A specific inositol 1, 2, 3-triphosphate receptor 1 gene and application thereof

Abstract

The present application relates to a kind of specific synthesis plant inulin of inulin sucrose enzyme mutant and its application, the mutant is the 300th aspartic acid of wild type inulin sucrose enzyme from Lactobacillus reuteri (Lactobacillus reuteri) Limosilactobacillus reuteri ) Mutated into alanine.It precisely cuts off the ability of wild type enzyme to synthesize million Dalton high molecular weight microbial inulin at molecular level, while perfectly retaining the synthesis ability of medium-low degree of polymerization inulin.This mutant fundamentally solves the problem of wild type enzyme product molecular weight distribution extremely wide, component complex, realizes the efficient biosynthesis of specific inulin.At the same time, it can greatly reduce the difficulty and energy consumption of downstream separation and purification process, significantly reduce production cost.In the high-quality inulin large-scale green manufacturing of food and health product industry, it has great application value.

Description

Technical Field

[0001] This invention belongs to the field of enzyme genetic engineering technology, specifically relating to a mutant inulin sucrase that specifically synthesizes plant inulin and its applications. Background Technology

[0002] Inulin, as a high-quality water-soluble dietary fiber and prebiotic, has extremely high commercial value in the food, health product, and pharmaceutical fields. Currently, commercially available inulin mainly relies on extraction from plants such as chicory and Jerusalem artichoke. The degree of polymerization (DP) of these plant-derived inulin typically ranges from 3 to 40, and the molecular weight is usually ≤10. 3 Inulin, with a concentration of g / mol, possesses excellent physicochemical properties and processing performance. However, plant extraction methods are limited by agricultural planting cycles and climatic conditions, and the extraction and purification processes are cumbersome, resulting in high production costs. In contrast, inulin is synthesized in vitro using inexpensive sucrose as a substrate through microbial enzymatic methods (such as inulosucrase). This method is not limited by raw materials, and the raw materials are inexpensive, making it a highly promising green alternative.

[0003] Lactobacillus reuteri ( Limosilactobacillus reuteri Wild-type inulin sucrase possesses the ability to efficiently convert sucrose. However, this wild-type enzyme exhibits broad product specificity during catalysis: it not only synthesizes products with a degree of polymerization similar to plant-derived inulin but also possesses extremely strong chain extension capabilities, continuously synthesizing high-molecular-weight microbial inulin with a degree of polymerization reaching millions of daltons (Da). The inulin synthesized by this wild-type enzyme has an extremely wide molecular weight distribution. On the one hand, the application scenarios for high-molecular-weight microbial inulin are yet to be developed, and it cannot directly compete with and replace existing plant-derived inulin products. On the other hand, the presence of complex mixtures greatly increases the difficulty and cost of downstream separation and purification of inulin with specific chain lengths, thus leading to serious limitations in industrial applications. Therefore, synthesizing plant-derived inulin-like products (inulin with a degree of polymerization ranging from 3 to 40) with a degree of polymerization similar to plant-derived polysaccharides is crucial to overcoming the limitations of microbial inulin in industrial applications.

[0004] Achieving the synthesis of inulin with specific chain lengths is currently a key technological challenge in the field of inulin synthesis using inulin sucrase. Therefore, it is crucial to develop improved mutants capable of specifically synthesizing plant-derived inulin by precisely controlling the catalytic properties of inulin sucrase at the molecular level through protein engineering, thereby blocking its ability to excessively extend and synthesize high molecular weight inulin. This would meet the food industry's demand for plant-derived inulin with medium to low polymerization degrees. Summary of the Invention

[0005] The purpose of this invention is to overcome the defects in the prior art and provide a specific inulin sucrase mutant that synthesizes plant-like inulin and its application. It can synthesize inulin with a degree of polymerization similar to that of plants, so as to replace plant inulin.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A specific inulin sucrase mutant for synthesizing plant-like inulin, the amino acid sequence of which is shown in SEQ ID NO.2.

[0007] As a further technical solution, the inulin sucrase mutant is derived from microorganisms. LimosiLactobacillus reuteri It was obtained by mutating asparagine to alanine at position 300 of the amino acid sequence of inulin sucrase (Genbank: AF459437.1, protein accession number: AF459437.1).

[0008] A gene encoding the inulin sucrase mutant, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0009] A recombinant vector carrying the gene.

[0010] A recombinant cell expressing the inulin sucrase mutant, carrying the gene, or carrying the recombinant vector, wherein the recombinant cell uses bacteria or fungi as the expression host.

[0011] As a further technical solution, the recombinant cells are... E. coli BL21(DE3) was used as the expression host, and pET-22b(+) was used as the expression vector.

[0012] As a further technical solution, the method for preparing the recombinant cells includes the following steps: (1) Utilizing sources LimosiLactobacillus reuteri Protein sequence homology modeling of inulin sucrase was used to identify mutation sites. (2) Design site-directed mutagenesis primers for mutants, and use the vector pET-22b(+)-LrIS carrying the inulin sucrase gene as a template to construct the mutant plasmid pET-22b(+)-N300A by site-directed mutagenesis; (3) Transform Escherichia coli with the mutant plasmid pET-22b(+)-N300A. E. coli BL21(DE3) was used to select positive monoclonal antibodies to obtain recombinant cells.

[0013] A method for preparing the inulin sucrase mutant involves inducing and culturing the recombinant cells to produce the inulin sucrase mutant.

[0014] As a further technical solution, the fermentation broth of the induced culture needs to be centrifuged, the cells collected and resuspended, then ultrasonically disrupted, and finally purified by nickel ion affinity chromatography to obtain the inulin sucrase mutant.

[0015] The application of the inulin sucrase mutant in the catalytic production of plant-like inulin from sucrose, wherein the degree of polymerization of the plant-like inulin is 3-40 (medium to low degree of polymerization).

[0016] As a further technical solution, the molecular weight of the plant-like inulin is [missing information]. .

[0017] As a further technical solution, inulin is produced by enzymatic reaction using sucrose as a substrate under the action of an inulin sucrase mutant. The enzymatic reaction temperature is 30℃, pH 6.5, and the reaction time is 12h.

[0018] As a further technical solution, in the enzymatic reaction system, the concentration of the substrate sucrose is 300 g / L, and the amount of inulin sucrase mutant added is 10 μg / mL.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The Lactobacillus reuteri-derived inulin sucrase mutant N300A provided by this invention precisely interrupts the wild-type enzyme's ability to synthesize high molecular weight microbial inulin (million daltons) at the molecular level, while perfectly retaining the ability to synthesize plant-based inulin (medium to low degree of polymerization). This mutant fundamentally solves the problem of the wild-type enzyme's products having an extremely wide molecular weight distribution and complex composition, achieving efficient biosynthesis of specific inulin; at the same time, it can greatly reduce the difficulty and energy consumption of downstream separation and purification processes, significantly reducing production costs.

[0020] 2. Utilizing the mutant N300A of this invention, inexpensive and readily available sucrose can be directly used industrially as a single substrate to achieve all-weather, high-efficiency inulin production through in vitro enzymatic catalysis. This helps to overcome the limitations imposed by plant growth cycles, climate change, and land resources, providing a technical solution for the large-scale, green, and low-carbon manufacturing of high-quality inulin. Attached Figure Description

[0021] Figure 1 This is a chromatogram of the degree of polymerization of a plant inulin standard in one embodiment of the present invention; Figure 2 This is a chromatogram of the degree of polymerization of the reaction product of the wild-type enzyme LrIS in one embodiment of the present invention; Figure 3 This is a chromatogram of the degree of polymerization of the reaction product of the mutant enzyme N300A in one embodiment of the present invention; Figure 4This is a molecular weight distribution diagram of the reaction products of the wild-type enzyme LrIS in one embodiment of the present invention; Figure 5 This is a molecular weight distribution diagram of the reaction product of the mutant enzyme N300A in one embodiment of the present invention; Figure 6 This is a comparison diagram of the relative enzyme activities of wild-type inulin sucrase and mutant enzyme N300A in one embodiment of the present invention. Detailed Implementation

[0022] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Additionally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0024] In this invention, 1. Culture medium LB liquid medium: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride.

[0025] LB solid medium: LB liquid medium supplemented with 15 g / L agar 2. The detection methods involved are as follows: 1) Average molecular weight of polysaccharides: Determined using high performance size exclusion chromatography (HPSEC). The HPSEC was equipped with a differential refractive index detector (Waters 2410) and an Ultrahydrogel-Liner column (7.8 × 300 mm). The mobile phase was 0.1 mol / L sodium nitrate, the flow rate was 0.5 mL / min, and the column temperature was 40 °C. The degree of polymerization of polysaccharides was determined using high performance anion exchange chromatography (HPAEC). The HPAEC was equipped with a pulsed amperometric detector and a Dionex CarboPac PA200 column. Mobile phase A was 150 mmol / L sodium hydroxide, and mobile phase B was 600 mmol / L sodium acetate and 150 mmol / L sodium hydroxide, the flow rate was 0.5 mL / min, and the column temperature was 30 °C. Polysaccharides with different degrees of polymerization were separated using a linear gradient of 0–70% buffer B.

[0026] 2) Glucose production: Determined by high-performance liquid chromatography (HPLC). The instrument used was a Waters e2695 HPLC system equipped with a Waters 2414 differential refractive index detector and a Sugar-Pak I column (6.5 mm × 300 mm, Waters). Ultrapure water was used as the mobile phase, and the flow rate and column temperature were set to 0.4 ml / min and 85 °C, respectively. One unit of total enzyme activity was defined as the amount of enzyme required to catalyze the release of 1 μmol of glucose per minute. The activity of wild-type inulin sucrase was set to 100%.

[0027] 3. Unless otherwise specified, all raw materials used in this invention are commercially available.

[0028] 4. Plant-like inulin: In this invention, plant-like inulin refers to inulin produced by microbial enzymatic methods, and whose degree of polymerization is the same as or similar to that of inulin produced by plant extraction.

[0029] Example 1: Preparation of inulin sucrase (Lare121-IS) mutant plasmid 1. Construction of recombinant plasmid pET-22b(+)-LrIS: derived from LimosiLactobacillus reuteri The inulin sucrase gene accession number is GenBank: AF459437.1, and the protein accession number is AF459437.1. The N-terminal 120 amino acid residues (corresponding to 360 bases) and C-terminal 97 amino acid residues (corresponding to 291 bases) were truncated, retaining amino acids from positions 121 to 696. The truncated recombinant enzyme was named LrIS and is considered wild-type inulin sucrase (abbreviated as wild-type enzyme). The wild-type inulin sucrase gene fragment was ligated to the pET-22b(+) restriction site. Nde I and Xho Between I and II, the wild-type enzyme recombinant plasmid pET-22b(+)-LrIS was obtained.

[0030] 2. Construction of the wild-type inulin sucrase mutant plasmid pET-22b(+)-LrIS-N300A (hereinafter referred to as mutant plasmid): Using the wild-type enzyme recombinant plasmid pET-22b(+)-LrIS as a template, N300A site-directed mutations were introduced by PCR. Sequencing verification results showed that no random mutations occurred except for the required mutation site. Therefore, the mutant plasmid pET-22b(+)-LrIS-N300A was successfully constructed. The nucleotide sequence of the inulin sucrase mutant is shown in SEQ ID NO. 1, and the amino acid sequence is shown in SEQ ID NO. 2.

[0031] The N300A mutant primers are shown below: (Underlined text indicates mutants) N300A forward mutation primer: 5'-GGTGCCGAT GCT ATTGCAATGCGTGATGC -3'; N300A reverse mutation primer: 5'-GCATTGCAAT AGC ATCGGCACCTTTGTTAG -3'; The composition of the PCR reaction system is shown in Table 1, using the cloning vector pET-22b(+)-LrIS carrying the inulin sucrase target gene as a template.

[0032] Table 1: Composition of the PCR reaction system

[0033] The PCR amplification conditions were as follows: 95 °C pre-denaturation for 3 min; followed by 95 °C denaturation for 0.5 min, 56 °C annealing for 0.5 min, and 72 °C extension for 3.5 min, for 26 cycles; and finally, incubation at 72 °C for 5 min.

[0034] The PCR amplification products were detected by agarose gel electrophoresis and purified by gel extraction.

[0035] The PCR amplification products, purified by agarose gel electrophoresis, were then subjected to restriction endonuclease... Nde I and Xho After digestion with enzyme I, the cells were ligated into the vector pET-22b(+) and transformed into E. coli DH5α competent cells. After overnight culture in LB solid medium containing 50 µg / mL ampicillin, single clones were picked and cultured in LB liquid medium containing 50 µg / mL ampicillin. Then, the mutant plasmid pET-22b(+)-LrIS-N300A was extracted and transformed into host E. coli BL21(DE3) competent cells. The mutant plasmid was identified as the correct mutation by sequencing, and the plasmid containing the mutant, namely the mutant plasmid pET-22b(+)-LrIS-N300A, was prepared.

[0036] Example 2: Expression and purification method of inulin sucrase mutant N300A 1. The pET-22b(+)-LrIS prepared in Example 1 and the pET-22b(+)-LrIS-N300A containing the mutant plasmid were transformed into Escherichia coli BL21(DE3) cells. Positive transformants were picked and cultured overnight in LB liquid medium at 37 °C and 200 rpm in shake flasks. Then, they were inoculated into LB liquid medium and cultured at 37 °C until the OD value reached 0.6~0.8. The temperature was then lowered to 28 °C, and IPTG was added to a final concentration of 1 mM to induce fermentation for 6 h to obtain the fermentation broth.

[0037] 2. Centrifuge the prepared fermentation broth at 4 °C and 10,000 rpm for 20 min, and collect the precipitate (cells). Add 20 mL of buffer (50 mM PIPES, 200 mM NaCl, pH adjusted to 7 with HCl) to fully resuspend the cells. Then place the centrifuge tube in an ice bath and place it in an ultrasonic cell disruptor. The ultrasonic disruption conditions are: working time 1 ls, stop time 2 s, for a total of 20 min. Centrifuge the obtained disrupted solution at low temperature and high speed at 4 °C and 10,000 rpm for 30 min to obtain the crude enzyme solution. Filter it through a 0.45 μm microporous membrane for later use.

[0038] Crude enzyme solutions containing wild-type enzyme LrIS and crude enzyme solutions containing inulin sucrase mutant N300A (hereinafter referred to as mutant enzyme N300A) were prepared respectively.

[0039] 3. Purification of crude enzyme solution To prepare a nickel ion affinity chromatography column, first, at room temperature, use a constant flow pump to pump deionized water into the column to rinse it (approximately 6-12 column volumes). Then, equilibrate the column environment with buffer A (500 mmol / L NaCl, 50 mM PIPES, pH 7.5). When the pH of the effluent from the bottom of the column matches that of buffer A pumped into the column (approximately 5 column volumes of buffer are required), add the crude enzyme solution obtained in step (2) into the column. First, wash the contaminating proteins with buffer B (500 mmol / L NaCl, 50 mmol / L imidazole, 50 mM PIPES, pH 7.5) until baseline equilibrium is reached. Then, elute with elution buffer containing a high concentration of imidazole (500 mmol / L NaCl, 500 mmol / L imidazole, 50 mM PIPES, pH 7.5). Collect the eluent with the absorption peak to obtain the target protein. Dilute the prepared pure enzyme solution to a final concentration of 1 mg / mL to obtain pure enzyme solutions of wild-type enzyme LrIS and mutant enzyme N300A.

[0040] Example 3: Enzymatic synthesis of inulin Using a 1 mL reaction system with 300 g / L sucrose as substrate, the wild-type enzyme LrIS and the mutant enzyme N300A prepared in Example 2 were added respectively, with an enzyme addition amount of 3 U / mL. The enzyme reaction pH was 6.5, the reaction temperature was 30 °C, and the reaction time was 12 h to obtain reaction solutions containing inulin, which were the reaction products of the wild-type enzyme LrIS and the mutant enzyme N300A, respectively.

[0041] Example 4: Determination of the degree of polymerization of the product The degree of polymerization of plant inulin standards, as well as the reaction products of the wild-type enzyme LrIS obtained in Example 3 and the mutant enzyme N300A, was determined. The results are shown in the figure. Figure 1-3 .

[0042] The degree of polymerization of inulin was determined using high-performance anion exchange chromatography (HPAEC). The HPAEC was equipped with a pulsed amperometric detector and a Dionex CarboPac PA200 column. Mobile phase A consisted of 150 mmol / L sodium hydroxide, and mobile phase B consisted of 600 mmol / L sodium acetate and 150 mmol / L sodium hydroxide. The flow rate was 0.5 mL / min, and the column temperature was 30 °C. A linear gradient of 0–70% buffer B was used to separate inulin at different degrees of polymerization.

[0043] Figure 1-3 The results showed that, compared with the wild-type enzyme LS, the mutant enzyme N300A had essentially the same ability to produce inulin, and while blocking the synthesis of high molecular weight inulin, it still retained the ability to synthesize inulin with a low degree of polymerization (3-40). Figure 2 and 3 ).

[0044] Example 5: Determination of product molecular weight The molecular weights of the reaction products of the wild-type enzyme LrIS and the mutant enzyme N300A obtained in Example 3 were determined, and the results are shown in [Figure 1]. Figure 4-5 ; The molecular weight of inulin was determined using high performance size exclusion chromatography (HPSEC). The HPSEC was equipped with a differential refractive index detector (Waters 2410) and an Ultrahydrogel-Liner column (7.8 × 300 mm). The mobile phase was 0.1 mol / L sodium nitrate, the flow rate was 0.5 mL / min, and the column temperature was 40 °C.

[0045] Figure 4-5 The results showed that the wild-type enzyme could produce enzymes with a molecular weight of [missing value]. and The inulin synthesized by the mutant enzyme N300A has a molecular weight range of [missing information - likely related to molecular weight]. It precisely cuts off the ability of wild-type enzymes to synthesize high molecular weight microbial inulin with a molecular weight of one million daltons, while perfectly preserving the ability to synthesize inulin with a low degree of polymerization.

[0046] Example 6: Determination of relative enzyme activity of inulin sucrase before and after mutation The catalytic enzyme activity of the reaction products of the wild-type enzyme LrIS and the mutant enzyme N300A obtained in Example 3 was determined, and the results are shown in [Figure 1]. Figure 6 ; The amount of glucose produced reflects the catalytic activity of inulin sucrase. The amount of glucose was determined by high-performance liquid chromatography (HPLC) using a Waters e2695 HPLC system equipped with a Waters 2414 differential refractive index detector and a Sugar-Pak I column (6.5 mm × 300 mm, Waters). Ultrapure water was used as the mobile phase, with the flow rate and column temperature set at 0.4 ml / min and 85 °C, respectively. One unit of total enzyme activity was defined as the amount of enzyme required to catalyze the release of 1 μmol of glucose per minute. The activity of wild-type inulin sucrase was set to 100%.

[0047] Figure 6 The results showed that the relative enzyme activity of the mutant enzyme N300A was 70.7% of that of the wild-type enzyme.

[0048] The embodiments described above are merely preferred embodiments of the present invention, and not an exhaustive list of all possible implementations of the present invention. Any obvious modifications made by those skilled in the art without departing from the principles and spirit of the present invention should be considered to be included within the scope of protection of the claims of the present invention.

Claims

1. A mutant inulinase that specifically synthesizes plant-like inulin, characterized in that, The amino acid sequence of the inulin sucrase mutant is shown in SEQ ID NO.

2.

2. The inulin sucrase mutant according to claim 1, characterized in that, The inulin sucrase mutant is derived from microorganisms. LimosiLactobacillus reuteri It was obtained by mutating asparagine at position 300 of the amino acid sequence of inulin sucrase to alanine.

3. A gene encoding the inulin sucrase mutant of claim 1.

4. A recombinant vector carrying the gene of claim 3.

5. A recombinant cell expressing the inulin sucrase mutant of claim 1, carrying the gene of claim 3, or carrying the recombinant vector of claim 4, characterized in that, The recombinant cells use bacteria or fungi as expression hosts.

6. The recombinant cell according to claim 5, characterized in that, The recombinant cells are E. coli BL21(DE3) was used as the expression host, and pET-22b(+) was used as the expression vector.

7. A method for preparing the inulin sucrase mutant as described in claim 1, characterized in that, The recombinant cells of claim 5 or 6 are induced and cultured to produce inulin sucrase mutants.

8. The application of the inulin sucrase mutant as described in claim 1 in the catalytic production of inulin from sucrose-producing plants, characterized in that, The degree of polymerization of the plant-like inulin is 3-40.

9. The application according to claim 8, characterized in that, Inulin was produced by enzymatic reaction using sucrose as a substrate under the action of inulin sucrase mutant. The reaction temperature was 30℃, pH 6.5, and the reaction time was 12h.

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