Escherichia coli capable of producing 6-alpha-glucosyltransferase and application of escherichia coli in production of highly branched starch

Abstract

The invention relates to Escherichia coli for producing 6-alpha-glucosyltransferase and application of the Escherichia coli in production of highly branched starch, and belongs to the technical field of enzyme engineering. According to the invention, a pET20b (+) vector is used for heterologously expressing a 6-alpha-glucosyltransferase coding gene derived from Sporosarcina globispora C11 in escherichia coli, and a signal peptide is designed at the upstream of the gene, so that the secretion and activity of enzyme can be promoted. The Escherichia coli and the microbial agent constructed based on the method can convert high amylose starch into highly branched starch, the reaction condition is mild, and the operation is simple and convenient. By directionally introducing an exogenous gene and optimizing an expression system, the hydrolytic activity of the recombinant 6-alpha-glucosyltransferase reaches 80-100U / mL, the proportion of a starch branched chain structure is remarkably increased, the problems of low modification efficiency and high cost of a traditional enzyme method are solved, and an efficient biological catalysis tool is provided for the fields of food, materials and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of enzyme engineering, in particular to Escherichia coli producing 6-α-glucosyltransferase and its application in the production of highly branched starch. Background Art

[0002] Starch is the main carbon and energy storage polysaccharide in nature, a rich carbohydrate, mainly present in the leaves, seeds, roots and tubers of many plants. Starch is the main energy source for the human body. According to its hydrolysis rate in the gastrointestinal tract, starch can be divided into rapidly digestible starch, slowly digestible starch, and resistant starch.

[0003] Since rapidly digestible starch can be quickly digested and absorbed by the human body, it will cause a sharp rise in postprandial blood glucose levels, which in turn leads to metabolic diseases such as obesity and type II diabetes. The content of rapidly digestible starch in natural starch is generally high, and during the gelatinization process of natural starch, its digestion rate will increase sharply, while the content of slowly digestible starch and resistant starch will decrease significantly. Highly branched starch has unique effects in maintaining blood glucose homeostasis and preventing chronic diseases, and can meet the needs of consumers for food nutrition and health. In recent years, it has become a research hotspot in aspects such as food nutrition. Therefore, modifying natural starch to prepare slowly digestible highly branched starch is crucial for human health. Biological enzyme modification has become a research hotspot in recent years due to its unique advantages. In current research, hydrolases and glycosyltransferases are commonly used enzymes for biological enzyme modification. After modification with hydrolases such as α-amylase, β-amylase, and pullulanase, the content of rapidly digestible starch in the products all decreases, and the content of slowly digestible starch and resistant starch increases to varying degrees.

[0004] 6-α-glucosyltransferase is an enzyme with the functions of catalyzing the exohydrolase and transglycosylation of the non-reducing end of α-1,4-glucan. When acting on starch, it will first excise glucose at the non-reducing end of α-1,4-glucan and connect 2-5 α-1,6 bonds through continuous transglycosylation to produce isomaltooligosyl-α-1,4-glucan. The highly branched starch produced by its transglycosylation has a variety of functional activities and has broad application prospects in the food and pharmaceutical industries. However, in the prior art, the application of 6-α-glucosyltransferase mainly focuses on the production of cyclic alternans and its branched derivatives. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides Escherichia coli producing 6-α-glucosyltransferase and its application in the production of highly branched starch. In the present invention, the pET20b(+) vector is used to heterologously express the 6-α-glucosyltransferase-encoding gene derived from Sporosarcina globispora C11 in Escherichia coli, and it is applied to modify high amylose starch, changing the chain length distribution of the product.

[0006] The first object of the present invention is to provide an Escherichia coli expressing 6-α-glucosyltransferase, which heterologously expresses the 6-α-glucosyltransferase-encoding gene derived from Sporosacina globispora C11.

[0007] Further, the Escherichia coli uses the pET20b(+) vector to express the 6-α-glucosyltransferase-encoding gene.

[0008] Further, the nucleotide sequence of the 6-α-glucosyltransferase-encoding gene is as shown in SEQ ID NO.1.

[0009] Further, a signal peptide is included upstream of the 6-α-glucosyltransferase-encoding gene.

[0010] Further, the sequence of the signal peptide is as shown in SEQ ID NO.3.

[0011] The second object of the present invention is to provide a microbial inoculant containing the above Escherichia coli.

[0012] The third object of the present invention is to provide the application of the above Escherichia coli or the above microbial inoculant in the production of highly branched starch.

[0013] The fourth object of the present invention is to provide a method for producing highly branched starch, using high amylose starch as a substrate and adding the above Escherichia coli or the above microbial inoculant to the reaction system.

[0014] In one embodiment of the present invention, a maltotriose solution is prepared with sodium acetate buffer solution, the maltotriose substrate is taken and kept at a constant temperature in a water bath, the enzyme solution is added, and after the reaction, it is inactivated by boiling water bath.

[0015] Further, the reaction temperature is 40 - 50 °C.

[0016] In one embodiment of the present invention, the reaction temperature is 45 °C.

[0017] Further, the reaction pH value is 6 - 8.

[0018] The beneficial effects of the present invention:

[0019] By heterologously expressing the 6-α-glucosyltransferase from Sporosacina globispora C11 in Escherichia coli, the present invention endows Escherichia coli with the ability to efficiently catalyze the branching of starch, realizes the safe and efficient production of 6-α-glucosyltransferase, and the hydrolysis activity of the recombinant 6-α-glucosyltransferase reaches 80-100 U / mL. The recombinant 6-α-glucosyltransferase secreted and expressed by the recombinant Escherichia coli is applied to the production of highly branched starch. The reaction system does not require complex purification steps, has a high substrate conversion rate, a significant increase in the degree of branching of the product, and the optimal reaction conditions of the recombinant enzyme are mild, the optimal reaction pH value is close to neutral, the reaction conditions reduce energy consumption, meet the requirements of industrial production, and have good application prospects. Description of the Drawings

[0020] In order to make the content of the present invention easier to be clearly understood, the following further details the present invention according to the specific embodiments of the present invention in combination with the drawings, where

[0021] Figure 1 Agarose gel electrophoresis detection of recombinant pET-20b(+) positive clones in the examples of the present invention; among them, M: DNA Marker; Lane 1: recombinant pET-28a(+) / sp positive clone;

[0022] Figure 2 Effect of temperature on the activity of recombinant 6-α-glucosyltransferase in the examples of the present invention;

[0023] Figure 3 Effect of pH value on the activity of recombinant 6-α-glucosyltransferase in the examples of the present invention;

[0024] Figure 4 pH stability of recombinant 6-α-glucosyltransferase in the examples of the present invention;

[0025] Figure 5 HPAEC-PAD detection results of the product of recombinant 6-α-glucosyltransferase from Sporosacina globispora C11 in the examples of the present invention;

[0026] Figure 6 Peak delineation of the HPAEC-PAD detection results of the product of recombinant 6-α-glucosyltransferase from Sporosacina globispora C11 in the examples of the present invention;

[0027] Figure 7 Peak delineation of the HPAEC-PAD detection results of the product of recombinant 6-α-glucosyltransferase from paenibcillus sp 598k in the examples of the present invention;

[0028] Figure 8 This is for the analysis of the transglycosylation mechanism of the recombinant 6-α-glucosyltransferase in the embodiments of the present invention. Detailed implementation manners

[0029] The present invention will be 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 be able to implement it, but the examples given are not intended to limit the present invention.

[0030] The culture media involved in the following examples are as follows:

[0031] LB medium: 1% tryptone, 0.5% yeast extract, 1% sodium chloride, pH = 7.0;

[0032] TB medium: 1.2% tryptone, 2.4% yeast extract, 0.4% glycerol, 17 mM KH2PO4, 72 mM K2HPO4, pH = 6.0.

[0033] Example 1: Construction of an Escherichia coli secretion expression system

[0034] Primers were designed according to the 6-α-glucosyltransferase encoding gene (shown in SEQ ID NO.1) from Sporosacina globispora C11, where:

[0035] The upstream primer is 5’-CCATGGCCTATGTGAGCAG-3’ (SEQ ID NO.4);

[0036] The downstream primer is 5’-GCGTGACCAAACAGCTCGA-3’ (SEQ ID NO.5).

[0037] Primers were designed according to the 6-α-glucosyltransferase encoding gene (shown in SEQ ID NO.2) from paenibcillus sp 598k, where:

[0038] The upstream primer is 5’-CCATGGCCGCGGGCCT-3’ (SEQ ID NO.6);

[0039] The downstream primer is 5’-GCGCGCGCCGCTCGAG-3’ (SEQ ID NO.7).

[0040] Primers were designed according to the pET-20b(+) vector sequence, where:

[0041] The upstream primer is 5’-TGCCCAGCCGGCGATGGCCATGGCCTATGTGA-3’ (SEQ ID NO.8);

[0042] The downstream primer is 5’-CTCGAGCACCACCACCACCACCACTGAGATCCGGC TGC-3’ (SEQ ID NO.9).

[0043] The signal peptide sequence of the pET-20b(+) vector is shown as SEQ ID NO.3.

[0044] Clone the target genes of 6-α-glucosyltransferase from Sporosacina globispora C11 and paenibcillus sp598k containing the coding signal peptide sequence and the pET20b(+) vector. The PCR system for the 6-α-glucosyltransferase gene is as follows: 25 μL of 2×phanta Max Master Mix (Dye plus), 2 μL of forward primer (20 μM), 2 μL of reverse primer (20 μM), 1 μL of template DNA, and add double-distilled water to 50 μL. The PCR amplification conditions are: pre-denaturation at 95°C for 3 min; then perform 30 cycles (95°C for 15 s, 60°C for 15 s, 72°C for 3.5 min); finally, incubate at 72°C for 5 min. The PCR system for the pET20b(+) vector is 25 μL of 2×phanta Max Master Mix (Dye plus), 2 μL of forward primer (20 μM), 2 μL of reverse primer (20 μM), 1 μL of template DNA, and add double-distilled water to 50 μL. The PCR amplification conditions are: pre-denaturation at 95°C for 3 min; then perform 30 cycles (95°C for 15 s, 60°C for 15 s, 72°C for 4 min); finally, incubate at 72°C for 5 min.

[0045] The PCR products of the 6-α-glucosyltransferase gene from different sources and the pET20b(+) vector were subjected to nucleic acid electrophoresis and then the gel was cut and recovered. The agarase gene and the pET20b(+) vector were ligated using homologous recombination method. The ligation system was as follows: 2 μL of purified 6-α-glucosyltransferase PCR fragment (50 ng / μL), 1 μL of purified pET20a(+) vector PCR fragment, 4 μL of 5×CEⅡ Buffer, 2 μL of ExnaseⅡ, and 10 μL of ddH2O. The homologous recombination conditions were: incubate at 37 °C for 30 min, then transform Escherichia coli JM109, spread on an LB plate containing 20 μg / mL ampicillin, pick the transformants for sequencing and nucleic acid electrophoresis verification, and obtain the expression vector pET-20b(+) / sp containing the 6-α-glucosyltransferase gene. The expression vector was transformed into Escherichia coli BL21(DE3) to obtain the genetically engineered strains E. coli BL21(DE3)(pET-20b(+) / sp) and E. coli BL21(DE3)(pET-20b(+) / 598k).

[0046] The expression vector pET-20b(+) / sp containing the 6-α-glucosyltransferase gene was verified by nucleic acid electrophoresis, and the results were as Figure 1 . The target gene fragment of pET-20b(+) / sp was sequenced, and the sequencing result was correct.

[0047] Example 2: Fermentation production of recombinant 6-α-glucosyltransferase

[0048] 100 μL of the glycerol stock solution containing the genetically engineered strain E. coli BL21(DE3)(pET-20b(+) / sp) was inoculated into 50 mL of LB medium containing 20 μg / mL ampicillin, and cultured overnight in a shaker at 37 °C and 200 rpm; transferred to 50 mL of TB medium containing 20 μg / mL ampicillin at an inoculation amount of 4%, and enriched and cultured in a shaker at 25 °C and 200 rpm for 6 h, then IPTG with a final concentration of 0.05 mM was added, and the induction was continued in a shaker at 25 °C (200 r / min) for 36 h. After fermentation, the fermentation broth was centrifuged to collect the cells, and the cells were resuspended in 50 mM PBS (pH = 7.0) buffer in equal proportion, sonicated, and then centrifuged at high speed to collect the supernatant, which was the crude enzyme solution of recombinant 6-α-glucosyltransferase.

[0049] According to the same method as above, the crude enzyme solution of recombinant 6-α-glucosyltransferase from the source of paenibcillussp 598k was obtained by fermenting E. coli BL21(DE3)(pET-20b(+) / 598k).

[0050] Example 3: Determination of the Transglycosylation Activity of Recombinant 6-α-Glucosyltransferase at Different Temperatures and pH Values

[0051] Prepare a 0.125% (w / v) high amylose starch substrate with sodium hydroxide solution. Add 0.1 mL of the crude enzyme solution of recombinant 6-α-glucosyltransferase from different sources to 0.9 mL of the substrate, react at 45 °C for 10 min, cool in an ice bath after boiling water bath for 10 min, add 1 mL of iodine working solution, and measure the absorbance at 660 nm after reacting for 5 min. Enzyme activity definition: The amount of enzyme required to reduce the absorbance by 1% per minute compared with the inactivated enzyme is 1 enzyme activity unit (U). The fermentation crude enzyme solution of recombinant 6-α-glucosyltransferase was prepared according to Example 2, where the induction temperature was 25 °C and the fermentation time was 36 h.

[0052] The activities of the recombinant 6-α-glucosyltransferase from Sporosacina globispora C11 at different temperatures and pH values are respectively as Figure 2 and Figure 3 shown. It can be seen that the enzyme has the highest activity at 45 °C; it has the highest activity when the pH is 7 and has relatively high enzyme activity within the range of pH 6 - 8.

[0053] Under the optimal conditions (45 °C; pH = 7), compared with the 6-α-glucosyltransferase from paenibcillus sp 598k, the hydrolysis activity of the 6-α-glucosyltransferase solution of Sporosacina globispora C11 reaches 80 - 100 U / mL, while the 6-α-glucosyltransferase from paenibcillus sp 598k can only reach 50 - 70 U / mL.

[0054] Example 4: Determination of the Stability of Recombinant 6-α-Glucosyltransferase at Different pH Values The fermentation crude enzyme solution of recombinant 6-α-glucosyltransferase was prepared according to Example 2, where the induction temperature was 25 °C and the fermentation time was 36 h. Dilute the enzyme solution 1:1 with buffers of different pH values, incubate at 4 °C for 2 h, and then measure the residual enzyme activity of the enzyme, with the activity of the unincubated enzyme solution being 100%. The transglycosylation activities of recombinant 6-α-glucosyltransferase at different pH values are as Figure 4 shown. It can be seen that the enzyme has strong stability in buffers with pH = 6 - 8.

[0055] Example 5: Analysis of the Transglycosylation Products of Recombinant 6-α-Glucosyltransferase

[0056] The method for determining the transglycosylation products of recombinant 6-α-glucosyltransferase is as follows: Using 0.125% (w / v) high amylose starch as the substrate, add an appropriate amount of 6-α-glucosyltransferase solution (the volume ratio of the substrate to the enzyme solution is 10:1), place it in a constant temperature shaking water bath at 45°C for reaction. After 12h and 24h, take samples, boil at high temperature for 30min to inactivate the enzyme. The reaction solution is preheated in a water bath shaker at 37°C for 10min, and then 100 μL of isoamylase (10000U / mL) is added for debranching for 24h, and boil in a water bath for 30min to terminate the reaction. Centrifuge and take the supernatant. Then dilute it 100 times, pass through a 0.22 μm aqueous filter membrane, and detect it by HPAEC-PAD. Use HPAEC-PAD to detect the chain length distribution in the product,

[0057] The results are as Figure 5 and Figure 6 shown. After the recombinant 6-α-glucosyltransferase from Sporosacina globispora C11 reacts with the high amylose starch solution, the chain length distribution of the product changes significantly. Among them, the number of chain segments with DP of 13 - 36 increases significantly, and the long chains with DP > 36 decrease significantly. This shows that 6-α-glucosyltransferase can make the branched chains of high amylose starch more, producing highly branched starch.

[0058] Compared with the 6-α-glucosyltransferase from Paenibcillus sp 598k (the results are shown in Figure 7 ), the recombinant 6-α-glucosyltransferase from Sporosacina globispora C11 has a lower proportion of small molecule chain segments generated after 24h of reaction, stronger anti-digestion ability, and better modification effect.

[0059] Table 1 Transglycosylation products of 6-α-glucosyltransferase from Sporosacina globispora C11 and high amylose starch

[0060]

[0061] Table 2 Transglycosylation products of 6-α-glucosyltransferase from Paenibcillus sp 598k and high amylose starch

[0062]

[0063]

[0064] Example 6: Analysis of the mechanism of transglycosylation products of recombinant 6-α-glucosyltransferase

[0065] Prepare a 2% (W / V) maltotriose solution using 50 mM sodium acetate buffer as the solvent. Take 200 μL of the maltotriose substrate and incubate it in a water bath at 45 °C for 10 min. Add 200 μL of the enzyme solution and react at 45 °C for 1 h. After the reaction, inactivate the enzyme by boiling in a water bath for 10 min.

[0066] Add 800 μL of disodium hydrogen phosphate - citric acid buffer (pH = 7), incubate at 40 °C for 10 min, add 3 μL of malt phosphorylase and react for 1 h. Inactivate the enzyme by boiling in a water bath for 10 min. Take 18 μL of the reaction solution and add 1.8 mL of GOD-POD reagent, incubate at 37 °C for 10 min, and measure the absorbance at a wavelength of OD 520 nm. Its transglycosylation mechanism is as Figure 8 shown, indicating that 6-α-glucosyltransferase can accurately hydrolyze and transfer a single glucose molecule.

[0067] Obviously, the above examples are only for clear illustration and not limitations on the implementation methods. For those of ordinary skill 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 implementation methods here. And the obvious changes or modifications derived therefrom are still within the protection scope of this invention.

Claims

1. An Escherichia coli expressing 6-α-glucosyltransferase, characterized in that: The Escherichia coli heterologously expresses the 6-α-glucosyltransferase-encoding gene derived from Sporosacina globispora C11.

2. The Escherichia coli according to claim 1, characterized in that: The Escherichia coli uses the pET20b(+) vector to express the 6-α-glucosyltransferase-encoding gene.

3. The Escherichia coli according to claim 1, characterized in that: The nucleotide sequence of the 6-α-glucosyltransferase-encoding gene is as shown in SEQ ID NO.

1.

4. The Escherichia coli according to claim 1, characterized in that: The upstream of the 6-α-glucosyltransferase-encoding gene contains a signal peptide.

5. The Escherichia coli according to claim 4, characterized in that: The sequence of the signal peptide is as shown in SEQ ID NO.

3.

6. A microbial inoculum comprising the Escherichia coli according to any one of claims 1-5.

7. Use of the Escherichia coli according to any one of claims 1-5 or the microbial inoculum according to claim 6 in the production of highly branched starch.

8. A method for producing hyperbranched starch, characterized in that: Using high amylose starch as a substrate, adding the Escherichia coli according to any one of claims 1-5 or the microbial inoculum according to claim 6 to the reaction system.

9. The method according to claim 8, characterized in that: The reaction temperature is 40-50 °C.

10. The method according to claim 8, wherein: The reaction pH value is 6-8.

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