A starch branching enzyme mutant with enhanced extracellular secretion amount and secretion rate

Abstract

The present invention discloses a starch branching enzyme mutant with enhanced extracellular secretion amount and secretion speed, belonging to the technical field of genetic engineering and enzyme engineering. The starch branching enzyme mutant of the present invention is obtained by truncating and mutating the amino acids of the first 6 positions of the starch branching enzyme shown in the amino acid sequence of SEQ ID NO.1. The present invention separates and purifies the starch branching enzyme mutant enzyme protein by a two-step purification method of first a hydrophobic column and then an ion column. The extracellular enzyme activity of the starch branching enzyme mutant ΔNloop obtained by the present invention is increased by 17.29% at 72h, and the extracellular secretion speed is greatly improved. The intracellular target protein can be completely secreted to the extracellular space within 48h, further promoting the efficient expression of starch branching enzyme in a food-grade Bacillus subtilis expression system.

Description

Technical Field

[0001] The invention relates to a starch branching enzyme mutant with enhanced extracellular secretion amount and secretion speed, belonging to the technical field of gene engineering and enzyme engineering. Background Art

[0002] Starch branching enzyme (1,4-α-glucan branching enzyme, GBE) is a type of glucosyltransferase that uses glucan molecules as substrates and has the three functions of "hydrolysis", "transfer" and "synthesis". Based on the similarity of the amino acid sequence, starch branching enzymes are classified as glycoside hydrolases (Glycoside hydrolases, GHs). GBE can hydrolyze the α-1,4-glycosidic bonds in starch and connect the sugar chains with non-reducing ends to the receptor chains in the form of α-1,6-glycosidic bonds to form new α-1,6-branching points, increase the branching degree of starch, and improve the resistance and slow digestibility of starch. Due to the unique property of GBE to increase the branching degree of starch, it can be used to prepare highly branched starch and starch derivatives, and has great industrial application prospects.

[0003] Starch branching enzymes are generally derived from plants, animals and microorganisms. Plant-derived starch branching enzymes are the most common, but they involve the coordinated catalysis of multiple isozymes in the process of starch modification, which is relatively complex and therefore not suitable for industrial production; animal-derived starch branching enzymes are relatively difficult to heterologously express during fermentation production. In comparison, microbial-derived starch branching enzymes have a higher optimal reaction temperature, strong specificity, a large degree of branching, a simple catalytic method, and are easy to genetically manipulate, so they have great advantages in industrial production. Starch branching enzymes in microorganisms are generally derived from bacteria (such as cyanobacteria, thermophilic cocci, Escherichia coli) and fungi (Saccharomyces cerevisiae, Aspergillus nidulans).

[0004] Generally, natural strains have low ability to produce branching enzymes, and the enzyme production conditions need to be optimized to achieve a higher level of enzyme production. The industrial application of enzymes is often limited by factors such as low enzyme expression and complex enzyme separation processes. In recent years, many studies at home and abroad have achieved heterologous expression of starch branching enzymes through genetic engineering technology, significantly improving the expression of starch branching enzymes. However, most reports use Escherichia coli as the host, and Escherichia coli is easy to form inclusion bodies, making separation more difficult. At the same time, the endotoxins contained in Escherichia coli itself seriously limit the application of starch branching enzymes in the food field.

[0005] Therefore, it is worth exploring the direction of safely expressing starch branching enzymes with good application prospects and exploring a more targeted and efficient expression strategy. The efficient expression and separation of starch branching enzymes through genetic engineering and enzyme engineering and their application in the food field are of great significance for the industrial application of starch branching enzymes. Summary of the invention

[0006] In order to solve the problems of poor extracellular secretion ability of starch branching enzyme in the prior art, the present invention obtains the starch branching enzyme by truncating and mutating the starch branching enzyme encoded by Geobacillus thermoglucosidans STB02.

[0007] The present invention provides a mutant ΔNloop with significantly improved extracellular expression amount and secretion rate, wherein the extracellular enzyme activity is increased by 17.29% at 72 h, and the extracellular secretion rate is greatly improved, further promoting the efficient expression of starch branching enzyme in a food-grade Bacillus subtilis expression system.

[0008] The present invention provides a starch branching enzyme mutant ΔNloop, wherein the starch branching enzyme mutant is obtained by truncating and mutating the first 6 amino acids of the starch branching enzyme with an amino acid sequence as shown in SEQ ID NO.1.

[0009] In one embodiment of the present invention, the amino acid sequence of the starch branching enzyme mutant ΔNloop is shown as SEQ ID NO.3.

[0010] In one embodiment of the present invention, the amino acid sequence of the starch branching enzyme mutant ΔNloop is shown as SEQ ID NO.4.

[0011] In one embodiment of the present invention, the starch branching enzyme parent enzyme is derived from Geobacillus thermoglucosidans STB02, and the nucleotide sequence encoding the starch branching enzyme parent enzyme is shown in SEQ ID NO.2.

[0012] The present invention also provides a gene encoding the starch branching enzyme mutant ΔNloop.

[0013] The invention also provides a recombinant vector carrying the gene.

[0014] In one embodiment of the present invention, the recombinant vector is pP43NMK as an expression vector.

[0015] The present invention also provides a recombinant cell carrying the above gene or the above recombinant vector.

[0016] In one embodiment of the present invention, bacteria or fungi are used as host cells.

[0017] In one embodiment of the present invention, the recombinant cell uses Bacillus subtilis or Escherichia coli as a host cell.

[0018] The present invention also provides a recombinant bacterium, wherein the recombinant bacterium uses Bacillus subtilis or Escherichia coli as a host and expresses the starch branching enzyme mutant ΔNloop.

[0019] In one embodiment of the present invention, Bacillus subtilis WB600 is used as the host.

[0020] In one embodiment of the present invention, pP43NMK is used as the expression vector.

[0021] The present invention also provides a method for increasing the extracellular secretion amount and secretion speed of starch branching enzyme, wherein the first 6 amino acids of the starch branching enzyme whose amino acid sequence is shown in SEQ ID NO.1 are subjected to truncation mutation.

[0022] The present invention also provides a method for synthesizing amylopectin, characterized in that the starch branching enzyme mutant or the recombinant cell is added to a reaction system containing amylose to prepare starch with an increased degree of branching.

[0023] In one embodiment of the present invention, the amylose is 0.1% (w / v) potato amylopectin.

[0024] In one embodiment of the present invention, the added amount of the starch branching enzyme mutant is: 0.1 mg / mL.

[0025] In one embodiment of the present invention, the reaction conditions are: 40-60° C. for 1-2 hours.

[0026] The present invention also provides a method for purifying starch branching enzyme, which comprises purifying the wild type or mutant by a two-step method of first passing through a hydrophobic column and then passing through an ion column.

[0027] Beneficial Effects

[0028] (1) The present invention significantly improves the extracellular expression level of starch branching enzyme derived from Geobacillus thermoglucosidans STB02 by truncating the first 6 amino acids of the starch branching enzyme. The method of the present invention is safe and efficient. The activity of the crude enzyme liquid containing starch branching enzyme mutant ΔNloop fermented for 72 hours is increased by 17.29% compared with the original enzyme. The intracellular enzyme can be completely secreted to the extracellular space after 48 hours, which significantly improves the extracellular secretion rate. The recombinant enzyme obtained by the method of the present invention is an extracellular enzyme, which is conducive to extraction and purification for large-scale production in the later stage, and the product meets food requirements.

[0029] (2) The present invention promotes the extracellular secretion expression of starch branching enzyme in Bacillus subtilis, and the expression level is relatively high, which to a certain extent innovates the existing expression level of starch branching enzyme in the food-grade Bacillus subtilis expression system and can further promote the production and application of starch branching enzyme in the food industry.

[0030] (3) The present invention can effectively obtain pure enzyme protein of starch branching enzyme through a two-step purification method, i.e., first hydrophobic column purification and then ion column purification. Without using a nickel column, it is more suitable for application in the food field and has a high application potential in the food field. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 : SDS PAGE electrophoresis analysis of crude extracellular and intracellular proteins of wild type and mutants, where band marker: protein standard molecular weight; ΔNloop, ΔN-15aa, ΔN-20aa, ΔN-30aa are mutants of Gt-GBE.

[0032] Figure 2 : Extracellular and intracellular crude enzyme activities of the wild type and mutants after 72h fermentation. ΔNloop, ΔN-15aa, ΔN-20aa, and ΔN-30aa are mutants of Gt-GBE.

[0033] Figure 3 : SDS PAGE electrophoresis analysis of wild-type and mutant crude enzyme solutions, wherein band Marker: protein standard molecular weight; Gt-GBE-WT: wild-type Gt-GBE; Gt-GBE-ΔNloop: mutant Gt-GBE.

[0034] Figure 4 : Extracellular and intracellular enzyme activity curves of the wild type and mutant during the entire 72h fermentation process, where WT is the wild type and ΔNloop is the mutant. DETAILED DESCRIPTION

[0035] The embodiments of the present invention are only used as further explanations of the present invention and cannot be used as limiting the content or scope of the present invention.

[0036] The maltodextrin solution disclosed in the following examples was purchased from Shandong Baolingbao Biological Co., Ltd., and the isoamylase involved in the following examples was purchased from Sigma, USA.

[0037] The culture medium involved in the following examples is as follows:

[0038] LB liquid medium: yeast powder 5 g / L, tryptone 10 g / L, NaCl 10 g / L, pH 7.0.

[0039] LB solid medium: yeast powder 5 g / L, tryptone 10 g / L, NaCl 10 g / L, pH 7.0, 1.5% (w / v) agar.

[0040] TB liquid culture medium: yeast powder 24 g / L, trypsin 12 g / L, glycerol 5 g / L, KH2PO4 17 mM, K2HPO4 72 mM, pH 7.0.

[0041] The detection methods involved in the following embodiments are as follows:

[0042] Detection of starch branching enzyme activity:

[0043] Prepare 0.25% (w / v) potato amylopectin solution with 10mM phosphate buffer (pH 7.5), add 0.5mg / mL starch branching enzyme, mix well and place in a 50℃ water bath for accurate reaction for 15min. After the reaction, inactivate the enzyme in a boiling water bath. Take 300μL of the reaction mixture, add 5.0mL of color development solution (0.05% (w / v) KI, 0.005% (w / v) I2, pH7.5), mix well and color at room temperature for 15min in the dark. After 15min of color development, measure the absorbance at 530nm.

[0044] One unit of enzyme activity is defined as the amount of enzyme required to reduce the absorbance by 1% per minute at 530 nm.

[0045] Example 1: Preparation of starch branching enzyme mutants

[0046] The specific steps are as follows:

[0047] (1) Chemically synthesizing the starch branching enzyme gbe as shown in SEQ ID NO.1, designing the complementary primer chains required for the experiment (see Table 1), using PCR to p-PCR the starch branching enzyme gene, and connecting it with the commercial vector plasmid pP43NMK by homologous recombination to construct the recombinant plasmid pP43NMK-gbe;

[0048] (2) Using the recombinant plasmid pP43NMK-gbe prepared in step (1) as a template, the complementary primer chains required for the experiment were designed (see Table 1). The primers were synthesized by GENEWISE Biotechnology Co., Ltd. and site-directed mutagenesis was performed according to the method described in the instructions of the STAR Primer GXL kit of TaKaRa Company. Recombinant plasmids of the genes of the mutants ΔNloop (the first 6 amino acids were truncated), ΔN15aa (the first 15 amino acids were truncated), ΔN20aa (the first 20 amino acids were truncated), and ΔN30aa (the first 30 amino acids were truncated) were prepared.

[0049] Table 1: Introduction of mutation sites of starch branching enzyme

[0050]

[0051]

[0052] The PCR reaction system was based on the conditions specified in the STAR Primer Kit instructions: 5× PrimeSTAR Buffer (Mg 2+ Plus) 10μL, template DNA 1μL, forward and reverse primers (10μM) 1μL, PrimeSTAR HSDNA Polymerase (2.5U / μL) 1μL, dNTPs (2.5mM each) 4μL, and finally ultrapure water 32μL. PCR amplification conditions are: pre-denaturation at 98℃ for 3min; then 98℃ 10s, 60℃ 15s, 68℃ 5min as one cycle, 35 cycles under the above conditions; 68℃ 10min, and finally incubation at 4℃ for 10min.

[0053] The PCR product obtained after the reaction was digested with DpnI at 37°C for 1 h, and then the treated PCR product was transferred into E. coli JM109 according to the E. coli JM109 competent transformation method to prepare transformants, and the transformants were spread on LB solid culture medium containing 50 μg / mL ampicillin, and the spread LB solid culture medium was inverted in a 37°C constant temperature incubator for 10-12 h, and the positive single clone was picked and inoculated into LB liquid culture medium containing 50 μg / mL ampicillin and cultured at 37°C for 10-12 h, and the plasmid was extracted and sent to the company for sequencing to obtain the recombinant plasmid containing the original gene: pP43NMK-gbe; and the recombinant plasmids containing mutant genes: pP43NMK-ΔNloop, pP43NMK-ΔN15aa, pP43NMK-ΔN20aa, pP43NMK-ΔN30aa.

[0054] Example 2: Construction of genetically engineered bacteria containing a gene expressing a starch branching enzyme mutant and secretory expression and separation and purification of the mutant

[0055] The specific steps are as follows:

[0056] (1) The recombinant plasmids pP43NMK-gbe and pP43NMK-ΔNloop, pP43NMK-ΔN15aa, pP43NMK-ΔN20aa, and pP43NMK-ΔN30aa obtained in Example 1 were respectively introduced into the competent host of Bacillus subtilis WB600 to prepare B. subtilis WB600 / pP43NMK-gbe and B. subtilis WB600 / pP43NMK-ΔNloop, B. subtilis WB600 / pP43NMK-ΔN15aa, B. subtilis WB600 / pP43NMK-ΔN20aa, and B. subtilis WB600 / pP43NMK-ΔN30aa recombinant strains;

[0057] (2) The recombinant strains were streaked on LB solid medium and cultured in a 37°C constant temperature incubator for 10 to 12 hours. The positive single colonies were picked and inoculated into a 250 mL Erlenmeyer flask containing 50 mL LB liquid medium. The Erlenmeyer flask was placed in a rotary shaker at 200 r / min and cultured at 37°C for 10 to 12 hours to prepare a seed solution;

[0058] (3) The seed solution was inoculated at an inoculum rate of 2% (v / v) into a 250-mL Erlenmeyer flask containing 50 mL of TB liquid culture medium, and the flask was placed in a rotary shaker and induced at a rate of 200 r / min at 25°C for 72 h to prepare crude enzyme solutions containing wild-type gbe, ΔNloop, ΔN15aa, ΔN20aa, and ΔN30aa, respectively; kanamycin was added to each culture medium at a final concentration of 50 μmg / mL before use.

[0059] Every 24 hours, the crude enzyme solution was analyzed by SDS-PAGE, and the intracellular cells were broken and analyzed by SDS-PAGE. The results were as follows: Figure 1 As shown, the results showed that both the original enzyme and the mutant enzyme were expressed.

[0060] (4) Detection of the activity of the original enzyme and mutant enzyme

[0061] The extracellular protein secretion of the wild type and mutant of Gt-GBE at 72 h was measured by iodine colorimetry and analyzed by SDS-PAGE. The results are shown in Table 2. Figure 1 and Figure 2 As shown:

[0062] Table 2: Extracellular crude enzyme activity of wild type and mutant GBE at 72h

[0063] Wild type / mutant <![CDATA[Extracellular crude enzyme activity (U / mL) 1 > Wild type WT 750.92±11.56 Mutant ΔNloop 882.77±38.74 Mutant ΔN15aa 553.46±12.23 Mutant ΔN20aa 144.75±2.04 Mutant ΔN30aa 150.86±12.23

[0064] The results showed that the enzyme proteins of each mutant and wild type were expressed extracellularly after 72 hours of fermentation. The extracellular secretion of the mutant ΔNloop increased by about 17.60% compared with the wild type, while the extracellular secretion of the mutants ΔN15aa, ΔN20aa, and ΔN30aa decreased to varying degrees compared with the wild type, and the extracellular secretion decreased significantly after truncating 20 and 30 amino acids, respectively.

[0065] (5) Purification of starch branching enzyme mutants:

[0066] Gt-GBE was purified by initial purification using a hydrophobic HiTrap Phenyl HP column (CV 5 mL) and then by a second-step purification using a HiTrap QHP column (CV 5 mL).

[0067] 1. Hydrophobic column:

[0068] Balanced solution A1: 20 mM Tris-HCl + 5% ammonium sulfate, pH 8.0;

[0069] Eluent B1: ultrapure water;

[0070] Dilute the crude enzyme solution 1:1 with solution A1 and gradually add solid ammonium sulfate to a final concentration of 5%;

[0071] All the above solutions were filtered through a 0.45um microporous membrane, the flow rate was set to 2.5mL / min, and the chromatography column was equilibrated with 4 column volumes of A1 solution. After equilibration, the sample was loaded, and after the UV line ran flat for about 4 column volumes, the B1 solution was used for elution, and the elution peak was collected to obtain the preliminary purified enzyme solution.

[0072] 2. Strong anion column:

[0073] Balanced solution A2: 20 mM Tris-HCl, pH 8.0;

[0074] Eluent B2: 20 mM Tris-HCl + 1 M NaCl, pH 8.0;

[0075] Diluent C: 40 mM Tris-HCl, pH 8.0;

[0076] Dilute the above eluate with solution C 1:1 and adjust the pH to 8.0 with HCl;

[0077] All the above solutions were filtered with a 0.45um microporous membrane, the flow rate was set to 2.5mL / min, and the chromatography column was balanced with 4 column volumes of A2 solution. After the balance was completed, the sample was loaded, and after the UV line ran flat for about 4 column volumes, it was replaced with B2 solution for gradient elution (concentrations were 0.15M, 0.25M, and 0.4M NaCl, respectively), and the elution peak was collected to obtain the second-step purified enzyme solution. The eluate was ultrafiltered and concentrated with an ultrafiltration concentration tube and stored at -80°C.

[0078] Pure enzymes containing wild-type gbe, pure enzymes containing ΔNloop, pure enzymes containing ΔN15aa, pure enzymes containing ΔN20aa, and pure enzymes containing ΔN30aa were prepared.

[0079] 3. The specific enzyme activities of the pure enzyme of wild-type gbe, the pure enzyme of ΔNloop, the pure enzyme of ΔN15aa, the pure enzyme of ΔN20aa, and the pure enzyme of ΔN30aa were detected respectively. The results are shown in Table 3:

[0080] Table 3: Specific enzyme activities (extracellular) of wild-type and mutant GBE

[0081] Wild type / mutant <![CDATA[Specific enzyme activity (U / mg) 1 > Wild type WT 1675.16±43.17 Mutant ΔNloop 1711.42±13.05 Mutant ΔN15aa 1653.23±23.22 Mutant ΔN20aa 1558.87±25.38 Mutant ΔN30aa 1553.53±15.45

[0082] 1 Each value is the average of 3 parallel experiments.

[0083] The results showed that the N-terminal mutation had little effect on the specific enzyme activity of Gt-GBE. Combined with the relevant data of crude enzyme activity in Table 2, it was proved that the ΔNloop mutant could effectively increase the extracellular secretion of Gt-GBE.

[0084] Example 3: Effect of starch branching enzyme mutants on protein secretion rate of genetically engineered bacteria

[0085] The specific steps are as follows:

[0086] (1) According to the method of Example 2, B. subtilis WB600 / pP43NMK-gbe and B. subtilis WB600 / pP43NMK-ΔNloop recombinant strains were prepared.

[0087] (2) The recombinant strains were streaked on LB solid medium and cultured in a 37°C constant temperature incubator for 10 to 12 hours. The positive single colonies were picked and inoculated into a 250 mL Erlenmeyer flask containing 50 mL LB liquid medium. The Erlenmeyer flask was placed in a rotary shaker at 200 r / min and cultured at 37°C for 10 to 12 hours to prepare a seed solution;

[0088] (3) The seed solution was inoculated at a 2% (v / v) inoculum into a 250 mL Erlenmeyer flask containing 50 mL of TB liquid medium and placed in a rotary shaker at 200 r / min at 25°C for 72 h;

[0089] The extracellular and intracellular enzyme activities of the recombinant strains of B. subtilis WB600 / pP43NMK-gbe and B. subtilis WB600 / pP43NMK-ΔNloop were detected respectively. The results are shown in Figure 4 As shown in Table 4, the SDS-PAGE results are as follows Figure 3 As shown:

[0090] Table 4: Extracellular and intracellular enzyme activity data of original enzyme and mutant enzyme at different fermentation times

[0091]

[0092] 1 Each value is the average of 3 parallel experiments.

[0093] The results showed that the mutant ΔNloop could almost completely secrete intracellular proteins to the extracellular space within 48 hours, while at this time some intracellular proteins of the wild-type WT had not yet been secreted to the extracellular space. Therefore, the protein secretion rate of the mutant ΔNloop was greatly improved compared to that of the wild-type WT.

[0094] Although the present invention has been disclosed as above in the form of a preferred embodiment, 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 starch branching enzyme mutant that increases the extracellular secretion amount and secretion rate of starch branching enzyme, characterized in that: The amino acid sequence of the starch branching enzyme mutant is shown in SEQ ID NO.

3.

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

3. A recombinant vector carrying the gene according to claim 2.

4. A recombinant cell carrying the gene according to claim 2 or the recombinant vector according to claim 3.

5. The recombinant cell according to claim 4, characterized in that Bacteria are used as host cells.

6. The recombinant cell according to claim 5, characterized in that The host cell is Bacillus subtilis or Escherichia coli.

7. Use of the starch branching enzyme mutant according to claim 1 in increasing the extracellular secretion amount and secretion rate of starch branching enzyme.

8. A method for synthesizing amylopectin, characterized in that: The method comprises adding the starch branching enzyme mutant according to claim 1 or the recombinant cell according to any one of claims 4 to 6 to a reaction system containing amylose, and reacting to prepare starch with an increased degree of branching.

9. Use of the mutant according to claim 1, or the gene according to claim 2, or the recombinant vector according to claim 3, or the recombinant cell according to claims 4 to 6 in synthesizing amylopectin.

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