Expression vector of cyclodextrin glucosyltransferase and construction and application of bacillus subtilis

Abstract

The invention discloses an expression vector of cyclodextrin glucosyltransferase and construction and application of bacillus subtilis, belongs to the technical field of enzyme engineering, and is characterized in that the amino acid sequence of the cyclodextrin glucosyltransferase is shown as SEQ ID NO. 1; the nucleotide sequence for coding the amino acid sequence of the cyclodextrin glucosyltransferase is as shown in SEQ ID NO. 2; the construction method of the expression vector of the cyclodextrin glucosyltransferase comprises the following steps: carrying out whole gene synthesis on an amino acid sequence as shown in SEQ ID NO.1 to obtain a target gene, and connecting the target gene with a vector plasmid pWB980-ori to obtain an expression vector pWB980-ori-CGT; the method has the beneficial effects that the expression vector pWB980-orii-CGT is obtained, the bacillus subtilis is constructed, the cyclodextrin glucosyltransferase can be stably and efficiently expressed, the bacillus subtilis can be directly treated by full-fermentation reaction enzyme liquid without separation, and the constructed genetically engineered bacterium is shortened in fermentation period and has high enzyme activity and high conversion rate.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme engineering, and more specifically relates to the construction and application of an expression vector of cyclodextrin glucosyltransferase and Bacillus subtilis. Background Art

[0002] Cyclodextrin Glycosyltransferase (CGTase, EC 2.4.1.19) is a major member of the α-amylase family (GH13). CGTase has the activity to catalyze both cyclization and coupling reactions, which is why the product gradually shifts from α-cyclodextrin to β-cyclodextrin over time during the starch catalysis process. When CGTase catalyzes starch hydrolysis, it cleaves the amylose molecules and transfers both ends to water molecules to complete the hydrolysis reaction; CGTase's activity in catalyzing hydrolysis is relatively weak.

[0003] After years of screening, our company has developed an alkaliphilic Bacillus that produces cyclodextrin glucosyltransferase. The strain has stable passage and good bacterial growth. However, the alkaline Bacillus has obvious disadvantages such as: 1. Slow growth rate and long reaction cycle; 2. Sensitive to pH; pH fluctuations may inhibit growth or product synthesis; 3. Risk of bacterial contamination. Although an alkaline environment can inhibit some bacteria, some alkali-resistant bacteria (such as halophilic alkali bacteria) may compete for nutrients; 4. The enzyme yield of cyclodextrin glucosyltransferase is low, and the yield of β-cyclodextrin is generally 38%; At present, there are reports on engineered bacteria that produce cyclodextrin glucosyltransferase. Among them, Escherichia coli has been studied the most, especially the Escherichia coli engineered bacteria constructed with pWB980-ori as the plasmid. It can effectively solve the problems of slow growth rate of alkaliphilic Bacillus, sensitivity to pH, easy contamination and low enzyme production. However, since Escherichia coli is a harmful bacterium, the cyclodextrin glucosyltransferase produced cannot be used directly in food processing and needs to be extracted and purified, which greatly affects production efficiency and applicability. Summary of the Invention

[0004] To solve the above problems and overcome the shortcomings of the existing technology, the present invention provides an expression vector for cyclodextrin glucosyltransferase and the construction and application of Bacillus subtilis, which can effectively solve the problems of slow growth rate, sensitivity to pH, susceptibility to bacterial contamination and low enzyme yield of alkaliphilic Bacillus. It also solves the problem that Escherichia coli is a harmful bacterium and the cyclodextrin glucosyltransferase produced cannot be directly used in food processing and needs to be extracted and purified, which greatly affects production efficiency and applicability.

[0005] The specific technical solution of the present invention to solve the above technical problems is: An expression vector for cyclodextrin glucosyltransferase, characterized in that the amino acid sequence of the cyclodextrin glucosyltransferase is shown in SEQ ID NO. 1; The 182nd amino acid in the amino acid sequence of the cyclodextrin glucosyltransferase is glutamine; The nucleotide sequence encoding the amino acid sequence of the cyclodextrin glucosyltransferase is as shown in SEQ ID NO. 2.

[0006] Furthermore, the method for constructing the expression vector of the cyclodextrin glucosyltransferase is as follows: the target gene obtained by whole gene synthesis of the amino acid sequence of SEQ ID NO.1 is connected with the vector plasmid pWB980-ori to obtain the expression vector pWB980-ori-CGT; The construction of Bacillus subtilis, using the expression vector of the cyclodextrin glucosyltransferase, is characterized in that the constructed expression vector pWB980-ori-CGT is introduced into the host cell Bacillus subtilis to obtain recombinant Bacillus subtilis, comprising the following steps: (1) Preparation of Bacillus subtilis competent cells: (2) Introduce the expression vector pWB980-ori-CGT into Bacillus subtilis competent cells to obtain recombinant Bacillus subtilis.

[0007] Furthermore, the preparation of Bacillus subtilis competent cells includes: streaking Bacillus subtilis onto an antibiotic-free LB plate and culturing overnight at 35-38°C. A single colony is inoculated from the plate into 50 mL of LB medium containing erythromycin (in a 250 mL Erlenmeyer flask), incubated at 37°C on a shaker for 8-12 hours, and then adding LB medium preheated to 37°C containing erythromycin. The culture is diluted to an A600 of 1.0. D-xylose is added to the dilution at an initial concentration of 1% (w / v). The cells are cultured at 37°C at 200 rpm for 2 hours to obtain competent cells.

[0008] Furthermore, the method of introducing the expression vector pWB980-ori-CGT into Bacillus subtilis competent cells to obtain recombinant Bacillus subtilis includes: The expression vector pWB980-ori-CGT was mixed with competent cells and cultured in a shaker at 37°C and 200 rpm for 1.5 h to complete the transformation. Positive colonies were gently mixed with 40% glycerol and stored in a −80°C refrigerator.

[0009] The invention relates to the application of Bacillus subtilis, and the Bacillus subtilis is used to express and prepare cyclodextrin glucosyltransferase liquid through fermentation.

[0010] The beneficial effects of the present invention are: The present invention creatively uses genetic engineering means to introduce nucleotides encoding the amino acid sequence of the cyclodextrin glucosyltransferase using the plasmid pWB980-ori as a vector, obtains the expression vector pWB980-ori-CGT, and constructs Bacillus subtilis. The cyclodextrin glucosyltransferase can be stably and efficiently expressed, and the Bacillus subtilis can be directly processed using the whole fermentation reaction enzyme liquid without isolation. The constructed genetically engineered bacteria has a shortened fermentation cycle, high enzyme activity and high conversion rate, saves production costs, and has potential application prospects.

[0011] The present invention creatively discovered a nucleotide sequence encoding the amino acid sequence of the cyclodextrin glucosyltransferase, wherein the 182nd amino acid in the amino acid sequence of the cyclodextrin glucosyltransferase is glutamine; the enzyme exhibits high enzymatic activity (1224 U / mL) in a heterologous expression system and shortens the fermentation cycle by 44%, and in particular, can be stably recombined and expressed with the plasmid pWB980-ori, and has relatively stable passage performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is the enzyme electrophoresis diagram of the pWB980-ori-CGT recombinant plasmid constructed in the present invention; Figure 2 This is the enzyme electrophoresis diagram of the pWB980-ori-Q9UWN2 recombinant plasmid constructed in Comparative Example 1; Figure 3 This is the enzyme electrophoresis diagram of the pWB980-ori-S5NU90 recombinant plasmid constructed in Comparative Example 2; Figure 4 This is a plate map of Bacillus subtilis transformed with pWB980-ori-CGT of the present invention; Figure 5 This is a plate image of Bacillus subtilis transformed with pWB980-ori-S5NU90 in Comparative Example 2; Figure 6 This is a plate image of Bacillus subtilis transformed with pWB980-ori-Q9UWN2 in Comparative Example 1; Figure 7This is a diagram showing the expression results of pWB980-ori-CGT constructed in the present invention in Bacillus subtilis; Figure 8 This is a diagram showing the expression results of pWB980-ori-S5NU90 constructed in Comparative Example 2 in Bacillus subtilis; Figure 9 This is the commercially available pWB980-ori plasmid map; Figure 10 This is the map of the pWB980-ori-CGT recombinant plasmid constructed by the present invention; Figure 11 This is the map of the pWB980-ori-Q9UWN2 recombinant plasmid constructed in Comparative Example 1; Figure 12 This is the map of the pWB980-ori-S5NU90 recombinant plasmid constructed in Comparative Example 2; DETAILED DESCRIPTION Specific implementation of the present invention: In order to better understand the present invention, a specific embodiment is used for illustration. It is worth emphasizing that the effects of this embodiment are not substantially different from those of various embodiments within the scope of protection of the present invention, including the respective reagents and the content ratios of the reagents. All of them can achieve the effects described in the present invention and solve the above-mentioned problems. Other combinations are not described here. (1) Construction of expression vector for cyclodextrin glucosyltransferase: The target gene obtained by whole gene synthesis of the amino acid sequence of SEQ ID NO. 1 was ligated with the vector plasmid pWB980-ori to obtain the expression vector pWB980-ori-CGT; (2) Construction of Bacillus subtilis: (2.1) Preparation of Bacillus subtilis competent cells: Streak Bacillus subtilis onto antibiotic-free LB plates and incubate overnight at 35-38°C. Inoculate a single colony from the plate into 50 mL of LB medium containing erythromycin (in a 250 mL Erlenmeyer flask) and incubate at 37°C in a shaker for 8-12 hours. Add LB medium preheated to 37°C containing erythromycin and dilute the culture to an A600 of 1.0. Add D-xylose to the dilution at an initial concentration of 1% (w / v). Incubate at 37°C at 200 rpm for 2 hours to obtain competent cells.

[0013] (2.2) Introduce the expression vector pWB980-ori-CGT into Bacillus subtilis competent cells to obtain recombinant Bacillus subtilis.

[0014] The expression vector pWB980-ori-CGT was mixed with competent cells and cultured in a shaker at 37°C and 200 rpm for 1.5 h to complete the transformation. Positive colonies were gently mixed with 40% glycerol and stored in a −80°C refrigerator.

[0015] (3) Application of Bacillus subtilis Bacillus subtilis is used to express and prepare cyclodextrin glucosyltransferase liquid by fermentation. Specifically: (3.1) Strain activation Thaw a tube of frozen recombinant Bacillus subtilis and streak it onto a Kan+ LB plate. Incubate at 35-38°C overnight. Pick a single colony and inoculate it into 4-5 mL Kan+ LB liquid medium. Incubate at 37°C, 200 rpm, for 10-12 hours.

[0016] (3.2) Seed tank culture The activated recombinant Bacillus subtilis suspension was inoculated into Kan+LB liquid medium at a 6% inoculum volume and cultured at 37°C for 18-20 hours.

[0017] (3.3) Fermentation tank culture Inoculate the seed solution into Kan+LB liquid medium at a 6% inoculum volume and culture at 37°C for 36-40 hours.

[0018] In order to more intuitively demonstrate the process advantages of the present invention, the construction and application method of the present invention using the expression vector of cyclodextrin glucosyltransferase and Bacillus subtilis is compared with the method using equivalent replacement of the same process. Comparative Example 1: The preparation method is the same as that in the example, except that the pWB980-ori plasmid is ligated with the target gene Q9UWN2 to obtain a recombinant plasmid; Comparative Example 2: The preparation method is the same as that in the example, except that the pWB980-ori plasmid is ligated with the target gene S5NU90 to obtain a recombinant plasmid; Testing and verification methods: 1. The electrophoresis diagram of enzyme digestion identification of recombinant plasmid is shown in ( Figure 1-3 ), Figure 1 This is the enzyme electrophoresis diagram of the pWB980-ori-CGT recombinant plasmid of the present invention; Among them: 1: pWB980-ori-CGT lane, 2: pWB980-ori-CGT digested with EcoRI and BamHI, M: KB Ladder DNA Marker; Figure 2This is the enzyme electrophoresis diagram of the pWB980-ori-Q9UWN2 recombinant plasmid in Comparative Example 1; Among them: 1: pWB980-ori-Q9UWN2 lane, 2: pWB980-ori-Q9UWN2 digested with EcoRI and BamHI, M: KB Ladder DNA Marker; Figure 3 The enzyme electrophoresis diagram of the pWB980-ori-S5NU90 recombinant plasmid in Comparative Example 2; Among them: 1: pWB980-ori-S5NU90 lane, 2: pWB980-ori-S5NU90 digested with EcoRI and BamHI, M: KB Ladder DNA Marker; For enzyme digestion identification of the recombinant plasmid, the gel bands at 2172bp, 2232bp and 2172bp corresponding to CGT, Q9UWN2 and S5NU90 were recovered and sequenced, and the obtained sequences were aligned with the codon-optimized CGT, Q9UWN2 and S5NU90 sequences through NCBI, with a matching value of 100%, indicating that the recombinant vector was successfully constructed.

[0019] 2. The recombinant plasmid was transformed into Bacillus subtilis, and the transformed competent cells were serially diluted. The dilutions were then spread on Kan+ LB plates and cultured at 37°C for 8-12 hours. It was found that Bacillus subtilis transformed with pWB980-ori-CGT of the present invention and pWB980-ori-S5NU90 of Comparative Example 2 grew on the Kan+ LB plate, while Bacillus subtilis transformed with pWB980-ori-Q9UWN2 of Comparative Example 1 did not grow on the plate, indicating that pWB980-ori-CGT and pWB980-ori-S5NU90 were successfully transformed into Bacillus subtilis, while the pWB980-ori-Q9UWN2 recombinant plasmid failed to be transformed. The plate culture results are shown as follows: Figure 4-6 .

[0020] 3. Expression Verification of Recombinant Engineering Bacteria The engineered bacterial seed liquid was transferred into 30 mL Kan + LB medium and cultured at 37°C for 24 h, 48 h, and 72 h. The enzyme solution was collected and centrifuged at 5000 rpm for 20 min. The obtained bacteria, the supernatant of the ultrasonically broken bacteria, and the fermentation supernatant (without concentration) were subjected to SDS-PAGE to detect the expression of the target protein.

[0021] The CGT target gene band was observed by SDS-PAGE gel electrophoresis. Figure 7It can be seen that there is a clear band at 78 kDa in the fermentation supernatant lane, which is the same as the protein molecular weight of pWB980-ori-CGT, indicating that the target gene can be secreted and expressed in the recombinant Bacillus subtilis. in, Figure 7 Expression results of pWB980-ori-CGT in Bacillus subtilis, T: total bacterial protein; S: supernatant of bacterial cell disruption; FS: fermentation supernatant (unconcentrated for loading); Depend on Figure 8 It can be seen that the expression results of pWB980-ori-S5NU90 in Bacillus subtilis, where T: total bacterial protein; S: supernatant of bacterial disruption; FS: fermentation supernatant (unconcentrated and loaded), no target band was observed in the SDS-PAGE image of S5NU90, indicating that S5NU90 may not be expressed in Bacillus subtilis or the expression level is low.

[0022] As a supplement, the genetically engineered bacteria constructed by the present invention were used as an experimental group, and the preparation method was the same as in the example, except that the amino acid at position 182 of the amino acid sequence of the cyclodextrin glucosyltransferase was glutamic acid. The genetically engineered bacteria constructed were used as a control group for comparison; 4.1 Determination of bacterial content Table 1: Determination of bacterial cell content by spectrophotometry

[0023] The bacterial content of the parallel experimental group was higher than that of the control group, proving that the recombinant Bacillus subtilis can reach the required bacterial content after a short period of cultivation.

[0024] 4.2 Determination of β-CGTase activity Table 2: Determination of β-CGTase activity using the blue value method

[0025] The enzyme activity of the parallel experimental group was 1.6 times higher than that of the control group, with an average enzyme activity of 1224u / ml. The fermentation time was shortened by 32h, a 44% reduction in time, saving costs and improving enzyme activity.

[0026] 4.3 Determination of cyclodextrin glucosyltransferase conversion rate Through the conversion experiment, the yield of the recombinant Bacillus subtilis fermentation broth and the original production strain fermentation broth were calculated using the conversion rate formula. Figure 6 It can be seen that the average yield of the recombinant Bacillus subtilis fermentation broth is 45.5%, which is 7.3% higher than the average yield of the control group.

[0027] Table 3: Cyclodextrin Glucosyltransferase Conversion Rate

[0028] In summary, the present invention creatively uses genetic engineering methods to introduce nucleotides encoding the amino acid sequence of the cyclodextrin glucosyltransferase using the plasmid pWB980-ori as a vector, obtains the expression vector pWB980-ori-CGT, and constructs Bacillus subtilis, which can stably and efficiently express the cyclodextrin glucosyltransferase. The yield of β-cyclodextrin generated at the same level is as high as 44.3%, and the Bacillus subtilis does not need to be isolated and can be directly used for treatment using the whole fermentation reaction enzyme liquid. The constructed genetically engineered bacteria has a shortened fermentation cycle, high enzyme activity and high conversion rate, saves production costs, and has potential application prospects.

[0029] The present invention creatively discovered a nucleotide sequence encoding the amino acid sequence of the cyclodextrin glucosyltransferase, wherein the 182nd amino acid in the amino acid sequence of the cyclodextrin glucosyltransferase is glutamine; the enzyme exhibits high enzymatic activity (1224 U / mL) in a heterologous expression system and shortens the fermentation cycle by 44%, and in particular, can be stably recombined and expressed with the plasmid pWB980-ori, and has relatively stable passage performance.

Claims

1. An expression vector for cyclodextrin glucosyltransferase, characterized in that The amino acid sequence of the cyclodextrin glucosyltransferase is shown in SEQ ID NO. 1; The nucleotide sequence encoding the amino acid sequence of the cyclodextrin glucosyltransferase is as shown in SEQ ID NO.

2.

2. The expression vector of cyclodextrin glucosyltransferase according to claim 1, characterized in that The method for constructing the expression vector of the cyclodextrin glucosyltransferase is as follows: the target gene is obtained by whole gene synthesis of the amino acid sequence of SEQ ID NO. 1, and the target gene is connected with the vector plasmid pWB980-ori to obtain the expression vector pWB980-ori-CGT.

3. Construction of Bacillus subtilis using the expression vector for cyclodextrin glucosyltransferase according to claim 1 or 2, characterized in that: The constructed expression vector pWB980-ori-CGT is introduced into the host cell Bacillus subtilis to obtain the recombinant Bacillus subtilis, comprising the following steps: (1) Preparation of Bacillus subtilis competent cells: (2) Introduce the expression vector pWB980-ori-CGT into Bacillus subtilis competent cells to obtain recombinant Bacillus subtilis.

4. The construction of Bacillus subtilis according to claim 3, characterized in that The preparation of the Bacillus subtilis competent cells includes: streaking Bacillus subtilis on a plate, inoculating a single colony from the plate into a culture medium, culturing on a shaking platform, adding preheated LB culture medium containing erythromycin, diluting the culture solution, and adding D-xylose for culturing to obtain competent cells.

5. The construction of Bacillus subtilis according to claim 3, characterized in that The method of introducing the expression vector pWB980-ori-CGT into Bacillus subtilis competent cells to obtain recombinant Bacillus subtilis comprises: The expression vector pWB980-ori-CGT was mixed with competent cells and cultured in a shaker at 37°C and 200 rpm for 1.5 h to complete the transformation. Positive colonies were gently mixed with 40% glycerol and stored in a −80°C refrigerator.

6. Use of Bacillus subtilis, using the Bacillus subtilis described in claims 3-5 to express and prepare cyclodextrin glucosyltransferase solution by fermentation.

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