Method for improving thermal stability of glycogen branching enzyme and use thereof

Abstract

Disclosed are a method for improving the thermal stability of a glycogen branching enzyme and the use thereof, which belong to the field of genetic engineering techniques and enzyme engineering. Disclosed is a glycogen branching enzyme mutant with an improved thermal stability. The enzyme activity of a single mutant is 1.14-1.45 times that of a wild-type enzyme, and the apparent melting temperature thereof is improved by 0.3-3.4°C. After incubation at 95°C for 15 h, a combined mutant retains 100% of an original enzyme activity. For a modified starch prepared by means of using the glycogen branching enzyme mutant disclosed, the ratio of the α-1,6 glycosidic bond in the modified starch prepared by means of using the mutant is 1.08-1.92 times higher than that of a modified starch prepared by mean of using a wild-type enzyme, and is 1.39-2.68 times higher than that of the original corn starch. The mutant provided has an increased optimal temperature and thermal stability, and a significantly increased degree of starch branching, and can be used to realize industrial production of a branched starch at a high temperature of 70°C, so that the branching reaction and starch swelling can be performed simultaneously.

Description

A method for improving the thermal stability of glycogen branching enzyme and its application Technical Field

[0001] The present invention relates to a method for improving the thermal stability of glycogen branching enzyme and its application, in particular to a glycogen branching enzyme mutant with improved thermal stability and its application in starch modification, belonging to the fields of genetic engineering technology and enzyme engineering. Background Art

[0002] Starch is a naturally abundant polymer synthesized by plants during photosynthesis. It is the largest energy storage substance and primary energy source in plants. Its diverse sources and wide range of applications offer promising applications. Natural starch possesses unique properties not found in similar substances like cellulose and pectin, such as viscosity, rheology, and gelling properties. It can be used as a thickener, stabilizer, and colloid gelling agent, finding widespread application in the biopharmaceutical, construction, and food industries. In the food industry, starch is a key raw material and ingredient, playing an irreplaceable role in the texture and sensory properties of processed foods. However, during food processing and application, starch undergoes a series of structural and property changes caused by processing techniques, which can irreversibly damage the quality, taste, and appearance of food. Therefore, it is necessary to modify natural starch to address its inherent shortcomings. Modified highly branched starch can significantly improve the solubility, viscosity, retrogradation, rheology, and digestibility of native starch.

[0003] There are four basic methods for modifying starch to achieve high branching: chemical, physical, genetic, and enzymatic. Physical starch modification is simple, economical, environmentally friendly, and highly safe. However, the approach is limited. Chemical starch modification requires the use of chemical reagents to alter the solubility, gelatinization, and aging properties of the native starch. However, the safety concerns associated with the use of these chemical reagents warrant attention. Genetic modification, primarily based on transgenic technology, results in increased starch branching and decreased amylose content. However, genetic modification is difficult to control and therefore not widely used. Enzymatic modification, due to its wide variety of sources (e.g., microorganisms, plants, and animals), strong substrate specificity, environmental friendliness, high yield, and high product yield, is gaining popularity in industrial modification. Due to the high gelatinization temperature of starch, the starch modification process requires enzymes to maintain good activity at high temperatures. Therefore, there is an urgent need to identify glycogen branching enzymes with high heat resistance and activity. However, most natural glycogen branching enzymes are heat-sensitive, have poor thermal stability, and are sensitive to environmental changes, making them difficult to fully meet the needs of industrial development. Therefore, it is of great significance to improve the thermal stability of glycogen branching enzymes through enzyme engineering technology.

[0004] Among the existing technologies, there are methods to improve the thermal stability of α-1,4-glucan branching enzyme by genetic engineering: for example, Ban, Liu and Zhang et al. obtained T by truncating the C-terminus of α-1,4-glucan branching enzyme from Geobacillus thermoglucosidans STB02. mThe mutant GBEΔC (Thermostabilization of a thermophilic 1,4-α-glucan branching enzyme through C-terminal truncation. International Journal of Biological Macromolecules, 107, 1510–1518.) was improved by 4°C; Ban, Wang and Fan et al. obtained the mutant K137E by constructing salt bridges through B-factor analysis of α-1,4 glucan branching enzyme from Geobacillus thermoglucosidans STB02, and the half-life at 60°C was increased by about 36.6% (Thermostability and catalytic ability enhancements of 1,4-α-glucan branching enzyme by introducing salt bridges at flexible amino acid sites. International Journal of Biological Macromolecules, 224, 1276–1282.); Li, Ban et al. obtained the mutant K137E by constructing salt bridges through B-factor analysis of α-1,4 glucan branching enzyme from Geobacillus thermoglucosidans STB02, and the half-life at 60°C was increased by about 36.6% (Thermostability and catalytic ability enhancements of 1,4-α-glucan branching enzyme by introducing salt bridges at flexible amino acid sites. International Journal of Biological Macromolecules, 224, 1276–1282.); Li, Ban et al. obtained the mutant K137E by constructing salt bridges through B-factor analysis of α-1,4 glucan branching enzyme from Geobacillus thermoglucosidans STB02 The rational design of disulfide bonds in 1,4-glucan branching enzyme resulted in mutants V75C and T77C, which showed a 1.5-fold improvement in heat resistance at 65°C (Rational Design of Disulfide Bonds for Enhancing the Thermostability of the 1,4-α-Glucan Branching Enzyme from Geobacillus thermoglucosidans STB02. Journal of Agricultural and Food Chemistry, 68(47), 13791–13797.). However, due to the high gelatinization temperature of starch, existing glycogen branching enzymes cannot meet the industrial requirements of long time and high temperature. Therefore, the development of highly heat-resistant glycogen branching enzymes will significantly improve the application value of glycogen branching enzymes in starch modification, and will be more conducive to the industrial production of branched starch, improve the digestibility, solubility, viscosity and degree of retrogradation of native starch, and broaden its application in the food field.

[0005] Summary of the Invention

[0006] [Technical Issues]

[0007] During starch modification, the starch gelatinization temperature is high, and the existing glycogen branching enzymes cannot meet the requirements of industrial production of highly branched starch, which requires a long time, high temperature and high enzyme activity.

[0008] [Technical solution]

[0009] The inventors previously discovered that the glycogen branching enzyme (PDB: 5WU7) from Pyrococcus horikoshii OT3 has a large enzyme production and good enzyme activity, and its optimal temperature is 60°C, which is close to the starch gelatinization temperature.

[0010] The present invention characterizes the wild-type glycogen branching enzyme from Pyrococcus horikoshii OT3 and the mutants of the present invention, and finds that the single or combined mutants greatly improve the heat resistance of the glycogen branching enzyme, and the apparent melting temperature (T m ) increased by up to 3.4°C, and the optimum temperature increased by up to 10°C. Enzyme activity also increased to varying degrees. This is beneficial for improving the degree of starch modification and increasing the yield of highly branched starch, potentially enabling the application of branched starch with slow digestion, delayed retrogradation, and high solubility in the food industry. Furthermore, 70°C has reached the gelatinization temperature of ordinary starch, which can intensify the swelling of starch granules, facilitate the entry and contact of enzymes with the starch interior, maximize the branching effect, and achieve simultaneous branching modification and gelatinization swelling, simplifying the modification process and saving industrial energy consumption.

[0011] The present invention provides a glycogen branching enzyme mutant with improved thermal stability and an engineered Escherichia coli bacterium capable of expressing the glycogen branching enzyme mutant.

[0012] The present invention provides a glycogen branching enzyme mutant, which is obtained by mutating at least one amino acid among positions 140, 206, 331, 334, 415, 427, 444, 484 and 508 of the glycogen branching enzyme.

[0013] The glycogen branching enzyme is a glycogen branching enzyme having an amino acid sequence as shown in SEQ ID NO.1; or a glycogen branching enzyme having at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the amino acid sequence shown in SEQ ID NO.1; or a glycogen branching enzyme gene having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% sequence identity with the coding sequence shown in SEQ ID NO.2.

[0014] In one embodiment of the present invention, the mutation comprises substitution, deletion or addition.

[0015] In one embodiment of the present invention, the mutant has at least one of the following amino acid substitutions in the amino acid sequence of SEQ ID NO. 1:

[0016] (1) mutating valine at position 140 to isoleucine;

[0017] (2) mutating lysine at position 206 to proline;

[0018] (3) mutating glycine at position 331 to phenylalanine;

[0019] (4) mutating leucine at position 334 to glutamic acid;

[0020] (5) mutating histidine at position 415 to tryptophan;

[0021] (6) mutating proline at position 427 to arginine;

[0022] (7) mutating tyrosine at position 444 to leucine;

[0023] (8) Mutation of methionine at position 484 to arginine;

[0024] (9) The threonine at position 508 was mutated to lysine.

[0025] In one embodiment of the present invention, the mutant is a mutant in which the valine at position 140 of the glycogen branching enzyme shown in the amino acid sequence of SEQ ID NO. 1 is mutated to isoleucine; the mutant is named: V140I.

[0026] In one embodiment of the present invention, the mutant is a mutant in which the lysine at position 206 of the glycogen branching enzyme shown in the amino acid sequence of SEQ ID NO. 1 is mutated to proline; the mutant is named: K206P.

[0027] In one embodiment of the present invention, the mutant is obtained by mutating the glycine at position 331 of the glycogen branching enzyme with the amino acid sequence as shown in SEQ ID NO. 1 to phenylalanine; the mutant is named: G331F.

[0028] In one embodiment of the present invention, the mutant is a mutant in which the leucine at position 334 of the glycogen branching enzyme shown in the amino acid sequence of SEQ ID NO. 1 is mutated to glutamic acid; the mutant is named: L334E.

[0029] In one embodiment of the present invention, the mutant is a mutant in which the histidine at position 415 of the glycogen branching enzyme shown in the amino acid sequence of SEQ ID NO. 1 is mutated to tryptophan; the mutant is named: H415W.

[0030] In one embodiment of the present invention, the mutant is a mutant in which the proline at position 427 of the glycogen branching enzyme shown in the amino acid sequence of SEQ ID NO. 1 is mutated to arginine; the mutant is named: P427R.

[0031] In one embodiment of the present invention, the mutant is a mutant in which the tyrosine at position 444 of the glycogen branching enzyme shown in the amino acid sequence of SEQ ID NO. 1 is mutated to leucine; the mutant is named: Y444L.

[0032] In one embodiment of the present invention, the mutant is a mutant in which the methionine at position 484 of the glycogen branching enzyme shown in the amino acid sequence of SEQ ID NO.1 is mutated to arginine; the mutant is named: M484R.

[0033] In one embodiment of the present invention, the mutant is a mutant in which the threonine at position 508 of the glycogen branching enzyme shown in the amino acid sequence of SEQ ID NO. 1 is mutated to lysine; the mutant is named: T508K.

[0034] In one embodiment of the present invention, the mutant is a glycogen branching enzyme whose amino acid sequence is as shown in SEQ ID NO.1, wherein the valine at position 140 is mutated to isoleucine and the histidine at position 415 is mutated to tryptophan; it is named: V140I+H415W.

[0035] In one embodiment of the present invention, the mutant is a glycogen branching enzyme whose amino acid sequence is as shown in SEQ ID NO.1, wherein the valine at position 140 is mutated to isoleucine and the threonine at position 508 is mutated to lysine; and is named: V140I+T508K.

[0036] In one embodiment of the present invention, the mutant is a glycogen branching enzyme whose amino acid sequence is as shown in SEQ ID NO.1, wherein the histidine at position 415 is mutated to tryptophan and the threonine at position 508 is mutated to lysine; and is named: H415W+T508K.

[0037] In one embodiment of the present invention, the mutant is a glycogen branching enzyme whose amino acid sequence is as shown in SEQ ID NO.1, wherein the valine at position 140 is mutated to isoleucine, the histidine at position 415 is mutated to tryptophan, and the threonine at position 508 is mutated to lysine; and is named: V140I+H415W+T508K.

[0038] The present invention provides a gene encoding the above mutant.

[0039] The present invention provides a recombinant vector for expressing the mutant or the gene.

[0040] In one embodiment of the present invention, the expression vector is a pET series, Duet series, pGEX series, pHY300, pHY300PLK, pPIC3K or pPIC9K series vector.

[0041] In one embodiment of the present invention, the expression vector is pET29b(+).

[0042] The present invention provides a recombinant cell expressing the mutant, or containing the gene, or containing the recombinant vector.

[0043] In one embodiment of the present invention, the recombinant cell uses a prokaryotic cell or a eukaryotic cell as an expression host.

[0044] In one embodiment of the present invention, the prokaryotic host cell can be any Gram-positive or Gram-negative bacteria. Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Thuringia, Neisseria, Pseudomonas, Salmonella, and Ureaplasma; the eukaryotic cell is a fungal cell.

[0045] In one embodiment of the present invention, the recombinant cell uses Escherichia coli BL 21 (DE3) as an expression host.

[0046] The present invention provides a method for preparing the above mutant, the method comprising the following steps:

[0047] (1) Designing mutation primers for the mutation site based on the mutation site, performing site-directed mutagenesis using a vector carrying the glycogen branching enzyme encoding gene as a template, and constructing an expression vector containing the gene encoding the mutant;

[0048] (2) transforming the expression vector containing the gene encoding the mutant into E. coli BL 21 (DE3) as the expression host;

[0049] (3) Select positive clones for expansion culture, collect cells by centrifugation, and use a cell disruptor to crush the cells. The supernatant is the crude enzyme solution of the glycogen branching enzyme mutant;

[0050] (4) The crude enzyme solution of glycogen branching enzyme is injected into a protein purifier, and the pure enzyme solution is obtained by gradient elution.

[0051] The present invention provides a method for improving the thermal stability of glycogen branching enzyme, wherein the method comprises the following steps: mutating the isoleucine at position 38 of the glycogen branching enzyme of the amino acid sequence as shown in SEQ ID NO.1 to leucine; mutating the valine at position 140 of the glycogen branching enzyme of the amino acid sequence as shown in SEQ ID NO.1 to isoleucine; or mutating the lysine at position 206 of the glycogen branching enzyme of the amino acid sequence as shown in SEQ ID NO.1 to proline; or mutating the glycine at position 331 of the glycogen branching enzyme of the amino acid sequence as shown in SEQ ID NO.1 to phenylalanine; or mutating the leucine at position 334 of the glycogen branching enzyme of the amino acid sequence as shown in SEQ ID NO.1 to glutamic acid; or mutating the histidine at position 415 of the glycogen branching enzyme of the amino acid sequence as shown in SEQ ID NO.1 to tryptophan; or mutating the proline at position 427 of the glycogen branching enzyme of the amino acid sequence as shown in SEQ ID NO.1 to arginine; or mutating the tyrosine at position 444 of the glycogen branching enzyme of the amino acid sequence as shown in SEQ ID NO.1 to leucine; or mutating the tyrosine at position 446 of the glycogen branching enzyme of the amino acid sequence as shown in SEQ ID NO.1 to leucine. The methionine at position 484 of the glycogen branching enzyme shown in SEQ ID NO.1 is mutated to arginine; or the threonine at position 508 of the glycogen branching enzyme shown in SEQ ID NO.1 is mutated to lysine.

[0052] The present invention provides a composition containing the glycogen branching enzyme mutant.

[0053] In one embodiment of the present invention, the composition contains the glycogen branching enzyme as the main enzyme component and one or more adjuvants.

[0054] In one embodiment of the present invention, the adjuvant includes but is not limited to an enzyme stabilizer.

[0055] In one embodiment of the present invention, the enzyme stabilizer comprises mannitol, DTT, trehalose or vitamin C.

[0056] The present invention provides a method for preparing branched starch, which comprises adding the glycogen branching enzyme mutant into amylose.

[0057] In one embodiment of the present invention, the reaction is carried out at 60-70° C. for 10-30 minutes.

[0058] The present invention provides use of the mutant, the gene, the recombinant vector, or the recombinant cell in preparing highly branched starch.

[0059] The present invention provides applications of the mutant, gene, recombinant vector, or recombinant cell in the fields of biochemistry and food. Beneficial effects:

[0060] 1. The present invention discloses a series of glycogen branching enzyme mutants with improved thermal stability. The method of the present invention is used to obtain single or combined mutants with improved thermal stability, which are the glycogen branching enzyme mutants with the highest optimal temperature increase in existing reports. The single mutants of the present invention have improved thermal stability while also improving their own enzyme activity, and the combined mutants have improved thermal stability while retaining the original enzyme activity of the wild type. The enzyme activity of the single mutants V140I, H415W, T508K, K206P, G331F, L334E, P427R, Y444L, and M484R is 1.14 to 1.45 times that of the wild type enzyme, and the apparent melting temperature (T m ) increased by 0.3 to 3.4℃.

[0061] 2. The mutants provided by the present invention have an optimal temperature of 60-70°C and significantly improved thermal stability. The single mutants V140I, H415W, and T508K, and the combined mutants V140I / H415W, V140I / T508K, H415W / T508K, and V140I / H415W / T508K retained 100% of their original enzyme activity after incubation at 95°C for 15 hours. The single mutant T508K retained 10% of its original activity after incubation at 95°C for 60 hours, and the combined mutant H415W / T508K retained 80% of its activity after incubation at 95°C for 60 hours. The remaining combined mutants retained more than 30% of their original enzyme activity after incubation at 95°C for 60 hours. The wild-type enzyme completely lost its activity after incubation at 95°C for 60 hours.

[0062] 3. The λ of amylose after the mutant glycogen branching enzyme provided by the present invention max Compared with wild-type λ max The absorbance also decreased significantly, which catalyzed the production of more branched chains in amylose.

[0063] 4. In the modified starch prepared using the mutant glycogen branching enzyme provided by the present invention, the mutant has a 1.08-1.92-fold higher ratio of α-1,6 glycosidic bonds than the modified starch prepared using the wild enzyme, and a 1.39-2.68-fold higher ratio of α-1,6 glycosidic bonds than the original corn starch.

[0064] 5. Compared with the wild enzyme, the mutant provided by the present invention has improved the optimal temperature and thermal stability, and significantly increased the branching degree of starch, enabling industrial production of branched starch at a high temperature of 70°C, so that the branching reaction and starch swelling can proceed simultaneously, thereby simplifying the modification steps, reducing the water consumption required for production and the energy consumption required for drying, and has good industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1: SDS-PAGE of wild-type glycogen branching enzyme and mutant enzymes after purification; wherein, M in the figure is a protein molecular weight standard, 1: purified wild-type glycogen branching enzyme, 2-17: purified mutants, in order: F24H, I38L, R83E, G109K, V140I, N165I, K206P, G331F, L334E, A367V, H415W, P427R, Y444L, E465Q, M484R, T508K, 2-5: V140I / H415W, V140I / T508K, H415W / T508K, V140I / H415W / T508K

[0066] Figure 2: T of wild-type glycogen branching enzyme and its mutants m and enzyme activity characterization.

[0067] Figure 3: Characterization of the optimal reaction temperature of wild-type glycogen branching enzyme and its single mutants.

[0068] Figure 4: Characterization of the optimal reaction temperature of wild-type glycogen branching enzyme and its combined mutants.

[0069] Figure 5: Characterization of the optimal reaction pH of wild-type glycogen branching enzyme and its single mutants.

[0070] Figure 6: Characterization of the optimal reaction pH of wild-type glycogen branching enzyme and its combined mutants.

[0071] Figure 7: Characterization of the thermal stability of wild-type glycogen branching enzyme and its mutants at 95°C.

[0072] Figure 8: Analysis of iodine binding capacity of wild-type glycogen branching enzyme and its mutants. DETAILED DESCRIPTION

[0073] Technical terms:

[0074] Glycogen branching enzyme: The term "glycogen branching enzyme" refers to an enzyme in the EC 2.4.1.18 class as defined in the enzyme nomenclature. For the purposes of the present invention, the activity of "glycogen branching enzyme" is determined according to the procedures described in the Examples. In one aspect, the glycogen branching enzyme of the present invention is a glycogen branching enzyme having an amino acid sequence as set forth in SEQ ID NO. 1; or a glycogen branching enzyme having at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 1; or a glycogen branching enzyme gene having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to the coding sequence set forth in SEQ ID NO. 2.

[0075] Expression: The term "expression" includes any step involved in the production of the glycogen branching enzyme mutant including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0076] Expression vector: The term "expression vector" means a linear or circular DNA molecule comprising a polynucleotide encoding a glycogen branching enzyme mutant of the present invention and operably linked to control sequences that provide for its expression.

[0077] Host cell: The term "host cell" means any cell type that is susceptible to transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term "host cell" encompasses any progeny of a parent cell that is not completely identical to the parent cell due to mutations that occur during replication.

[0078] The host cell can be any cell useful in the recombinant production of a glycogen branching enzyme mutant, such as a prokaryotic or eukaryotic cell.

[0079] Prokaryotic host cells can be any gram-positive or gram-negative bacteria. Gram-positive bacteria include but are not limited to: Bacillus, Clostridium, Enterococcus, Geobacillus (Geobacillus), Lactobacillus, Lactococcus, Ocean Bacillus, Staphylococcus, Streptococcus and Streptomyces. Gram-negative bacteria include but are not limited to Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Silene Bacillus, Neisseria, Pseudomonas, Salmonella and Ureaplasma.

[0080] The host cell may also be a eukaryotic organism, such as a mammalian, insect, plant, or fungal cell.

[0081] Amylose: It is a linear long-chain molecule composed of hundreds to thousands of glucose monomers connected by α-D-1,4 glycosidic bonds (99%), and contains a small amount of α-D-1,6 branches (1.0%). The α-D-1,6 glycosidic bonds at the branch points account for 0.3% to 0.5% of the total glycosidic bonds, and its degree of polymerization is 324 to 4920.

[0082] Branched starch: "Branched starch" and "modified starch" can be used interchangeably. It is a multi-branched soluble polymer carbohydrate composed of D-glucose. The main chain is composed of D-glucose groups connected by α-1,4 glycosidic bonds. There is a branch for every 24 to 30 D-glucose groups, and the branch points are connected to the main chain by α-1,6 glycosidic bonds.

[0083] The main reagents involved in the following examples: E. coli BL21 (DE3) and BCA concentration determination kit were purchased from Beyotime Biotechnology Co., Ltd., amylose was purchased from Sigma, corn starch was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., and other commonly used reagents were all domestically produced analytical grade.

[0084] The culture media involved in the following examples are as follows: The culture media were all prepared with ultrapure water and sterilized in an autoclave at 121° C. for 20 min after preparation.

[0085] LB liquid medium: yeast extract 0.5 g / 100 mL, tryptone 1.0 g / 100 mL, NaCl 1.0 g / 100 mL.

[0086] LB solid medium: Based on LB liquid medium, agar powder 1.5g / 100mL.

[0087] LB liquid resistance medium: Based on LB liquid medium, kanamycin was added to 100 mL of liquid medium to a final concentration of 30 μg mL -1 .

[0088] LB solid medium: Based on LB solid medium, kanamycin was added to 100 mL of solid medium to a final concentration of 30 μg mL -1 .

[0089] The purified solutions of the enzymes involved in the following examples are as follows:

[0090] (1) Loading buffer: Weigh 2.42 g Tris-HCl, 29.22 g NaCl, and 1.36 g imidazole, dissolve in ultrapure water, and adjust the volume to 1 L (pH = 7.4).

[0091] (2) Elution buffer: Weigh 2.42 g Tris-HCl, 29.22 g NaCl, and 34.04 g imidazole, dissolve in ultrapure water, and adjust the volume to 1 L (pH = 7.4).

[0092] (3) Resuspension: Weigh 2.42 g Tris-HCl and 29.22 g NaCl, dissolve in ultrapure water and adjust the volume to 1 L (pH = 7.4).

[0093] The loading buffer is used to wash away impurities, and the eluent is used to elute and collect the target protein to obtain the purified enzyme.

[0094] The detection methods involved in the following embodiments are as follows:

[0095] Enzyme activity assay:

[0096] Take 200 μl of 1 mg / mL amylose solution in a 5 ml centrifuge tube and place it in a water bath shaker for 5 minutes. Then add 14 μg of enzyme and shake it in a water bath shaker at 150 rpm. Under certain temperature and pH conditions (enzyme activity is determined according to the optimal temperature and optimal pH of the corresponding enzyme measured in the following examples), react for 10 minutes. Add 500 μl of 1 mmol / L hydrochloric acid to quench the reaction. Then add 1 ml of iodine solution and color it in the dark for 5 minutes. Measure the absorbance change at 660 nm and calculate the enzyme activity.

[0097] Enzyme activity was defined as the amount of enzyme that caused the absorbance to decrease by 0.01 optical density units per minute at 660 nm.

[0098] Iodine solution preparation: 0.5 mL of stock iodine solution (containing 0.26 g I2 and 2.6 g KI in water) and 1 mL of 1 mol / L HCl were diluted to 130 mL using ultrapure water.

[0099] T m Value determination:

[0100] A differential scanning microcalorimeter (Nano DSC) was used. The purified enzyme solution was diluted to 0.5 mg / ml and degassed with mercury at 635 nm for 10 minutes. 300 μl of the enzyme solution was added to the Nano DSC sample chamber. The scanning temperature was set to 60-120°C and the temperature was increased at a rate of 1°C / min. The temperature corresponding to the observed peak was fitted using Nano analysis software and was obtained as T. m value.

[0101] Thermal stability determination:

[0102] Place the enzyme at an appropriate concentration in a water bath at 95°C for 15, 30, 45 and 60 hours, respectively. Measure the enzyme activity at the corresponding time periods using the above enzyme activity determination steps.

[0103] Determination of amylose and iodine complexes:

[0104] Take 200 μl of 1 mg / mL amylose solution (pH = 6.0) in a 5 ml centrifuge tube, place it in a water bath shaker at 60°C or 70°C, keep warm for 5 minutes, add 200 μl of diluted enzyme solution, shake and mix, and react for 15 minutes in a water bath shaker at 60°C or 70°C, 150 rpm, add 500 μl of 1 mmol / L hydrochloric acid to quench the reaction, then add 1 ml of iodine solution, protect from light and develop color for 5 minutes, and then use a microplate reader to scan the absorption spectrum of the amylose-iodine complex at 500-800 nm and determine its maximum absorption wavelength.

[0105] Determination of α-1,6 glycosidic bond ratio:

[0106] Using nuclear magnetic resonance spectroscopy ( 1 H NMR) was used to determine the relative content of α-1,6 glycosidic bonds in corn starch and modified starch. 40 mg of corn starch and modified starch were weighed and dissolved in 0.5 mL of heavy water (D2O) and vortexed in a boiling water bath for 30 min. The gelatinized sample was freeze-dried for 72 h and then redissolved in 0.5 mL of heavy water (D2O) to prepare an 8% (w / v) solution. The solution was heated to 60°C. 1 The α-1,6 glycosidic bond content was determined by H NMR, which showed that the α-1,6 glycosidic bond content was around 4.97 ppm, and the α-1,4 glycosidic bond content was around 5.22 ppm.

[0107] Example 1: Construction of mutants

[0108] (1) Construction of a recombinant vector containing the wild-type glycogen branching enzyme Ph GBE gene

[0109] Based on the wild-type glycogen branching enzyme Ph GBE, NCBI accession number PDB: 5WU7 (amino acid sequence shown in SEQ ID NO. 1), the E. coli encoding gene was optimized and sent to Anshengda Biotechnology Co., Ltd. for gene synthesis and recombinant plasmid. The vector was plasmid pET29b(+), with restriction enzyme sites NdeI and XhoI, respectively, to obtain the wild-type recombinant plasmid pET29b-GBE.

[0110] (2) Construction of mutant recombinant vector

[0111] Site mutation primers were designed, and site-directed mutagenesis was performed using the wild recombinant plasmid obtained in step (1) as a template to obtain recombinant plasmids pET29b-F24H, pET29b-I38L, pET29b-R containing mutants F24H, I38L, R83E, G109K, V140I, N165I, K206P, G331F, L334E, A367V, H415W, P427R, Y444L, E465Q, M484R and T508K, respectively. 83E, pET29b-G109K, pET29b-V140I, pET29b-N165I, pET29b-K206P, pET29b-G331F, pET29b-L334E, pET29b-A367V, pET29b-H415W, pET29b-P427R, pET29b-Y444L, pET29b-E465Q, pET29b-M484R, and pET29b-T508K.

[0112] The primer sequences used for mutation are as follows:

[0113] The primers for introducing the F24H mutation point are:

[0114] F24H-F:5'-GCAAATGGCCACATGGTGAAGAATGGTTATTTGAAGCC-3';

[0115] F24H-R:5'-CACCATGTGGCCATTTGCCATGTTTGCGAACATACG-3';

[0116] The primers for introducing the I38L mutation point are:

[0117] I38L-F:5'-CGGAAAGCTACCTTCCACTGTTAATGGAGCTGG-3';

[0118] I38L-R:5'-GGAAGGTAGCTTTCCGCCATGGCTTCAAATAACC-3';

[0119] The primers for introducing the R83E mutation point are:

[0120] R83E-F:5'-GGAAGAGAAACTGAAAAGCATGGAAGAAGATCTGGAGC-3';

[0121] R83E-R:5'-CCATGCTTTTCAGTTTTCTCTCCATATATTTTTCAAATTCGCGC-3';

[0122] The primers for introducing the G109K mutation point are:

[0123] G109K-F:5'-TTTTATGATTAAGTACTTCAAGGATGTGTACAGCTACTGGAAGAGT-3';

[0124] G109K-R:5'-TCCTTGAAGTACTTAATCATAAAATTAATGGCCTCGCGCAGCTTTT-3';

[0125] The primers for introducing the V140I mutation point are:

[0126] V140I-F:5'-GTTATGTGGAAATTATTACCAGCGCGGCCACCCATGG-3';

[0127] V140I-R:5'-GGTAAATAACCATGGGTGGCCGCGCTGGTAATAATTTCC-3';

[0128] The primers for introducing the N165I mutation point are:

[0129] N165I-F:5'-CCCAACTGCTGATTGGCATTAAAGTGTATGAAAAATATTTTGGCCGC-3';

[0130] N165I-R:5'-CCAATCAGCAGTTGGGCCTCAATGGCTTCATCAC-3';

[0131] The primers for introducing the K206P mutation point are:

[0132] K206P-F:5'-CAAGTACCGGTGAAGTGAAGTGGCGCCCAGGCATTG-3';

[0133] K206P-R:5'-CCAAATTTTTTCAGGAAGTGCTCAATGCCTGGGCGCCAC-3';

[0134] The primers for introducing the G331F mutation point are:

[0135] G331F-F:5'-CCAAACATTTCATTTTCCTGGTTCTGAGC-3';

[0136] G331F-R:5'-GGAAAATGAAATGTTTGGCATGTTCATTCACG-3';

[0137] The primers for introducing the L334E mutation point are:

[0138] L334E-F:5'-CATTGGTCTGGTTGAGAGCATTCTGGAGAG-3';

[0139] L334E-R:5'-CTCAACCAGACCAATGAAATGTTTGGCATGTTCATTC-3';

[0140] The primers for introducing the A367V mutation point are:

[0141] A367V-F:5'-GGTTTGAAGGTGTGAAGTGGTTAAGTCGTGTG-3';

[0142] A367V-R:5'-CACACCTTCAAACCACCAGTGGCCAAATAAC-3';

[0143] The primers for introducing the H415W mutation point are:

[0144] H415W-F:5'-GCTGGGGTATGTTTGGCACCCATTGGACCTGGTG-3';

[0145] H415W-R:5'-CACTCAACCTCCGGATTCCACCAGGTCCAATGGG-3';

[0146] The primers for introducing the P427R mutation point are:

[0147] P427R-F:5'-GTGGACCTGGCGTATTATTCATAAAGCGGAAGATCG-3';

[0148] P427R-R:5'-GAATAATACGCCAGGTCCACTCAACCTCCG-3';

[0149] The primers for introducing the Y444L mutation point are:

[0150] Y444L-F:5'-GGTGAGTTTAGCCACCAAGTATTTAGGCAAAG-3';

[0151] Y444L-R:5'-CGGTCACCAAAACTTATCTTTGCCTAAATACTTGG-3';

[0152] The primers for introducing the E465Q mutation point are:

[0153] E465Q-F:5'-TATTATTACAAGCCAGTGATTGGCAGTTCTTAATGACCACGG-3';

[0154] E465Q-R:5'-CTGGCTTGTAATAATAATAACTCACGGGCCAGTTGGG-3';

[0155] The primers for introducing the M484R mutation point are:

[0156] M484R-F:5'-GTAAACGGCGCATTCTGGAGCATGCCC-3';

[0157] M484R-R:5'-GAATGCGCCGTTTACCATATTCTTTGGCTTGGCC-3';

[0158] The primers for introducing the T508K mutation point are:

[0159] T508K-F:5'-GCGGCAAGTTTGATGAAGTGGAACTGCTGAATGAAG-3';

[0160] T508K-R:5'-CCACTTCATCAAACTTGCCGCGTTCAAAATAACGTTC-3';

[0161] The PCR reaction system is as follows:

[0162] Mutation PCR reaction system (20 μl): 0.25 μl original plasmid template (2 ng / μl), 0.5 μl upstream primer F (10 μM), 0.5 μl downstream primer R (10 μM), 10 μl PrimeSTAR Max DNA Polymerase, 8.75 μl ddH2O.

[0163] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 3 min; followed by denaturation at 98°C for 10 s, annealing at 55°C for 5 s, and extension at 72°C for 3 min 30 s, for a total of 30 cycles.

[0164] Combination mutants: Based on the original plasmid template, the primer sequences were the same as above, and the method was the same as above to obtain the combination mutant engineering bacteria: pET29b-V140I / H415W, pET29b-V140I / T508K, pET29b-H415W / T508K and pET29b-V140I / H415W / T508K. Taking pET29b-V140I / H415W as an example, the single mutant plasmid pET29b-V140I was used as a template, and the primers for the mutation site H415W were used to perform whole-plasmid PCR to obtain the combination mutant plasmid pET29b-V140I / H415W.

[0165] According to the above method, the wild engineered bacteria plasmid expressing the original enzyme was extracted.

[0166] (3) Construction of engineered bacteria

[0167] The recombinant plasmid was verified by agarose gel electrophoresis, and the target fragment was recovered and transformed into competent E. coli BL21 (DE3). The transformants were then plated onto LB plates containing kanamycin (30 μg / mL) and incubated inverted at 37°C overnight. Once colonies emerged, individual colonies were transferred to liquid LB medium containing kanamycin (30 μg / mL) and incubated at 37°C, 200 rpm, for 10-16 hours. The culture suspension was then sent to Suzhou Anshengda Biotechnology Co., Ltd. for sequencing. This yielded engineered strains containing either mutants or wild-type enzymes containing the correct mutation point.

[0168] (4) Expression and purification

[0169] The engineered strain containing the recombinant plasmid constructed in step (3) was streaked onto a solid LB plate containing kanamycin (30 μg / mL), inverted and cultured at 37°C for 10 h, a single colony was picked and inoculated into LB liquid medium containing kanamycin (30 μg / mL), and cultured on a shaking platform at 37°C and 200 rpm for 10-16 h to obtain an activated strain. The activated strain was pipetted into LB liquid medium containing kanamycin (30 μg / mL) at a 1% (v / v) inoculum to expand the culture, and cultured on a shaking platform at 37°C and 200 rpm for about 2 h 20 min. After adding IPTG with a final concentration of 0.5 mmol / L and inducing at 30°C for 6 h, the bacteria were collected by centrifugation at 1440 × g for 10 min. Resuspend the cells with the resuspension solution, and ultrasonically disrupt them in an ice bath for 30 minutes at 40% power, ultrasonically for 2 seconds, and then stop for 3 seconds. After disruption, centrifuge at 10,000 rpm for 10 minutes to collect the supernatant, and pass it through a 0.45 μm aqueous membrane to obtain the crude enzyme solution.

[0170] Using His Trap HP 1mL Ni 2+ Protein was purified by column affinity chromatography, and Ni was first equilibrated with loading buffer. 2+The crude enzyme solution was loaded onto the affinity chromatography column at a flow rate of 1 mL / min. After loading, the pure mutant enzyme was obtained by gradient elution with elution buffer.

[0171] Pure enzyme solutions containing wild-type glycogen branching enzyme and various mutant enzymes were prepared respectively.

[0172] Example 2: Determination of enzyme activity and enzymatic properties

[0173] (1) The enzyme activities of the wild-type glycogen branching enzyme and the mutant enzymes F24H, I38L, R83E, G109K, V140I, N165I, K206P, G331F, L334E, A367V, H415W, P427R, Y444L, E465Q, M484R, T508K, V140I / H415W, V140I / T508K, H415W / T508K and V140I / H415W / T508K were determined. The wild-type enzyme activity was defined as 100%, and the relative enzyme activities of the mutant enzymes were detected. The results are shown in Tables 1 and 2 and Figure 2.

[0174] Table 1 Wild type and its mutant T m and relative enzyme activity

[0175] Table 2 ΔH and optimum temperature of wild type and its mutants

[0176] The results showed that the T of 21 sites in the wild-type glycogen branching enzyme and the mutant enzyme was determined by Nano DSC. m values, among which T508K (ΔT m =3.4℃)、H415W(ΔT m =2.1℃)、V140I(ΔT m =1.3℃)、H415W / T508K(ΔT m =2.8℃)、V140I / H415W(ΔT m =2.7°C), and except for the V140I mutant, the optimum temperature of the other mutants increased from 60°C to 70°C. The heat resistance was improved, as shown in Tables 1, 2 and Figure 3.

[0177] Although the enzyme activities of G331F and A367V mutants were significantly improved, m The growth is not obvious, or even decreases.

[0178] Combined with the mutation points that increased relative enzyme activity and optimum temperature, and in combination with starch modification reactions, single-point mutants V140I, H415W, and T508K with superior overall performance were selected, and combined mutants V140I / H415W, V140I / T508K, H415W / T508K, and V140I / H415W / T508K were selected for subsequent experiments.

[0179] (2) Optimum reaction temperature: In order to further explore the characteristics of the mutant enzymes, the single-point mutants V140I, H415W, T508K, and the combined mutants V140I / H415W, V140I / T508K, H415W / T508K, and V140I / H415W / T508K were further studied for their enzymatic properties. 1 mg / ml amylose substrate was prepared with 50 mmol / L sodium acetate buffer (pH = 6.0), and 14 μg of enzyme was added. The enzyme activity was measured after reacting at 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, 90°C, and 95°C for 10 min. As shown in Figures 3 and 4, the optimal reaction temperature for the wild-type enzyme and mutant V140I was 60°C, while the optimal reaction temperature for the other mutants was 70°C. All temperatures are shown in Table 2 and Figures 3 and 4.

[0180] (3) Optimal reaction pH: 1 mg / mL amylose substrate was prepared in the pH range of 4.0-5.0 using 50 mmol / L citrate buffer, 1 mg / mL amylose substrate was prepared in the pH range of 6.0-7.0 using 50 mmol / L sodium acetate buffer, 1 mg / mL amylose substrate was prepared in the pH range of 7.0-8.0 using 50 mmol / L Tris-HCl buffer, and 1 mg / mL amylose substrate was prepared in the pH range of 9.0 using glycine sodium hydroxide buffer. -1 14 μg of enzyme was added to the amylose substrate and the enzyme activity was determined at 60°C for 10 min. As shown in Figures 5 and 6, the optimal reaction pH of the wild enzyme and the mutant were both in the range of 5-7, and the properties were similar.

[0181] (4) Thermal stability of wild-type and mutant enzymes at 95°C

[0182] The results of the enzyme thermostability test at 95°C are shown in Figure 7. The thermostability of the combined mutants is generally higher than that of the single mutants.

[0183] At 95°C, the experimental results showed that after incubation for 15 hours, the wild glycogen branching enzyme retained 80% of its original enzyme activity, after incubation for 30 hours, it retained 50% of its original enzyme activity, and after incubation for 60 hours, it almost lost all its enzyme activity.

[0184] The single mutants V140I, H415W and T508K, and the combined mutants V140I / H415W, V140I / T508K, H415W / T508K and V140I / H415W / T508K retained 100% of the original enzyme activity after incubation at 95°C for 15 h.

[0185] After incubation at 95°C for 15 hours, the mutant T508K still retained 100% of the original enzyme activity. After incubation for 30 hours, it retained 70% of the original enzyme activity. After incubation for 60 hours, it still retained 10% of the original enzyme activity.

[0186] The H415W / T508K mutant retained 100% of its original enzyme activity after incubation at 95°C for 15 hours, 90% after 45 hours, and 80% after 60 hours. The remaining mutant combinations retained more than 30% of their original activity after incubation at 95°C for 60 hours.

[0187] The thermal stability of the combined mutation points is significantly improved. As shown in Tables 1 and 2, although T m The values ​​did not change compared with those of the single-point mutants, but the protein with the combined mutations required more energy (ΔH) to unfold, which may be the reason for its improved thermal stability.

[0188] (5) Kinetic parameters

[0189] Amylose substrates at concentrations of 0.05%, 0.1%, 0.2%, 0.3%, and 0.4% were prepared in 50 mmol / L sodium acetate buffer (pH = 6.0). 14 μg of enzyme was added and the reactions were performed at the optimal reaction temperature. The OD values ​​were then measured and the kinetic parameters of the wild type and mutants were fitted using the Lineweaver-Burk method (Table 3).

[0190] Table 3 Kinetic parameters

[0191] From the data in the table, we can see that the K m The value was lower than that of the wild-type enzyme, indicating that the single mutant had a stronger ability to bind to the substrate amylose. The K m The values ​​are similar to those of the wild-type enzyme, indicating that the combined mutants have similar binding abilities to the substrate amylose, which is consistent with the conclusion of relative enzyme activity. cat / K m They are all larger than those of the wild enzyme, indicating that the catalytic ability of the mutants is improved.

[0192] Example 3: Application of mutants

[0193] (1) Analysis of the combination of amylose and iodine

[0194] The wild-type and mutant enzymes were tested at their optimum temperatures according to the method of step (3) of Example 2. After the wild-type and mutant glycogen branching enzymes reacted with amylose, a 500-800 nm spectrum was scanned in a microplate reader. The results are shown in FIG8 and Table 4.

[0195] The results showed that the amylose-iodine complex λ max At around 646 nm, the absorbance was 0.579. After 15 min of action of the wild enzyme and the mutant enzyme, λ max The absorbance shifted from 646 nm to 581 nm, 574 nm, 569 nm, 577 nm, 565 nm, 569 nm, 570 nm, and 575 nm, respectively. The absorbance shifted from 0.579 to 0.275, 0.246, 0.24, 0.216, 0.229, 0.192, 0.22, and 0.213, respectively. max Compared with the wild enzyme max The absorbance also decreased. This may be because the thermostability of glycogen branching enzyme has increased, while the enzyme activity has not decreased. This results in the mutant enzyme's ability to react with starch at 70°C significantly enhanced compared to the wild-type enzyme, allowing it to better bind to amylose at high temperatures and exert a catalytic effect. This results in more branched chains in the structure.

[0196] Table 4 Analysis of the combination of amylose and iodine

[0197] (2) Preparation of modified starch

[0198] A 20% (w / w) corn starch emulsion was prepared with a 50 mmol / L sodium acetate solution (pH 6.0), to which the prepared wild-type or mutant glycogen branching enzyme was added. The mixture was reacted in a shaking water bath at 70°C at 200 rpm for 10 hours. After completion, the reaction was terminated by a 30-minute boiling water bath. The mixture was then frozen at -80°C for 12 hours, freeze-dried, ground into powder, and passed through a 100-mesh sieve to obtain modified corn starch for later use.

[0199] (3) Relative proportion of α-1,6 glycosidic bonds

[0200] The modified starch prepared in step (2) was taken to determine the relative proportion of its α-1,6 glycosidic bonds. The results showed (as shown in Table 5) that, except for V140I, the branching degree of the modified starch of the other mutant enzymes was significantly higher than that of the wild enzyme modified starch. Among them, the relative proportion of α-1,6 glycosidic bonds of the modified starch of mutant H415W and combined mutant V140I / H415W was 12.50%, which was 2.68 times more than that of ordinary starch and 1.92 times more than that of the wild enzyme modified starch.

[0201] Table 5 Relative proportions of α-1,6 glycosidic bonds

[0202] The above results show that the mutant glycogen branching enzyme has improved thermal stability and retained the original enzyme activity. It has stronger branching ability at high temperature and can improve the digestibility, solubility, viscosity and regeneration of native starch, providing certain help for its application in the food and pharmaceutical industries.

[0203] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A glycogen branching enzyme mutant, characterized in that: The glycogen branching enzyme mutant undergoes any one of the following mutations (1) to (7) on the amino acid sequence of the starting sequence shown in SEQ ID NO.1: (1) mutating the valine at position 140 to isoleucine; (2) mutating the histidine at position 415 to tryptophan; (3) mutating the threonine at position 508 to lysine; (4) mutating valine at position 140 to isoleucine and threonine at position 508 to lysine; (5) mutating valine at position 140 to isoleucine and histidine at position 415 to tryptophan; (6) mutating histidine at position 415 to tryptophan and threonine at position 508 to lysine; (7) The valine at position 140 was mutated to isoleucine, the histidine at position 415 was mutated to tryptophan, and the threonine at position 508 was mutated to lysine.

2. A gene encoding the glycogen branching enzyme mutant according to claim 1.

3. A recombinant vector expressing the glycogen branching enzyme mutant according to claim 1 or the gene according to claim 2.

4. The recombinant vector according to claim 3, characterized in that The expression vector is a pET series, Duet series, pGEX series, pHY300, pHY300PLK, pPIC3K or pPIC9K series vector.

5. A recombinant cell expressing the glycogen branching enzyme mutant according to claim 1, or containing the gene according to claim 2, or containing the recombinant vector according to claim 3 or 4; the recombinant cell is a non-plant cell.

6. The recombinant cell according to claim 5, characterized in that The recombinant cell uses prokaryotic cells or eukaryotic cells as expression hosts.

7. A method for improving the thermal stability of glycogen branching enzyme, characterized in that: The method comprises the following steps: mutating the valine at position 140 of the glycogen branching enzyme of the amino acid sequence as shown in SEQ ID NO. 1 to isoleucine, mutating the histidine at position 415 to tryptophan and / or mutating the threonine at position 508 to lysine.

8. A composition comprising the glycogen branching enzyme mutant according to claim 1.

9. The composition according to claim 8, characterized in that The composition comprises the glycogen branching enzyme according to claim 1 as a main enzyme component and contains one or more adjuvants.

10. The composition according to claim 9, characterized in that Such adjuvants include, but are not limited to, enzyme stabilizers.

11. A method for preparing branched starch, characterized in that: The method comprises adding the glycogen branching enzyme mutant according to claim 1 into amylose.

12. The method according to claim 11, characterized in that The method comprises reacting at 60-70° C. for 10-30 minutes.

13. Use of the glycogen branching enzyme mutant according to claim 1, or the gene according to claim 2, or the recombinant vector according to claim 3 or 4, or the recombinant cell according to claim 5 or 6 in the preparation of highly branched starch.

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