepared by known methods.

[0048] Example 1 Construction of a recombinant vector of the mutant PtGBE nucleotide sequence

[0049] (1) According to the nucleotide sequence SEQ ID NO.2 of the said PtGBE, using the method of mutant PtGBE primers and PCR, a DNA molecule encoding mutant PtGBE was artificially synthesized, and the following 4 primers were designed:

[0050] F1: 5’-TCTGgaaTTTTCTCACGATGAAGTTGTTCACG-3’

[0051] R1: 5’-CGTGAGAAAAttcCAGAATGAAGTTTTCAGAGTAAGCG-3’

[0052] F2: 5’-CATTCTGaaaATCGCAGAGGATAGCACCGAAT-3’

[0053] R2: 5’-CTGCGATtttCAGAATGCTCGGATCGTATGCG-3’

[0054] Using the PtGBE plasmid containing the nucleotide sequence SEQ ID NO.2 (constructed by Guangzhou Aiji Biotechnology Co., Ltd., with the basic plasmid pET-28a(+) and restriction enzyme sites NcoI at the 5' end and PspXI at the 3' end) as a template, PCR amplification was carried out with F1 / R1 and F2 / R2 as primers. 10 μL of 2×Pfu Max HiFi PCR ProMix (produced by Guangzhou Yingzan Biotechnology Co., Ltd., EnzyValley, product number P217), 0.4 μL of each upstream and downstream primer (10 μmol / L) F1 / R1, F2 / R2, and an appropriate amount of sterilized water were added for PCR amplification. The amplification conditions were: 98°C for 30 s; 98°C for 10 s, 60°C for 30 s, 72°C for 30 s, for a total of 30 cycles; 72°C for 10 min. The specific PCR results are as Figure 1 shown.

[0055] (2) The products after two rounds of PCR amplification were transformed into DH5α competent cells. Single colonies were picked for colony PCR identification, and the positive monoclonal clones were sent to a sequencing company for sequencing verification to confirm the correctness of the DNA molecule encoding the mutant PtGBE.

[0056] Example 2 Induced expression of starch branching enzyme in Escherichia coli

[0057] The PtGBE plasmid and the constructed mutant PtGBE plasmid were transformed into BL21(DE3). Single colonies were inoculated into LB medium containing 50 μg / mL kanamycin and cultured overnight in a shaker at 37°C; The overnight culture seed solution was inoculated into 200 mL of TB medium containing 50 μg / mL kanamycin at a ratio of 1:100 and continued to be cultured in a shaker at 37°C until the OD 600 was 1.0 - 1.2; IPTG was added to each bottle of bacterial solution to a final concentration of 0.01 mmol / L, and induction was continued at 25°C for 24 h; The induced bacteria were collected by centrifugation and weighed, and the wet weight of the bacteria was recorded.

[0058] Example 3 Isolation and purification of starch branching enzyme

[0059] 1. Ultrasonic disruption of recombinant bacteria

[0060] Take the PtGBE and mutant PtGBE bacterial cells stored at -20°C. According to the wet weight of the bacterial cells recorded in Example 2, resuspend the bacterial cells by adding 10 mL of lysis Buffer per gram of bacterial cells, and lyse the bacterial cells with an ultrasonic cell disruptor. The ultrasonic conditions are: power 195 W, ultrasound for 3.0 s, interval 3.0 s, and continuous for 20 min. Place the lysed bacterial cells in a high-speed refrigerated centrifuge and centrifuge at 10000 g at 4°C for 30 min. Adjust the supernatant to contain 500 mmol / L NaCl and 20 mmol / L imidazole for subsequent purification use, and filter with a 0.22 μm microporous membrane.

[0061] 2. Purification by nickel ion affinity chromatography

[0062] The chromatography column selected is HisTrap TM HP 5 mL (purchased from GE Healthcare). The binding buffer is buffer A: 10 mmol / L PBS, 20 mmol / L imidazole, 500 mmol / L NaCl, pH 6.0; the elution buffer is buffer B: 10 mmol / L PBS, 500 mmol / L imidazole, 500 mmol / L NaCl, pH 6.0. Filter with a 0.22 μm membrane for standby.

[0063] First, equilibrate the chromatography column with buffer A, then load the supernatant described in step 1 using a sample pump. After loading, wash the column with buffer A first, and then perform gradient elution with the elution buffer buffer B, and collect the elution peak.

[0064] Take a sample of the elution peak and detect it by SDS PAGE protein electrophoresis. The results are as Figure 2 shown. It can be seen from the SDS-PAGE results that both the PtGBE and mutant PtGBE eluates contain bands with molecular sizes close to the target protein. Although the bands are relatively light, the purity is high.

[0065] Example 4 Measurement of starch branching enzyme activity

[0066] The activity of starch branching enzyme was determined according to the following method. 0.1% (w / v) potato amylose was prepared with 10 mmol / L PBS buffer (pH 7.5). 150 μL of substrate and 150 μL of enzyme solution were added to a 1.5 mL centrifuge tube and reacted at 50 °C for 15 min. After the reaction, it was inactivated in a boiling water bath for 20 min. After complete cooling, it was centrifuged (10000 g, 2 min). 200 μL of the reaction supernatant was taken and mixed with 2 mL of the chromogenic solution (0.005% (w / v) I2, 0.05% (w / v) KI) by shaking. When the color was stable in the dark, the absorbance was measured at 660 nm. The enzyme activity was defined as: at 660 nm, the amount of enzyme required for the absorbance to decrease by 1% per minute was one enzyme activity unit.

[0067] The enzyme activities and yields of PtGBE and mutant PtGBE are shown in Table 1. The results show that the specific enzyme activity of PtGBE is 3238.50 U / mg, and the specific enzyme activity of mutant PtGBE is 7119.35 U / mg.

[0068] Table 1 Comparison of enzyme activities of mutants

[0069]

[0070] Example 5 Determination of the enzymatic properties of starch branching enzyme

[0071] 1. Determination of the optimal reaction pH

[0072] The enzyme activities of PtGBE and mutant PtGBE were determined under different pH conditions (4.0, 5.0, 6.0, 7.0, 7.5, 8.0, 9.0). Taking the one with the highest activity as 100%, their relative enzyme activities were calculated respectively. Among them, 50 mM citric acid-sodium citrate buffer was used for pH 4.0 - 6.0, 50 mM PBS buffer was used for pH 6.0 - 8.0, and 50 mM Tris-HCl buffer was used for pH 8.0 - 9.0.

[0073] The results are as Figure 3 shown. When the pH < 5.0, the activities of both were poor. As the pH increased, the activities of both showed a trend of first increasing and then decreasing, and the optimal pH remained unchanged. PtGBE and mutant PtGBE both had good activities in the range of pH 6.0 - 8.0, and mutant PtGBE showed higher activity than PtGBE.

[0074] 2. Determination of the optimal reaction temperature

[0075] The enzyme activities of PtG BE and mutant PtGBE were measured at 30 °C, 35 °C, 41 °C, 45 °C, 50 °C, 55 °C, 59 °C, and 65 °C respectively under the optimal pH condition, and the group with the highest enzyme activity was taken as 100%.

[0076] The results are as Figure 4 shown. With the increase of temperature, the activities of both PtGBE and mutant PtGBE showed a trend of first increasing and then decreasing, and the optimal temperature remained unchanged at 50 °C.

[0077] 3. Determination of thermal stability

[0078] The enzyme solutions were incubated at 30 °C, 35 °C, 41 °C, 45 °C, 50 °C, 55 °C, 59 °C, and 65 °C for 2 h respectively. After incubation, the residual activities of PtGBE and mutant PtGBE were measured at the optimal pH and optimal temperature, and the enzyme activity of the unincubated enzyme was defined as 100%.

[0079] The results are as Figure 5 shown in a. The remaining enzyme activities of both PtGBE and mutant PtGBE decreased with the increase of incubation temperature. After incubation at 50 °C for 2 h, the remaining enzyme activity of mutant PtGBE could still be maintained above 90%, while the remaining enzyme activity of PtGBE was only below 70%. When incubated at 65 °C for 0 - 25 min, the enzyme activities of both PtGBE and mutant PtGBE gradually decreased as Figure 5 shown in b. After incubation for 10 min, mutant PtGBE retained 51% of its activity, while PtGBE only retained 37% of its activity. Overall analysis showed that in the temperature range of 0 - 65 °C, mutant PtGBE had higher thermal stability than PtGBE.

[0080] 4. Determination of pH stability

[0081] The enzyme solutions were incubated at different pH conditions for 2 h. After incubation, they were adjusted to the optimal pH and then the residual activities of PtGBE and mutant PtGBE were measured at the optimal temperature. The enzyme activity of the unincubated enzyme was defined as 100%.

[0082] The results are as Figure 6 shown. After incubation at pH = 7.0, the remaining enzyme activities of both were the highest. When incubated at pH > 7.5 or pH < 6 for 2 h, the relative enzyme activity of mutant PtGBE remained above 70%, while the relative enzyme activity of PtGBE decreased to below 70%. Thus, it can be seen that the pH stability of mutant PtGBE was improved compared with that of PtGBE.

[0083] Example 6 Effect of starch branching enzyme on the modification of different starch samples

[0084] 1. Preparation of modified starch by enzymatic method

[0085] Accurately weigh 2.5 g of starch sample and dissolve it in 100 mL of PBS (pH 7.0, 10 mmol / L) buffer solution. Gelatinize it at 90 °C for 30 min under a heating magnetic stirrer. After gelatinization, incubate the starch in a constant temperature water bath shaker at 50 °C for 10 min, then add the mutant PtGBE prepared in Example 4 at 400 U / g and react for 2 h. After the reaction, terminate the reaction by heating and boiling for 20 min, and freeze-dry the reacted sample.

[0086] 2. In vitro digestion performance analysis

[0087] Add 0.5 g of porcine pancreatic α-amylase (9.4 U / mg) to 9 mL of sodium acetate solution (pH 5.2) and stir well at low temperature for 10 min. After stirring, centrifuge at 8000 g for 2 min, transfer the supernatant (900 μL) to a centrifuge tube, and then add 100 μL of amyloglucosidase solution (500 U / mL) to prepare a mixed enzyme solution. Weigh 0.2 g of the freeze-dried sample and add it to 10 mL of sodium acetate solution (pH 5.2). Take 1 mL and add 0.2 mL of the above enzyme solution, and react in an oscillating incubator at 37 °C (200 r / min). At 0 min, 20 min, and 120 min, respectively, take 200 μL and terminate the reaction in a boiling water bath. Centrifuge the reaction solution at 10000 g for 2 minutes to obtain the supernatant, and use the GOD-POD kit to measure the glucose content in the hydrolysis product after starch digestion. According to the measured data of G0 (free glucose content), G20 (glucose amount released within 20 min of digestion), and G120 (glucose amount released within 120 min of digestion), calculate the glucose content in the corresponding sample through the following formula:

[0088]

[0089] In the formula, A t is the absorbance value of the solution to be measured; V t is the total volume (mL) of the solution to be measured; C is the concentration of standard glucose (mg / mL); A s is the absorbance value of standard glucose; W t is the weight (mg) of the sample; D is the dilution factor.

[0090] Calculate the contents of the three components in the product. The specific calculation formulas are as follows:

[0091] RDS(%) = (G 20 - G0) × 0.9

[0092] SDS(%) = (G 120 - G 20 ) × 0.9

[0093] RS(%) = 100% - RDS(%) - SDS(%)

[0094] The results are shown in Table 2. Through the analysis of the RDS, SDS, and RS contents of corn starch, potato starch, and cassava starch after being treated with PtGBE, it is found that the RDS contents of the three samples all decreased, and the RS contents all increased significantly. Among them, the change in the RS content of cassava starch was the most obvious, and the RS content of cassava starch increased to 73.81%, indicating that the mutant PtGBE can significantly increase the content of resistant components in starch and is applicable to the food industry field for producing resistant starch.

[0095] Table 2 Effects of mutant PtGBE on starch modification

[0096]

[0097] Note: Different letters in the same column indicate significant differences between the data (P < 0.05).

[0098] Example 7 Effects of starch branching enzyme on dextrin modification

[0099] Dextrin is formed by the degradation and rearrangement of starch under the action of heat, acid, or enzyme, and is a key intermediate in the preparation of resistant dextrin. In this example, the effect of starch branching enzyme on dextrin was explored to further study the value of starch branching enzyme in the preparation of resistant dextrin.

[0100] Accurately weigh 2.5 g of dextrin sample, dissolve it in 100 mL of PBS (pH 7.0, 10 mmol / L) buffer solution by heating and dissolving. After incubating the dissolved dextrin in a constant temperature water bath shaker at 50 °C for 10 min, add 400 U / g of the mutant PtGBE prepared in Example 4 and react for 2 h. After the reaction is completed, heat it to boiling for 20 min to inactivate the reaction, and then freeze-dry the reacted sample.

[0101] Add 0.5 g of porcine pancreatic α-amylase (9.4 U / mg) to 9 mL of sodium acetate solution (pH 5.2) and stir well at low temperature for 10 min. After stirring, centrifuge at 8000 g for 2 min. Transfer the supernatant (900 μL) to a centrifuge tube, and then add 100 μL of amyloglucosidase solution (500 U / mL) to prepare a mixed enzyme solution. Weigh 0.2 g of the freeze-dried sample and add it to 10 mL of sodium acetate solution (pH 5.2). Take 1 mL and add 0.2 mL of the above enzyme solution, and react in an oscillating incubator at 37 °C (200 r / min). At 0 min, 20 min, and 120 min, take 200 μL and terminate the reaction in a boiling water bath. Centrifuge the reaction solution at 10000 g for 2 minutes to obtain the supernatant, and use the GOD-POD kit to measure the glucose content of the hydrolysis product after starch digestion, and calculate the content of each component.

[0102] The results are shown in Table 3. After treatment with PtGBE under the optimal conditions, the RS content of dextrin increased from 85.68% to 91.51%, the SDS content increased from 2.38% to 4.32%, and the RDS content decreased from 11.94% to 4.17%. The decrease in the RDS content while the SDS and RS contents increased indicates that the mutant PtGBE changed the structure of dextrin and reduced the hydrolysis effects of α-amylase and amyloglucosidase, suggesting that the mutant PtGBE is suitable for the food industry field of resistant dextrin.

[0103] Table 3 Effects of mutant PtGBE on dextrin

[0104]

[0105] Note: Different letters in the same column indicate significant differences between the data (P < 0.05).

[0106] The above embodiments are the preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. Starch branching enzyme, characterized in that: It is starch branching enzyme PtGBE or mutant starch branching enzyme PtGBE; wherein, the amino acid sequence of the starch branching enzyme PtGBE is shown in SEQ ID NO.1, and the amino acid sequence of the mutant starch branching enzyme PtGBE is shown in SEQ ID NO.

3.

2. The biological material related to the starch branching enzyme described in claim 1, characterized in that: It is any one of the following biological materials: (1) A DNA molecule encoding the starch branching enzyme PtGBE or mutant starch branching enzyme PtGBE; (2) An expression cassette containing the DNA molecule described in (1); (3) A recombinant vector containing the expression cassette described in (2); (4) A recombinant bacterium containing the recombinant vector described in (3).

3. The related biological material of the starch branching enzyme according to claim 2, wherein: The nucleotide sequence of the DNA molecule encoding the starch branching enzyme PtGBE is shown in SEQ ID NO.2; The nucleotide sequence of the DNA molecule encoding the mutant starch branching enzyme PtGBE is shown in SEQ ID NO.4; The expression vector used for the recombinant vector is pET-28a(+); The expression bacterium used for the recombinant bacterium is Escherichia coli BL21(DE3).

4. The preparation method of the starch branching enzyme described in claim 1, characterized in that: It includes the following steps: introducing the DNA molecule encoding the starch branching enzyme PtGBE or mutant starch branching enzyme PtGBE into the pET-28a(+) expression vector, then transforming it into Escherichia coli BL21(DE3), inducing the expression of mutant PtGBE, and purifying and preparing the starch branching enzyme PtGBE or mutant starch branching enzyme PtGBE by metal chelating chromatography.

5. The application of the starch branching enzyme described in claim 1 in modified starch.

6. The application of the starch branching enzyme according to claim 5 in modified starch, wherein: The application includes the following steps: dissolving starch in a buffer solution, heating and gelatinizing it under stirring, incubating the gelatinized starch, then adding the starch branching enzyme, reacting, heating and boiling to inactivate and terminate the reaction after the reaction is completed, and freeze-drying the reacted sample.

7. The application of the starch branching enzyme according to claim 6 in modified starch, wherein: The buffer solution is a PBS buffer solution with pH 7.0 and 10 mmol / L; The conditions for heating and gelatinizing are: temperature 90 ± 5°C, time 30 ± 5 min; The conditions for incubation are: water bath shaker, temperature 50 ± 5°C, time 10 ± 2 min; The conditions for the reaction are: time 2 ± 0.5 h; The conditions for inactivation are: time 20 ± 5 min; The addition amount of the starch branching enzyme is 400 ± 100 U / g; The starch is at least one of corn starch, cassava starch, and potato starch.

8. The application of the starch branching enzyme described in claim 1 in modified dextrin.

9. The application of the starch branching enzyme according to claim 8 in modified dextrin, wherein: The application described above includes the following steps: Dissolve dextrin in a buffer solution, incubate, then add the starch branching enzyme described above, react, and after the reaction is completed, heat to boiling for inactivation to terminate the reaction, and freeze-dry the reacted sample.

10. The application of the starch branching enzyme in modifying dextrin according to claim 9, characterized in that: The buffer solution is PBS buffer solution with pH 7.0 and 10 mmol / L; The conditions for incubation are: water bath shaker, temperature 50 ± 5 °C, time 10 ± 2 min; The conditions for the reaction are: time 2 ± 0.5 h; The conditions for inactivation are: time 20 ± 5 min; The addition amount of the starch branching enzyme is 400 ± 100 U / g.

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