A starch branching enzyme mutant with improved extracellular secretion ability and enzyme activity

Abstract

The present invention relates to a starch branching enzyme mutant with improved extracellular secretion ability and enzyme activity. The present invention performs site-directed mutation on starch branching enzyme derived from Geobacillus thermoglucosidans, wherein the mutation sites are lysine at positions 159, 374, 393, 426 and 432, and after detecting the crude enzyme activities of the mutants, it is found that the extracellular expression of mutants K159A and K159H in Bacillus subtilis is significantly improved compared with the wild type. The crude enzyme solution is further purified using a hydrophobic column and an anion column, and the pure enzyme specific enzyme activities of mutants K159A and K159H reach 1745.18U / mg and 1873.78U / mg, respectively. The starch branching enzyme mutant provided by the present invention has improved extracellular expression ability and specific enzyme activity on the basis of the wild type, and can be applied to the modification of potato amylopectin, which is beneficial to the industrial application of starch branching enzyme.

Description

Technical Field

[0001] The invention relates to the technical field of enzyme engineering, in particular to a starch branching enzyme mutant with improved extracellular secretion ability and enzyme activity. Background Art

[0002] Starch branching enzyme (1,4-α-glucan branching enzyme, GBE) is a functionally diverse, critical enzyme widely used in food, pharmaceuticals, biotechnology and other fields. GBE can recognize and hydrolyze α-1,4-glycosidic bonds in starch, and connect sugar chains with non-reducing ends to receptor chains in the form of α-1,6-glycosidic bonds, thereby forming new α-1,6-branching points, significantly increasing the branching degree of starch. This branching property gives starch significant advantages in digestibility and functionality, and therefore has important application prospects in the preparation of highly branched starch and its derivatives.

[0003] Starch branching enzyme plays an important role in the field of healthy food and special dietary processing. Starch branching enzyme can reduce the overall digestion rate of starch from different sources, and the degree of reduction is closely related to multiple factors such as starch source, relative crystallinity, chain length and branch density. The use of starch branching enzyme to modify corn starch can obtain short clustered maltodextrins with characteristics such as dense internal structure and short external chain. This short aggregate molecule can hinder the action of digestive enzymes and significantly reduce the digestibility. It can be used as a substitute for dietary starch and can effectively improve the level of postprandial blood sugar fluctuations, thus benefiting the health of diabetics. In addition, the functionality of modified starch is also significantly enhanced, such as improving the texture, stability, water retention and processing performance of food, so that it shows a wide range of application potential in multiple food fields such as baking, frozen food, beverages and capsule preparations. In the pharmaceutical industry, highly branched starch and its derivatives involved in starch branching enzymes are used as matrix materials for drug carriers due to their good biocompatibility and degradability, which helps to improve the solubility, stability and bioavailability of drugs and is an indispensable component in the design of drug sustained release systems.

[0004] At present, the existing starch branching enzyme has the problem of low catalytic efficiency, and its catalytic performance is difficult to meet the requirements of industrial production, and the fermentation yield is still low. This leads to an increase in enzyme usage and cost in industrial production, which seriously restricts the industrial application of GBE. Therefore, improving the catalytic performance of GBE is the key to solving the problem. In addition, the improvement of enzyme catalytic performance can also shorten the reaction time of industrial production, improve the output and quality of products, and thus meet the market demand for high-efficiency and high-quality products. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a starch branching enzyme mutant with improved extracellular secretion ability and enzyme activity. The present invention performs site-directed mutagenesis on starch branching enzyme derived from Geobacillus thermoglucosidans, and the mutation sites are lysine at positions 159, 374, 393, 426 and 432. After detecting the crude enzyme activity of the mutants, it is found that the extracellular expression of mutants K159A and K159H in Bacillus subtilis is significantly improved compared with the wild type. The crude enzyme solution is further purified using a hydrophobic column and an anion column, and the pure enzyme specific enzyme activities of mutants K159A and K159H reach 1745.18U / mg and 1873.78U / mg, respectively.

[0006] The first object of the present invention is to provide a starch branching enzyme mutant, wherein the starch branching enzyme mutant uses the amino acid sequence shown in SEQ ID NO.1 as the starting sequence, and the 159th lysine is mutated to alanine or histidine.

[0007] Furthermore, the amino acid sequence of the starch branching enzyme mutant is shown in SEQ ID NO.2 or SEQ ID NO.3.

[0008] The second object of the present invention is to provide a gene encoding the above starch branching enzyme mutant.

[0009] Furthermore, the nucleotide sequence of the gene is shown in SEQ ID NO.5 or SEQ ID NO.6.

[0010] The third object of the present invention is to provide a recombinant expression vector comprising the above gene.

[0011] Furthermore, the recombinant expression vector is constructed based on the pP43NMK expression vector.

[0012] Furthermore, the recombinant expression vector is a double promoter combination P HpaII -P srfa Start the expression of the target gene.

[0013] The fourth object of the present invention is to provide a host cell comprising the mutant, the gene or the recombinant expression vector, wherein the host cell is a non-plant cell.

[0014] Furthermore, the host cell is Bacillus subtilis.

[0015] Furthermore, the host cell is Bacillus subtilis WB600.

[0016] The fifth object of the present invention is to provide the use of the mutant, the gene, the recombinant expression vector or the host cell in the modification of potato amylopectin.

[0017] Beneficial effects of the present invention:

[0018] (1) The starch branching enzyme mutant provided by the present invention is expressed extracellularly in the host Bacillus subtilis, and the extracellular expression amount is increased by 13.4% (K159A) and 15.8% (K159H) respectively compared with the wild-type enzyme, that is, the mutant can be secreted outside the cell more effectively, thereby simplifying the subsequent enzyme extraction and purification steps and reducing the production cost;

[0019] (2) After two-step purification, the pure enzyme specific activities of mutants K159A and K159H reached 1745.18 U / mg and 1873.78 U / mg, respectively. Compared with the wild-type enzyme, their specific activities were increased by 6% (K159A) and 13.8% (K159H), respectively, which means that under the same conditions, these mutant enzymes can catalyze more substrates into products, thereby improving the efficiency of the bioconversion process. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to make the content of the present invention easier to understand, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0021] Figure 1 is the result of comparing the extracellular expression of the wild-type starch branching enzyme and 15 starch branching enzyme mutants in Example 3 of the present invention;

[0022] Figure 2 It is the result of SDS-PAGE electrophoresis analysis of the pure enzyme solution of the wild-type starch branching enzyme and the purified starch branching enzyme mutants K159R, K159A and K159H in Example 4 of the present invention, wherein band M is the standard protein molecular weight and WT is the wild-type starch branching enzyme. DETAILED DESCRIPTION

[0023] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0024] The culture medium used in the following examples is as follows:

[0025] LB liquid medium: yeast powder 5 g / L, tryptone 10 g / L, NaCl 10 g / L, pH 7.0.

[0026] LB solid medium: yeast powder 5 g / L, tryptone 10 g / L, NaCl 10 g / L, pH 7.0, 1.5% (w / v) agar.

[0027] 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.

[0028] The detection methods used in the following examples are as follows:

[0029] Detection of starch branching enzyme activity: 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 developing solution (0.05% (w / v) KI, 0.005% (w / v) I2, pH=7.5), mix well and color at room temperature for 15min in the dark. After 15min of color development, measure the absorbance at 530nm.

[0030] One unit of enzyme activity is defined as the amount of enzyme required to reduce the absorbance by 1% per minute at 530 nm.

[0031] Example 1: Preparation of starch branching enzyme mutants

[0032] (1) Construction of recombinant plasmid pP43NMK / gbe

[0033] The starch branching enzyme gene (derived from Geobacillus thermoglucosidans STB02) with the nucleotide sequence shown in SEQ ID NO.4 was connected to the vector pP43NMK and the double promoter combination P HpaII -P srfa The expression of the target gene starch branching enzyme gene was initiated to obtain the recombinant plasmid pP43NMK / gbe. For the specific construction process, please refer to the literature "Study on the secretory expression of starch branching enzyme gene derived from Bacillus thermoglucoside in Bacillus subtilis" (Xu Tao, 2023, Master's thesis of Jiangnan University).

[0034] (2) Construction of recombinant plasmid containing mutants

[0035] Previous studies have found that acetic anhydride modification of starch branching enzyme improves its catalytic performance, and acetic anhydride modifies lysine residues, so the mutation site is selected as the lysine site. Modification of lysine near the catalytic center is more likely to cause changes in catalytic performance, so K159, K374, K393, K426, and K432 are selected as mutation sites. Arginine, alanine, and histidine are selected as replacement mutations based on the different characteristics of the R group.

[0036] Using the expression vector pP43NMK / gbe as a template, the complementary primer chains required for the experiment were designed (see Table 1). The primers were synthesized by GeneWeizhi 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. The sites included positions 159, 374, 393, 426 and 432. The lysine at the above sites was mutated to arginine, alanine or histidine to prepare recombinant plasmids containing mutants.

[0037] PCR system: 5× PrimeSTAR GXL Buffer ((Mg 2+ Plus) 10μL, dNTP Mixture (2.5mM each) 4μL, forward primer (10μM) 1μL, reverse primer (10μM) 1μL, template DNA 1μL, PrimeSTAR GXL DNA Polymerase (1.25U / μL) 1μL, add double distilled water to 50μL. PCR amplification conditions are: 98℃ pre-denaturation for 3min; then 30 cycles (98℃ 10s, 60℃ 15s, 68℃ 8min); finally 68℃ incubation for 10min.

[0038] Table 1 Introduction of mutation sites of starch branching enzyme

[0039]

[0040]

[0041] Note: The bold underlined bases correspond to the mutated amino acids.

[0042] Example 2: Construction of genetically engineered bacteria

[0043] (1) The PCR product (i.e., the recombinant plasmid containing the mutant) and the recombinant plasmid pP43NMK / gbe obtained in Example 1 were treated with DpnI overnight at 37°C, and then the treated PCR product and the recombinant plasmid pP43NMK / gbe were transformed into E. coli JM109 to obtain transformants, respectively. The transformant bacterial liquid was spread on LB solid medium containing 10 μg / mL kanamycin, and cultured overnight in a 37°C incubator for 12 hours, and single colonies were selected from them;

[0044] (2) The single colonies obtained in step (1) were inoculated into LB liquid culture medium containing 10 μg / mL kanamycin, cultured at 37°C and 200 r / min for 12 h, and the plasmids were extracted for sequencing according to the method indicated in the instructions of the plasmid extraction kit.

[0045] (3) The successfully constructed recombinant plasmid was then transferred into the expression host B. subtilis WB600 competent cells by chemical transformation, and finally the recombinant genetically engineered bacteria B. subtilis WB600 / gbe and B. subtilis WB600 / gbe were obtained. K159R 、B.subtilis WB600 / gbe K159A 、B.subtilis WB600 / gbe K159H 、B.subtilis WB600 / gbe K374R 、B.subtilis WB600 / gbe K374A 、B.subtilis WB600 / gbe K374H 、B.subtilis WB600 / gbe K393R 、B.subtilis WB600 / gbe K393A 、B.subtilis WB600 / gbe K393H 、B.subtilis WB600 / gbe K426R 、B.subtilis WB600 / gbe K426A 、B.subtilis WB600 / gbe K426H 、B.subtilis WB600 / gbe K432R 、B.subtilis WB600 / gbe K432A 、B.subtilis WB600 / gbe K432H .

[0046] Example 3: Expression of starch branching enzyme mutants

[0047] (1) Cultivation of recombinant bacteria:

[0048] The recombinant genetically engineered bacteria obtained in Example 2 were streaked on LB solid culture medium and cultured in a 37°C constant temperature incubator for 12 h. Subsequently, positive single colonies were picked and inoculated into 250 mL conical flasks containing 50 mL LB liquid culture medium. 10 μg / mL kanamycin was added before inoculation. The conical flasks were placed in a rotary shaker at 200 r / min and cultured at 37°C for 8-12 h to prepare seed solutions.

[0049] (2) Fermentation culture:

[0050] The seed solution prepared in step (1) was transferred to a 250 mL conical flask containing 50 mL TB medium (added with 10 μg / mL kanamycin) at an inoculum volume of 4% (v / v), and cultured in a shaking incubator at 25°C for 60-90 h at a rotation speed of 200 r / min.

[0051] After the fermentation was completed, the fermentation broth was centrifuged at 4°C and 10,000 rpm for 20 min, and the supernatant was collected to obtain crude enzyme solutions containing wild-type gbe and crude enzyme solutions containing mutants K159R, K159A, K159H, K374R, K374A, K374H, K393R, K393A, K393H, K426R, K426A, K426H, K432R, K432A and K432H, respectively. The crude enzyme activities were detected for comparison of their extracellular expression levels. The results are shown in FIG. Figure 1 As shown, after the 159th lysine of the wild-type starch branching enzyme was mutated, the extracellular expression level was significantly higher than that of the mutants at other sites, so the mutants K159R, K159A, and K159H were selected for further study in the subsequent examples;

[0052] Example 4: Determination of expression level of starch branching enzyme mutants

[0053] (1) Determination of starch branching enzyme activity:

[0054] The extracellular expression levels of the starch branching enzyme wild type and mutants K159R, K159A and K159H prepared in Example 3 are shown in Table 2. The enzyme proteins of the mutants and the wild type achieved extracellular expression after 72 hours of fermentation, among which the activity and expression level of the crude enzyme solution of the mutants K159A and K159H were increased by approximately 13.51% and 15.83% respectively compared with the wild type.

[0055] Table 2 Extracellular expression levels of wild type and mutants after 72h

[0056] Wild type / mutant Extracellular expression level (U / mL) WT 883.26±13.06 K159R 859.14±8.63 K159A 1002.34±4.35 K159H 1022.78±6.68

[0057] (2) Purifying the crude enzyme solution containing wild-type gbe and the crude enzyme solution containing mutants K159R, K159A and K159H prepared in Example 3 respectively, the steps are as follows:

[0058] The product was initially purified using a hydrophobic HiTrap Phenyl HP column (CV 5 mL), followed by a second-step purification using a HiTrap Q HP column (CV 5 mL).

[0059] 1. Hydrophobic column:

[0060] Balanced solution A1: 20 mM Tris-HCl + 5% ammonium sulfate, pH 8.0;

[0061] Eluent B1: ultrapure water;

[0062] Dilute the crude enzyme solution 1:1 with solution A1 and gradually add solid ammonium sulfate to a final concentration of 5%;

[0063] 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.

[0064] 2. Strong anion column:

[0065] Balanced solution A2: 20 mM Tris-HCl, pH 8.0;

[0066] Eluent B2: 20 mM Tris-HCl + 1 M NaCl, pH 8.0;

[0067] Diluent C: 40 mM Tris-HCl, pH 8.0;

[0068] Dilute the above eluate with solution C 1:1 and adjust the pH to 8.0 with HCl;

[0069] All the above solutions were filtered with a 0.45 μm microporous membrane, the flow rate was set to 2.5 mL / 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 0.15 M, 0.25 M, and 0.4 M 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.

[0070] Pure enzymes containing wild-type gbe, pure enzymes containing mutant K159R, pure enzymes containing mutant K159A and pure enzymes containing mutant K159H were prepared.

[0071] The pure enzymes containing mutants K159R, K159A and K159H were analyzed by SDS-PAGE electrophoresis. Figure 2 shown.

[0072] 3. The specific enzyme activities of the pure enzyme of wild-type gbe, the pure enzyme of K159R, the pure enzyme of K159A, and the pure enzyme of K159H were detected respectively. The results are shown in Table 3:

[0073] Table 3 Specific enzyme activity of wild type and mutant pure enzymes

[0074] Wild type / mutant Specific enzyme activity (U / mg) WT 1647.26±26.03 K159R 1444.92±18.63 K159A 1745.18±23.35 K159H 1873.78±16.69

[0075] The results showed that mutants K159A and K159H not only improved the activity and expression level of crude enzyme, but also improved the specific enzyme activity of pure enzyme after purification of crude enzyme solution, which further indicated that mutants K159A and K159H could effectively improve the catalytic performance and extracellular fermentation amount of Gt-GBE.

[0076] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A starch branching enzyme mutant, characterized in that: The starch branching enzyme mutant uses the amino acid sequence shown in SEQ ID NO.1 as the starting sequence, and lysine at position 159 is mutated to alanine or histidine.

2. The starch branching enzyme mutant according to claim 1, characterized in that: The amino acid sequence of the starch branching enzyme mutant is shown in SEQ ID NO.2 or SEQ ID NO.

3.

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

4. The gene according to claim 3, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.5 or SEQ ID NO.

6.

5. A recombinant expression vector comprising the gene according to claim 3 or 4.

6. The recombinant expression vector according to claim 5, characterized in that: The recombinant expression vector is constructed based on the pP43NMK expression vector.

7. The recombinant expression vector according to claim 5, characterized in that: The recombinant expression vector is a double promoter combination P HpaII -P srfa Start the expression of the target gene.

8. A host cell comprising the mutant according to any one of claims 1-2, the gene according to any one of claims 3-4, or the recombinant expression vector according to any one of claims 5-7, wherein the host cell is a non-plant cell.

9. The host cell according to claim 8, characterized in that: The host cell is Bacillus subtilis.

10. Use of the mutant according to any one of claims 1-2, the gene according to any one of claims 3-4, the recombinant expression vector according to any one of claims 5-7 or the host cell according to any one of claims 8-9 in modifying potato amylopectin.

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