Starch branching enzyme mutant with improved thermal stability and its application

Abstract

The present invention relates to the field of biotechnology, and specifically, to a mutant of a starch branching enzyme derived from a microorganism and its use. The present invention discloses a mutant of a starch branching enzyme (GBE), wherein the amino acid sequence of the starch branching enzyme mutant is relative to the amino acid sequence of a wild-type starch branching enzyme, and one or more of the amino acid residues at positions 155, 280, 282, 408 or 536 are mutated into amino acids suitable for improving the thermal stability of the starch branching enzyme; the amino acid sequence of the wild-type starch branching enzyme includes the sequence shown in SEQ ID NO: 6. The mutant of the starch branching enzyme disclosed in the present invention has significantly improved thermal stability, which helps to solve the problem of GBE being unstable and inactivated at high temperatures during practical applications.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a mutant of starch branching enzyme and use thereof. Background Art

[0002] Starch branching enzyme (1,4-α-glucan branching enzyme, GBE, EC 2.4.1.18) belongs to the glycoside hydrolase 13 family (GH13). Starch branching enzyme can usually use different starches as substrates, catalyze the cleavage of α-1,4-glycosidic bonds, produce short chains with non-reducing ends, and under the action of transglycosidation, connect the short chains to the receptor chains and form α-1,6-glycosidic bonds. Starch modified by starch branching enzyme will have better application value, mainly manifested in better stability, better anti-digestion properties, better anti-regeneration, better slow digestibility, etc.

[0003] The fine structure changes of starch involved in starch modification require the accumulation of starch branching enzymes at higher temperatures for a longer period of time, which often requires starch branching enzymes to have good thermal stability. At the same time, the appropriate use of high temperature in the starch industrial modification process has the advantages of accelerating the reaction rate, reducing pollution, and increasing the solubility of reactants. Therefore, it is urgent to find a starch branching enzyme with better stability to meet the needs of starch industrial modification and help starch be better used in the food and pharmaceutical industries. Summary of the invention

[0004] In order to solve the problem of poor thermal stability of starch branching enzyme (1,4-α-glucan branching enzyme, GBE, EC2.4.1.18) in the prior art, the present invention takes GBE derived from Aquifex aeolicus VF5 as the target, and based on the amino acid sequence of starch branching enzyme GBE and the characteristics of its own natural structure, completes molecular modification from two aspects. First, taking the loop region of GBE as the target, combining the kinetic flexibility weakness of GBE through consensus mutation, the non-conservative amino acids at the conservative sites of the loop region are mutated into conservative amino acids to complete the determination of the mutation site (conservative amino acids refer to the amino acid sequences of 1372 starch branching enzymes in the database. After alignment, 70% of the amino acids at the same position are the same amino acid, and this type of amino acid is considered to be conservative amino acids). Secondly, in view of the fact that GBE has no natural disulfide bonds, the natural disulfide bond formation rules ((1) the number of residues between any two mutation sites is greater than 25; (2) the disulfide bond formed involves the loop region of GBE; (3) the χ3 torsion angle is near -87° and +97°; (4) the disulfide bond energy value is as small as possible and should not be greater than 2.2 kcal·mol-1; (5) the distance between the β carbon atoms of the two bonded Cys is generally within 2.5 km; (6) the distance between the β carbon atoms of the two bonded Cys is generally within 2.5 km; (7) the distance between the β carbon atoms of the two bonded Cys is generally within 2.5 km; (8) the distance between the β carbon atoms of the two bonded Cys is generally within 2.5 km; (9) the distance between the β carbon atoms of the two bonded Cys is generally within 2.5 km; ( (6) The α carbon atom of the two bonded Cys is within The mutation sites were determined by combining Disulfide by Design 2.0 semi-rational design, and finally the thermally stable mutants GBE2 and GBE5 were obtained. The thermal half-inactivation time t 1 / 2 The results showed that the enzyme activity of starch branching enzyme increased by 300% and 324% respectively, which further promoted the application of starch branching enzyme in food and medicine.

[0005] The above five mutation sites of the present invention are screened out by combining different algorithms. In the process of molecular modification in the prior art, it is easy to improve thermal stability after modification, but the catalytic activity decreases significantly, resulting in the mutant having little application value. The mutants obtained in the present invention, such as H408C-W536C, have improved catalytic activity and thermal stability, which is an innovation.

[0006] To achieve the above-mentioned object and other related objects, the present invention provides a mutant of starch branching enzyme, wherein the amino acid sequence of the mutant of starch branching enzyme is relative to the amino acid sequence of wild-type starch branching enzyme, and one or more of the amino acid residues at positions 155, 280, 282, 408 or 536 are mutated into amino acids suitable for improving the stability of starch branching enzyme; the amino acid sequence of the mutant of starch branching enzyme includes SEQ ID NO: 4 and SEQ ID NO: 5. The amino acid sequence of the wild-type starch branching enzyme includes the sequence shown in SEQ ID NO: 6.

[0007] In any embodiment of the present invention, the mutant of the starch branching enzyme has improved thermal stability compared with the wild-type starch branching enzyme.

[0008] In any embodiment of the present invention, compared with the wild-type starch branching enzyme, the amino acids at positions 155, 280 and 282 of the mutant of starch branching enzyme are replaced with amino acids suitable for forming a rigid structure of the protein.

[0009] In any embodiment of the present invention, compared with the wild-type starch branching enzyme, the 408th and 536th amino acids of the mutant of starch branching enzyme are replaced with amino acids suitable for forming a disulfide bond.

[0010] In any embodiment of the present invention, the nucleotide sequence of the mutant of starch branching enzyme includes those shown in SEQ ID NO: 2 and SEQ ID NO: 3.

[0011] The present invention also provides the use of the mutant of the starch branching enzyme in the preparation of modified starch products.

[0012] The present invention also provides a nucleotide encoding the mutant of starch branching enzyme described above. In a specific embodiment of the present invention, the nucleotide sequence of the mutant includes SEQ ID NO:2 and SEQ ID NO:3, and the amino acid sequence is shown in SEQ ID NO:4 and SEQ ID NO:5.

[0013] The present invention further provides a construct comprising the nucleotide described above, wherein the construct is a microorganism.

[0014] The present invention further provides a host, comprising the construct or gene in which the exogenous nucleotide is integrated, wherein the host is a microorganism, a cell or a virus.

[0015] The present invention further provides a method for producing starch branching enzyme, comprising culturing the host under conditions suitable for the expression of the mutant of the starch branching enzyme to obtain the starch branching enzyme.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: in view of the fact that GBE itself has no natural disulfide bonds, the present invention mutates some of the amino acids located in the ring region into cysteine ​​to form disulfide bonds by changing the amino acid sequence, and the formation of disulfide bonds increases the rigidity of the ring region in this area, further improving the stability of the starch branching enzyme mutant. The present invention takes the ring region of GBE as the transformation target, and mutates some of the non-conservative amino acids located at the conservative sites of the ring region into conservative amino acids by changing the amino acid sequence. The mutated amino acids increase the van der Waals force between different amino acids, improve the flexibility at the mutation site, and thus improve the stability of the starch branching enzyme mutant. By mutating the 408th and 536th positions of GBE to cysteine ​​to form disulfide bonds, the stability of the enzyme is improved, and the thermal half-inactivation time of the mutated starch branching enzyme is increased by about 300% compared with the wild-type starch branching enzyme. By mutating the amino acid at position 155, 280, 282, 408 or 536 of GBE, an excellent mutant V155L-V280I-D282N-H408C-W536C was obtained. The thermal half-inactivation time of the starch branching enzyme of this mutant was increased by about 324% compared with the wild-type starch branching enzyme. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Shown is a schematic diagram of the electrophoresis results of the H408C-W536C mutant purification;

[0018] Among them, lane 1 is the supernatant of H408C-W536C fermentation,

[0019] Lane 2 is the supernatant after ammonium sulfate precipitation of H408C-W536C.

[0020] Lane 3 is the re-dissolution of H408C-W536C ammonium sulfate precipitation.

[0021] Lane 4 is H408C-W536C before loading on the column, and lane 5 is H408C-W536C breakthrough.

[0022] Lane 6: H408C-W536C washed with 0.1M Nacl Tris-Hcl (pH=7.5).

[0023] Lane 7: H408C-W536C washed with 0.2M Nacl Tris-Hcl (pH=7.5).

[0024] Lane 8: H408C-W536C washed with 0.4M Nacl Tris-Hcl (pH=7.5).

[0025] Lane 9: H408C-W536C washed with 0.6M Nacl Tris-Hcl (pH=7.5).

[0026] Lane 10: H408C-W536C washed with 0.8M Nacl Tris-Hcl (pH=7.5).

[0027] Lane 11: H408C-W536C 1M Nacl Tris-Hcl (pH=7.5) wash.

[0028] Figure 2 Shown is a schematic diagram of the electrophoresis results of the V155L-V280I-D282N-H408C-W536C mutant purification;

[0029] Among them, lane 1 is the fermentation broth of V155L-V280I-D282N-H408C-W536C,

[0030] Lane 2 is the fermentation supernatant of V155L-V280I-D282N-H408C-W536C,

[0031] Lane 3 is the supernatant after ammonium sulfate precipitation of V155L-V280I-D282N-H408C-W536C.

[0032] Lane 4 is the re-dissolution of the ammonium sulfate precipitation of V155L-V280I-D282N-H408C-W536C.

[0033] Lane 5 is V155L-V280I-D282N-H408C-W536C penetration,

[0034] Lane 6: V155L-V280I-D282N-H408C-W536C washed with 0.1M Nacl Tris-Hcl (pH=7.5).

[0035] Lane 7: V155L-V280I-D282N-H408C-W536C washed with 0.2M Nacl Tris-Hcl (pH=7.5).

[0036] Lane 8: V155L-V280I-D282N-H408C-W536C washed with 0.4M Nacl Tris-Hcl (pH=7.5).

[0037] Lane 9: V155L-V280I-D282N-H408C-W536C washed with 0.6M Nacl Tris-Hcl (pH=7.5).

[0038] Lane 10: V155L-V280I-D282N-H408C-W536C washed with 0.8M Nacl Tris-Hcl (pH=7.5).

[0039] Lane 11: V155L-V280I-D282N-H408C-W536C 1M Nacl Tris-Hcl (pH=7.5) wash,

[0040] Lane 12: V155L-V280I-D282N-H408C-W536C 2M Nacl Tris-Hcl (pH=7.5) wash.

[0041] Figure 3 Shown is a schematic diagram of the relative enzyme activity assay results of starch branching enzyme mutants.

[0042] Figure 4 Lanes 1-4 are schematic diagrams of the purification electrophoresis results of the wild-type starch branching enzyme and its mutants V155L, V280I and D282N.

[0043] Figure 5 Lanes 1-6 are schematic diagrams of the purification electrophoresis results of V155L-V280I, V155L-D282N, V280I-D282N, V155L-V280I-D282N, H408C-W536C and V155L-V280I-D282N-H408C-W536C, respectively.

[0044] Figure 6 Schematic diagram showing the percentage of remaining enzyme activity of starch branching enzyme mutants and wild-type starch branching enzyme after incubation at 85°C for different time periods.

[0045] Figure 7 Graph showing the percentage of remaining enzyme activity of starch branching enzyme mutants and wild-type starch branching enzyme after incubation at 60-90°C for 10 min.

[0046] Figure 8 Shown are the results of an in vitro digestion simulation assay of potato starch modified with starch branching enzymes. DETAILED DESCRIPTION

[0047] In order to make the purpose of the invention, technical scheme and beneficial effects of the present invention clearer, the present invention is further described below in conjunction with examples. It should be understood that the following examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified, and those familiar with the technology can easily understand other advantages and effects of the present invention from the contents disclosed in this description.

[0048] The first aspect of the present invention provides a mutant of starch branching enzyme, wherein the amino acid sequence of the mutant of starch branching enzyme is relative to the amino acid sequence of wild-type starch branching enzyme, and one or more of the amino acid residues at positions 155, 280, 282, 408 or 536 are mutated into amino acids suitable for improving the stability of starch branching enzyme. The base sequence of wild-type starch branching enzyme includes SEQ ID NO: 1, and the amino acid sequence is SEQ ID NO: 6. Compared with the wild-type starch branching enzyme, the mutant of starch branching enzyme of the present invention can improve the stability of starch branching enzyme produced by Bacillus subtilis.

[0049] The stability of starch branching enzyme is characterized by the thermal half-inactivation time and thermal half-inactivation temperature after treatment at 85°C for 30 minutes. The thermal stability is expressed by the residual enzyme activity by measuring the reaction enzyme activity.

[0050] Specifically, the calculation formula of enzyme activity is:

[0051] Enzyme activity calculation formula:

[0052] Enzyme activity (U / mL) = (OD 660 - Experimental group OD 660 ) / control group OD 660 / reaction time × dilution factor * 100. Specifically, the calculation formula for the remaining enzyme activity is:

[0053] Thermal half-inactivation time (t 1 / 2): The wild-type starch branching enzyme and the mutant of starch branching enzyme were incubated at 85°C and samples were taken at regular intervals, and then quickly cooled on ice for 5 minutes. The residual enzyme activity of the wild-type starch branching enzyme and the mutant of starch branching enzyme was determined according to the above enzyme activity calculation formula, and the activity of the unincubated enzyme solution was set as 100%.

[0054] Thermal half-inactivation temperature (T 50 ): The wild-type starch branching enzyme and the mutants of starch branching enzyme were incubated at different temperatures (60-90°C) for 10 minutes, then samples were taken and quickly cooled on ice for 5 minutes. The residual enzyme activity of the wild-type starch branching enzyme and the mutants of starch branching enzyme was determined according to the above enzyme activity calculation formula, and the activity of the unincubated enzyme solution was set as 100%.

[0055] In the mutant of starch branching enzyme provided by the present invention, compared with the wild-type starch branching enzyme, the amino acids at positions 155, 280 and 282 are replaced with amino acids suitable for forming a rigid structure of protein. The amino acids suitable for forming a rigid structure of protein include a combination of one or more of leucine, isoleucine and asparagine.

[0056] In the mutant of starch branching enzyme provided by the present invention, compared with the wild-type starch branching enzyme, the 408th and 536th amino acids are replaced by amino acids suitable for forming disulfide bonds. The amino acid suitable for forming disulfide bonds is cysteine.

[0057] In a specific embodiment of the present invention, in the mutant of starch branching enzyme, compared with the wild-type starch branching enzyme, the histidine at position 408 is mutated to cysteine, and the tryptophan at position 536 is mutated to cysteine. The obtained mutant is GBE2 (H408C-W536). Compared with the wild-type starch branching enzyme, the stability of this mutant is improved by 300%, and the catalytic activity is increased to a certain extent, and it has good industrialization prospects.

[0058] In a specific embodiment of the present invention, in the mutant of starch branching enzyme, compared with the wild-type starch branching enzyme, the valine at position 155 is mutated to leucine, the valine at position 280 is mutated to isoleucine, the aspartic acid at position 282 is mutated to asparagine, the histidine at position 408 is mutated to cysteine, and the tryptophan at position 536 is mutated to cysteine. The obtained mutant is GBE5 (V155L-V280I-D282N-H408C-W536C). Compared with the wild-type starch branching enzyme, the mutant has a heat half-inactivation t of 85°C. 1 / 2 The catalyst activity increased by 324% and decreased slightly, so it has good industrialization prospects.

[0059] In a specific embodiment of the present invention, the vector is the commercial plasmid pHT01.

[0060] The second aspect of the present invention provides a nucleotide encoding the mutant of starch branching enzyme described in the first aspect. In a specific embodiment of the present invention, the nucleotide sequence of the mutant of starch branching enzyme includes SEQ ID NO: 2 and SEQ ID NO: 3, and the amino acid sequence is SEQ ID NO: 4 and SEQ ID NO: 5.

[0061] The third aspect of the present invention provides a construct comprising the nucleotides described above.

[0062] The fourth aspect of the present invention provides a host comprising the construct or gene described in the third aspect into which the exogenous nucleotide described in the second aspect is integrated, wherein the host is a microorganism.

[0063] The construct is transformed, transduced or transfected into the host by conventional methods in the art, such as chemical transformation by calcium chloride method, high voltage electric shock transformation. More preferably, the host is a genetically engineered bacterium commonly used in the art, such as Bacillus subtilis. In a specific embodiment of the present invention, Bacillus subtilis can be, for example, conventional hosts such as Bs168, DB403, and WB600. The above-mentioned Bacillus subtilis needs to be able to express a mutant of starch branching enzyme, so as to provide conditions for the existence of the mutant of starch branching enzyme. Suitable methods for constructing the above-mentioned Bacillus subtilis should be known to those skilled in the art.

[0064] The fifth aspect of the present invention provides a method for producing starch branching enzyme, comprising culturing the host described in the fourth aspect under conditions suitable for the expression of the starch branching enzyme mutant described in the first aspect to obtain starch branching enzyme. In a specific embodiment of the present invention, the production method comprises the following steps: inducing the host to express starch branching enzyme in the presence of the aforementioned starch branching enzyme mutant. Suitable induction expression methods should be known to those skilled in the art. The induction method of the present invention mainly ferments in LB liquid culture medium for 8-12 hours, then transfers to TB liquid culture medium with an inoculation amount of 2% and ferments for 48 hours, then centrifuges the TB culture medium fermented for 48 hours at 8000rpm for 10 minutes, discards the supernatant, and resuspends the bacteria with an equal volume of yeast culture medium, then ferments for 60-72 hours, and finally centrifuges at 12000rpm for 30 minutes to obtain crude starch branching enzyme.

[0065] The sixth aspect of the present invention provides the use of the mutant of the starch branching enzyme described in the first aspect in the preparation of modified starch products. The mutant of the starch branching enzyme provided by the present invention can improve the stability of the starch branching enzyme, especially the stability under high temperature conditions, thereby better helping to improve the utilization rate of starch.

[0066] The invention of the present invention is further described below by way of examples, but the scope of the present invention is not limited thereto.

[0067] Unless otherwise stated, the experimental methods, detection methods and preparation methods disclosed in the present invention all adopt conventional molecular biology, biochemistry, analytical chemistry, bacterial culture, recombinant DNA technology and related techniques in the technical field.

[0068] Example 1

[0069] Construction of starch branching enzyme site-directed mutant strain

[0070] Amplify the gene sequence GBE of starch branching enzyme: Using pHT01-GBE (the vector is pHT01, and the gene GBE is inserted into the vector through BamHI and SmaI restriction sites) constructed in the early stage of the laboratory as a template, design the primers required for the experiment (sequence see Table 1) for PCR amplification. The primers are synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.

[0071] Table 1 Primer names and sequence names

[0072]

[0073] 1 The underlined bases correspond to the corresponding mutated amino acids

[0074] The PCR reaction system was slightly modified from the conditions set in the STAR Primer Kit instructions: 5× PrimeSTARBuf fer (Mg 2+ Plus) 10μL, template DNA 1μL, forward and reverse primers (10μM) 4μL, PrimeSTAR HS DNA Polymerase (2.5U / μL) 0.5μL, dNTPs (2.5mM each) 4μL, and finally ultrapure water 26.5μL. PCR amplification conditions: 98℃ 1min30s; 18 cycles (98℃ 30s, 51℃ 15s, 72℃ 9min10s); 72℃ 5min. After electrophoresis verification and recovery of the electrophoresis gel, the purified gbe gene fragment was obtained.

[0075] The gbe gene fragment purified above was digested with Dpn I enzyme, and the digestion product was purified by column. The purified product was transformed into E. coli DH5α, coated on LB solid plate containing Amp (final concentration 100 μg / ml), cultured for 16 hours, and then subjected to colony PCR detection and sent to the company for sequencing.

[0076] Example 2

[0077] Fermentation of starch branching enzyme producing strains

[0078] The recombinant plasmid sequenced correctly in Example 1 of the present invention was used to transform Bacillus subtilis DB403. The single clone transformant was selected and placed in LB medium supplemented with chloramphenicol (25 mg / ml), cultured overnight at 37°C, and then transferred to TB medium and inoculated at a transfer volume of 2%. 600 When the protein grows to 0.6-0.8, IPTG (final concentration is 1mM) is added to induce expression. Then continue to culture for 24 hours, transfer the culture medium into a sterile centrifuge tube and centrifuge at 8000r / min for 10 minutes to precipitate the bacteria, discard the supernatant, resuspend the bacteria with YA medium, and continue to culture for 72 hours. Centrifuge at 12000r / min for 2 minutes to precipitate the bacteria, and the resulting supernatant is the crude enzyme solution, which is stored in a 4°C refrigerator.

[0079] The culture medium used was as follows:

[0080] LB medium: Tryptone: 10 g / L, Yeast extract: 5 g / L, NaCl: 10 g / L, Agar (added when preparing LB solid plates): 20 g / L, mixed with deionized water, and sterilized at 121°C for 20 min.

[0081] TB medium: Tryptone: 12 g / L, Yeast extract: 24 g / L, glycerol: 10 g / L, add deionized water and stir evenly, sterilize at 121°C and high temperature and high pressure for 20 min.

[0082] YA medium: Yeast extract: 5g / L, add deionized water and stir evenly, sterilize at 121℃ high temperature and high pressure for 20min.

[0083] The transformation method of Bacillus subtilis is as follows:

[0084] (1) Strain recovery: Take all Bacillus subtilis out of the -80°C freezer, streak them on LB solid plates with a sterile toothpick, and invert them in a 37°C incubator for 16 h.

[0085] (2) Culture: Pick a single clone and activate it in 10 mL LB liquid culture medium. Place it in a shaker at 37°C and culture at 200 rpm for 16 h.

[0086] (3) Take 200 μL of bacterial solution and add it to SPI medium. Place it in a 37°C shaker and culture at 200 rpm until OD600 = 0.8.

[0087] (4) Take 200 μL of bacterial solution and add it to SPII medium that has been pre-warmed at 37°C. Incubate the culture in a shaker at 37°C and 200 rpm for 90 min.

[0088] (5) Immediately add 20 μL of EGTA to the SPII medium of the culture solution and culture at 37°C, 200 rpm, and shake on a shaker for 10 min.

[0089] (6) Take 200 μL from step 5 and add it to a 1.5 mL EP tube that was previously incubated at 37°C and contained 0.1 μg-0.3 μg DNA, and incubate at 37°C, 200 r / min for 1 h. Negative control: Take 200 μL and add it to a 1.5 mL EP tube that was previously incubated at 37°C but contained DNA, and incubate at 37°C, 200 r / min for 1 h.

[0090] (7) Add 100 μL of liquid LB medium to the 1.5 mL EP tube in step 6 and culture at 37°C and 200 rpm for 1 h.

[0091] (8) Place the 1.5 mL EP tube from step 7 in a high-speed centrifuge and centrifuge at 5000 rpm for 5 min.

[0092] (9) Discard the supernatant and keep 100 μL of the bacterial suspension, pipette it onto a resistance plate (chloramphenicol plate with a final concentration of 25 μg / mL), spread it evenly with a glass coating rod, place the plate upside down in a 37°C incubator and culture it for 16 h. Count the transformants the next day.

[0093] Example 3

[0094] Purification of starch branching enzyme mutants and activity assay

[0095] According to the method described in Example 2 of the present invention, the fermented broth fermented for 72 hours was centrifuged at 8000 rpm for 30 minutes, the precipitate was removed and the supernatant was retained. Then, ammonium sulfate was slowly added to the supernatant at a ratio of 0.22 g / ml. The supernatant was placed at 4° C. for about 10 minutes. The mixed solution after adding ammonium sulfate was centrifuged at 8000 rpm for 30 minutes, the supernatant was removed, and the mixture was reconstituted with an equal volume of 20 mm Tris. After the pH of the complex solution was adjusted to 7.5, it was purified using an anion exchange column. The eluents used for purification were: 0.1M NaclTris-Hcl (pH = 7.5), 0.2M Nacl Tris-Hcl (pH = 7.5), 0.4M Nacl Tris-Hcl (pH = 7.5, 0.6M Nacl Tris-Hcl (pH = 7.5), 0.8M Nacl Tris-Hcl (pH = 7.5), 1M Nacl Tris-Hcl (pH = 7.5), and 2M Nacl Tris-Hcl (pH = 7.5). Pure samples of H408C-W536C and V155L-V280I-D282N-H408C-W536C proteins were obtained. The results are as follows: Figure 1 and2 As shown. Subsequently, the mutant proteins of the purified starch branching enzyme were subjected to enzymatic kinetic analysis and the structures are shown in Table 2. The catalytic activity determination and SDS-PAGE results of the purified starch branching enzyme mutants V155L, V280I, D282N, V155L-V280I, V155L-D282N, V280I-D282N, V155L-V280I-D282N, H408C-W536C and V155L-V280I-D282N-H498C-W536C are shown in Figures 3 to 5 .

[0096] Table 2 Enzymatic kinetics of wild-type starch branching enzyme and starch branching enzyme mutants

[0097]

[0098] The starch-iodine colorimetric method was used to determine the activity of the mutant of starch branching enzyme.

[0099] Definition of enzyme activity: The amount of enzyme required to reduce the absorbance by 1% per minute at 660 nm is one unit of enzyme activity.

[0100] The specific enzyme activity calculation formula is as follows:

[0101] Experimental group: For the experimental samples to be tested, centrifuge at 12000r / min for 2min, take 50μL of the supernatant, add 50μL of 0.4% potato starch, react in a constant temperature metal bath at 80℃ at 200r / min for 20min, and immediately add 2mL of iodine colorimetric solution to terminate the reaction; take 200ul of the sample and add it to a 96-well plate, and use an enzyme reader to measure the OD 660 .

[0102] Control group: 50 μL of 0.4% potato starch was mixed with 2 mL of iodine solution and reacted for 30 min in the dark. Then 50 μL of 0.4% potato starch solution was added and 200 μL was transferred to a 96-well plate. The OD was measured using an ELISA reader. 660 .

[0103] Enzyme activity (U / mL) = (OD 660 - Experimental group OD 660 ) / control group OD 660 / reaction time×dilution factor*100.

[0104] The enzymatic kinetics assay method is as follows:

[0105] Potato starch solutions with concentrations of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% and 10% (w / v) were prepared, and a Line Weaver-Burk equation curve was drawn with the reciprocal of the substrate concentration as the abscissa and the reciprocal of the maximum reaction rate as the ordinate according to the enzyme activity determination method, and Km, Kcat and Kcat / Km were calculated and compared.

[0106] Figure 3 The results showed that among different mutants, there was no significant difference in the relative enzyme activity of single-site (V155L and V280I) and starch branching enzyme GBE. The relative enzyme activity of other mutants, whether single-site (D282N), double-site (V155L-V280I, V155L-D282N and V280I-D282N), triple-site (V155L-V280I-D282N) or five-site (V155L-V280I-D282N-H408C-W536C) mutants, decreased by 15%, 23%, 28%, 33%, 27% and 15% respectively compared with GBE, while the relative enzyme activity of H408C-W536C was increased by 15% compared with GBE. It is further explained that the catalytic activity of the mutants H408C-W536C and V155L-V280I-D282N-H408C-W536C was not significantly reduced after molecular modification, and they were still able to maintain good catalytic activity. The higher catalytic activity is beneficial in reducing the increase in production costs caused by the increase in the amount of enzyme added in the catalytic reaction due to the decrease in enzyme catalytic activity in the actual production application process.

[0107] Example 4

[0108] Analysis of the thermostability of starch branching enzyme mutants

[0109] The method used to detect the stability of the mutants of starch branching enzyme is to determine the thermal half-inactivation time and the thermal half-inactivation temperature, wherein the thermal half-inactivation time is determined by warming the wild-type starch branching enzyme and the mutants of starch branching enzyme at 85°C and taking samples at certain intervals, quickly cooling them on ice for 5 minutes, and determining the residual enzyme activity of the wild-type starch branching enzyme and the mutants of starch branching enzyme according to the enzyme activity calculation formula in Example 3 of the present invention, and taking the activity of the uninsulated enzyme solution as 100%. The thermal half-inactivation temperature is determined by warming the wild-type starch branching enzyme and the mutants of starch branching enzyme at different temperatures (60-90°C) for 10 minutes, taking samples, quickly cooling them on ice for 5 minutes, and determining the residual enzyme activity of the wild-type starch branching enzyme and the mutants of starch branching enzyme according to the above enzyme activity calculation formula, and taking the activity of the uninsulated enzyme solution as 100%. The activity is used to obtain its stability, and the results are as follows: Figures 6-7 The specific changes are shown in Table 3.

[0110] The results showed that the mutants of starch branching enzyme V155L, V280I, D282N, V155L-V280I, V155L-D282N, V280I-D282N and V155L-V280I-D282N obtained by the present invention had a t 1 / 2 Compared with GBE, the t 1 / 2 Compared with GBE, the improvement is larger, which is 50min and 56min respectively. The T of the starch branching enzyme mutants V155L, V280I, D282N, V155L-V280I, V155L-D282N, V280I-D282N and V155L-V280I-D282N obtained by the present invention is 50 Compared with GBE, the T values ​​of the mutants H408C-W536C and V155L-V280I-D282N-H408C-W536C of starch branching enzyme obtained by the present invention were increased by 2.33℃, 1.69℃, 0.84℃, 2.45℃, 1.73℃, 2.02℃ and 2.7℃ respectively. 50 Compared with GBE, the improvement is larger, increasing by 3.55℃ and 4.15℃ respectively.

[0111] Table 3 Thermal half-inactivation temperature T of wild-type starch branching enzyme and starch branching enzyme mutants 50 Thermal half-inactivation time t 1 / 2 Changes

[0112]

[0113] In summary, the present invention applies computational biology and structural biology to predict the amino acids that may form disulfide bonds in starch branching enzymes and to find flexible weaknesses for design. The corresponding amino acids are mutated by point mutation. The stability of the mutants H408C-W536C and V155L-V280I-D282N-H408C-W536C is increased by 300% and 342%, respectively. It can better adapt to the needs of starch modification in the food field and has broad market prospects.

[0114] Example 5

[0115] In vitro digestion simulation of starch modified by starch branching enzyme

[0116] The in vitro enzymatic digestion simulation characteristics of potato starch and modified starch after the action of starch branching enzyme GBE were analyzed. The specific method is as follows: dilute 40 μL of glucosidase to 2.0 mL with deionized water; weigh 4.0 g of porcine pancreatic α-amylase and dissolve it in 26 mL of deionized water, stir and mix, centrifuge at 4 ° C (1000r, 5min), and transfer the supernatant to a pre-cooled beaker; mix 1.2 mL of deionized water, 1.8 mL of glucosidase solution and 18.0 mL of pancreatic amylase as the final enzyme solution, which is mixed and used immediately. Weigh 200 mg of sample and dissolve it in a reactor containing 15 mL of sodium acetate buffer (200 mM, pH 5.2). Keep warm at 37°C for 5 minutes, then add 5.0 mL of fresh mixed enzyme solution and react at 37°C and 150 rpm. Take out 0.5 mL of reaction solution every 20 minutes and add (four times the volume of ethanol for inactivation) or treat it in a boiling water bath for 10 minutes. The hydrolyzed glucose content is determined by a glucose kit. For specific operations, see the instructions. The contents of fast-digestible starch RDS, slowly-digestible starch SDS and resistant-digestible starch RS are calculated according to the following formula:

[0117] RDS(%)=(G20-FG)×0.9×100

[0118] SDS(%)=(G120-G20)×0.9×100

[0119] RS(%)=(TG-FG)×0.9×100-RDS(%)-SDS(%)

[0120] Among them: FG is the glucose content in the system before enzymatic hydrolysis, TG is the total glucose content in the system, G20 and G120 are the glucose contents in the system after 20 minutes and 120 minutes of enzymatic hydrolysis, respectively, and RDS, SDS, and RS are fast-digestible starch, slowly-digestible starch, and resistant starch, respectively. Figure 8 As shown, after modification, the contents of slowly digestible starch and resistant starch in potato starch increased by 4.5% and 12.75% respectively compared with the unmodified state, and the content of quickly digestible starch decreased by 16.75%.

[0121] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

[0122] The nucleotide sequence of GBE is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 6.

[0123]

[0124] The nucleotide sequence of GBE2 is shown in SEQ ID NO:2, and the amino acid sequence is shown in SEQ ID NO:4.

[0125]

[0126] The nucleotide sequence of GBE5 is shown in SEQ ID NO:3, and the amino acid sequence is shown in SEQ ID NO:5.

[0127] ATGAAAAAATTCTCTCTTATCTCTGATTACGATGTTTACCTTTTCAAAGAAGGCACACATACACGTCTTTACGATAAACTTGGCTCTCATGTTATCGAACTTAACGGCAAACGTTACACATTCTTCGCTGTTTGGGCTCCTCATGCTGATTACGTTTCTCTTATCGGCGATTTCAACGAATGGGATAAAGGCTCTACACCTATGGTTAAACGTGAAGATGGCTCTGGCATCTGGGAAGTTCTTCTTGAAGGCGATCTTACAGGCTCTAAATACAAATACTTCATCAAAAACGGCAACTACGAAGTTGATAAATCTGATCCTTTCGCTTTCTTCTGCGAACAACCTCCTGGCAACGCTTCTGTTGTTTGGAAACTTAACTACCGTTGGAACGATTCTGAATACATGAAAAAACGTAAACGTGTTAACTCTCATGATTCTCCTATCTCTATCTACGAAGTTCATCTTGGCTCTTGGCGTCGTGTTCCTGAAGAAGGCAACCGTTTCCTTTCTTACCGTGAACTTGCTGAATACCTTCCTTACTACGTTAAAGAAATGGGCTTCACACATGTTGAATTCCTTCCTGTTATGGAACATCCTTTCTACGGCTCTTGGGGCTACCAAATCACAGGCTACTTCGCTCCTACATCTCGTTACGGCACACCTCAAGATTTCATGTACCTTATCGATAAACTTCATCAAGAAGGCATCGGCGTTATCCTTGATTGGGTTCCTTCTCATTTCCCTACAGATGCTCATGGCCTTGCTTACTTCGATGGCACACATCTTTACGAATACGAAGATTGGCGTAAACGTTGGCATCCTGATTGGAACTCTTTCATTTTCAATTACGGCAAACCTGAAGTTCGTTCTTTCTTTCTTTCTTCTGCTCATTTCTGGCTTGATAAATACCATGCTGATGGCCTTCGTGTTGATGCTGTTGCTTCTATGCTTTACCTTGATTACT(GBE5,SEQ ID NO:3);

[0128] MKKFSLISDYDVYLFKEGTHTRLYDKLGSHVIELNGKRYTFFAVWAPHADYVSLIGDFNEWDKGSTPMVKREDGSGIWEVLLEGDLTGSKYKYFIKNGNYEVDKSDPFAFFCEQPPGNASVVWKLNYRWNDSEYMKKRKRVNSHDSPISIYEVHVGSWRRVPEEGNRFLSYRELAEYLPYYVKEMGFTHVEFLPVMEHPFYGSWGYQITGYFAPTSRYGTPQDFMYLIDKLHQEGIGVILDWVPSHFPTDAHGLAYFDGTHLYEYEDWRKRWHPDWNSFVFDYGKPEVRSFFLSSAHFWLDKYHADGLRVDAVASMLYLDYSRKEWVPNIYGGKENLEAIEFLRKFNESVYRNFPDVQTIAEESTAWPMVSRPTYVGGLGFGMKWNMGWMNDTLFYFSKDPIYRKYHCEVLTFSIWYAFSENFVLSLSHDEVVHGKGSLIGKMPGDYWQKFANLRALFGYMWAHPGKKLLFMGGEFGQFKEWDHETSLDWHLLEYPSYRGIQRLVKDLNEVYRREKALHETDFSPEGFEWVDFHDCEKSVISFLRKDKSGKEIILVVCNFTPVPCYDYRVGVPKGGYWREIMNTDAKEYWGSGMGNLGGKEADKIPWHGRKFSLSLTLPPLSVIYLKHEG(GBE2,SEQ ID NO:4);

[0129] MKKFSLISDYDVYLFKEGTHTRLYDKLGSHVIELNGKRYTFFAVWAPHADYVSLIGDFNEWDKGSTPMVKREDGSGIWEVLLEGDLTGSKYKYFIKNGNYEVDKSDPFAFFCEQPPGNASVVWKLNYRWNDSEYMKKRKRVNSHDSPISIYEVHLGSWRRVPEEGNRFLSYRELAEYLPYYVKEMGFTHVEFLPVMEHPFYGSWGYQITGYFAPTSRYGTPQDFMYLIDKLHQEGIGVILDWVPSHFPTDAHGLAYFDGTHLYEYEDWRKRWHPDWNSFIFNYGKPEVRSFFLSSAHFWLDKYHADGLRVDAVASMLYLDYSRKEWVPNIYGGKENLEAIEFLRKFNESVYRNFPDVQTIAEESTAWPMVSRPTYVGGLGFGMKWNMGWMNDTLFYFSKDPIYRKYHCEVLTFSIWYAF SENFVLSLSHDEVVHGKGSLIGKMPGDYWQKFANLRALFGYMWAHPGKKLLFMGGEFGQFKEWDHETSLDWHLLEYPSYRGIQRLVKDLNEVYRREKALHETDFSPEGFEWVDFHDCEKSVISFLRKDKSGKEIILVVCNFTPVPCYDYRVGVPKGGYWREIMNTDAKEYWGSGMGNLGGKEADKIPWHGRKFSLSLTLPPLSVIYLKHEG(GBE5,SEQ ID NO:5)。

[0130] MKKFSLISDYDVYLFKEGTHTRLYDKLGSHVIELNGKRYTFFAVWAPHADYVSLIGDFNEWDKGSTPMVKREDGSGIWEVLLEGDLTGSKYKYFIKNGNYEVDKSDPFAFFCEQPPGNASVVWKLNYRWNDSEYMKKRKRVNSHDSPISIYEVHVGSWRRVPEEGNRFLSYRELAEYLPYYVKEMGFTHVEFLPVMEHPFYGSWGYQITGYFAPTSRYGTPQDFMYLIDKLHQEGIGVILDWVPSHFPTDAHGLAYFDGTHLYEYEDWRKRWHPDWNSFVFDYGKPEVRSFLLSSAHFWLDKYHADGLRVDAVASMLYLDYSRKEWVPNIYGGKENLEAIEFLRKFNESVYRNFPDVQTIAEESTAWPMVSRPTYVGGLGFGMKWNMGWMNDTLFYFSKDPIYRKYHHEVLTFSIWYAFSENFVLPLSHDEVVHGKGSLIGKMPGDYWQKFANLRALFGYMWAHPGKKLLFMGGEFGQFKEWDHETSLDWHLLEYPSHRGIQRLVKDLNEVYRREKALHETDFSPEGFEWVDFHDWEKSVISFLRKDKSGKEIILVVCNFTPVPRYDYRVGVPKGGYWREIMNTDAKEYWGSGMGNLGGKEADKIPWHGRKFSLSLTLPPLSVIYLKHEG(GBE,SEQ ID NO:6)。

Claims

1. A mutant of starch branching enzyme, characterized in that The amino acid sequence of the starch branching enzyme mutant is shown in SEQ ID NO:4 or SEQ ID NO:

5.

2. A nucleotide, characterized in that It encodes the mutant of starch branching enzyme as claimed in claim 1.

3. The nucleotide according to claim 2, characterized in that The nucleotide sequence of the mutant of starch branching enzyme is shown in SEQ ID NO: 2 or SEQ ID NO:

3.

4. A construct, characterized in that Containing the nucleotide according to claim 2 or 3.

5. A host, characterized in that The construct or gene according to claim 4 contains an exogenous nucleotide according to claim 2 or 3, wherein the host is a microorganism, a cell or a virus.

6. A method for producing starch branching enzyme, characterized in that: The method comprises culturing the host according to claim 5 under conditions suitable for expressing the mutant of the starch branching enzyme according to claim 1 to obtain the starch branching enzyme.

7. Use of the mutant of starch branching enzyme as claimed in claim 1 in the preparation of modified starch products.

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