Endoinulinase Inu-B and Its Application in Degrading Inulin Fructan to Produce Prebiotic Inulotriose with High Activity and High Purity
The degradation of inulin fructose by endoinulinase Inu-B is solved, and the problem of low production efficiency of high-purity inulin trisaccharides in the prior art is achieved, and the efficient preparation of high-purity inulin trisaccharides is achieved, which simplifies the separation and purification operation and reduces production costs.
Patent Information
- Application Number
- CN202210914602.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Priority Date
- 2022-05-06
- Filing Date
- 2022-08-01
- Publication Date
- 2025-07-11
- Estimated Expiration
- 2042-08-01
AI Technical Summary
The prior art is difficult to efficiently produce high-purity inulin trisaccharides, resulting in complex separation and purification operations and low efficiency.
The endoinulinase Inu-B was obtained from the Paenibacillus sp.LX16 strain through heterologous expression. This enzyme was used to degrade inulin fructose in buffer to prepare high-active and high-purity inulin trisaccharide.
The purity of inulin trisaccharide reaches 92.3%, simplifies separation and purification operations, reduces production costs and energy consumption, and provides a convenient industrial production solution.
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Figure CN115838708B_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of enzyme engineering and relates to an endoinulinase Inu-B and its application in degrading inulin fructan to produce prebiotic inulotriose with high activity and high purity. Specifically, it relates to a heterologous expression method of an endoinulinase Inu-B derived from Paenibacillus sp. LX16 strain that can degrade inulin fructan and can produce prebiotic inulotriose with high activity and high purity by degrading inulin fructan. Background Art
[0002] Inulin is a polyfructose linked by β-1,2 glycosidic bonds, with a glucose residue at its reducing end connected by a sucrose-type glycosidic bond, presenting a linear structure, and its abbreviated formula is GFn. Inulin is the second most abundant plant polysaccharide in nature after starch. It is an ideal functional food ingredient and also a good raw material for producing products such as fructooligosaccharides, polyfructose, high fructose syrup, and crystalline fructose.
[0003] Inulinase, also known as β-fructanase, is a glycoside hydrolase that catalyzes the β-2,1-fructofuranoside bonds in inulin. It mainly comes from microorganisms, and mammals lack enzymes to decompose inulin in their bodies. According to the different ways inulinase acts on substrates, it is further divided into endoinulinase (EC 3.2.1.7) and exoinulinase (EC 3.2.1.26). Different types of inulinase produce different products after degrading inulin. Among them, endoinulinase randomly acts on the glycosidic bonds inside the inulin chain, and the hydrolysis products are mainly a mixture of fructooligosaccharides with different degrees of polymerization (DP).
[0004] As a prebiotic, fructooligosaccharides mainly have the functions of promoting the proliferation of Bifidobacterium in the intestine, maintaining a good intestinal environment, regulating blood sugar and blood lipids, promoting the absorption of mineral elements by the human body, improving intestinal health, and enhancing resistance. Its excellent physiological functions have made it a widely popular functional food base in the international food market in the past decade, with applications in more than 500 kinds of foods, health products, and pharmaceuticals, and it is known as the new healthy sugar source in the 21st century.
[0005] Inulotriose is formed by connecting three fructose through β-2,1 bonds and is the fructooligosaccharide with the smallest molecular weight. Studies have shown that in addition to having most of the functions of fructooligosaccharides, inulotriose also has its own characteristics. As a prebiotic, due to its small molecular weight, its utilization efficiency is higher than that of long-chain or branched fructooligosaccharides, and it is more conducive to being absorbed by the intestinal flora. Inulotriose also has very important applications in the scientific research field. It is used as a standard product for analytical instruments such as thin-layer chromatography, ion chromatography, and high-performance liquid chromatography in the laboratory, and also as an enzyme reaction substrate in the process of enzyme characterization. Summary of the Invention
[0006] The endo-inulinase Inu-B provided by the present invention can achieve a purity of up to 92.3% of the inulotriose obtained after degradation of the inulin substrate.
[0007] To achieve a high proportion of inulotriose in the product, the endo-inulinase Inu-B is: a protein consisting of the amino acid sequence shown in SEQ ID NO.1.
[0008] The present invention also provides a gene encoding the above-mentioned endo-inulinase Inu-B.
[0009] In an embodiment of the present invention, the nucleotide sequence of the gene is as shown in SEQ ID NO.2.
[0010] The present invention also provides a recombinant plasmid carrying the above gene.
[0011] In an embodiment of the present invention, the vector of the recombinant plasmid is the pET28a(+) vector.
[0012] The present invention also provides a host cell carrying the above gene or the above recombinant plasmid.
[0013] In an embodiment of the present invention, the host cell is Escherichia coli.
[0014] The present invention also provides a method for preparing the above-mentioned endo-inulinase Inu-B. Add the host cell to the medium and culture it until OD 600 = 0.5 - 0.6, then add IPTG with a final concentration of 0.1 mM to the fermentation broth, and continue to induce culture at 20°C for 16 h to obtain endo-inulinase Inu-B.
[0015] In an embodiment of the present invention, the medium contains LB medium
[0016] In an embodiment of the present invention, the temperature of the culture is 20°C and the rotation speed is 200 rpm.
[0017] In an embodiment of the present invention, the concentration of IPTG in the fermentation broth is 0.1 mM.
[0018] The present invention also provides an endo-inulinase prepared by the above method.
[0019] The present invention also provides the application of the above-mentioned endo-inulinase Inu-B, or the above gene, or the above recombinant plasmid, or the above host cell, or the above preparation method, or the endo-inulinase obtained by applying the above preparation method in degrading inulin fructan and producing highly active and highly pure prebiotic inulotriose.
[0020] The present invention also provides a method for degrading inulin fructan and producing prebiotic inulin trisaccharide with high activity and high purity. The above-mentioned endo-inulinase Inu-B is added to a buffer solution containing inulin for conversion to obtain prebiotic inulin trisaccharide with high activity and high purity.
[0021] In one embodiment of the present invention, the buffer solution is a phosphate buffer solution.
[0022] In one embodiment of the present invention, the concentration of the phosphate buffer solution is 50 mM.
[0023] In one embodiment of the present invention, the pH of the phosphate buffer solution is 7.0 - 8.0, and further, the pH is 7.0.
[0024] In one embodiment of the present invention, the temperature of the conversion is 25 - 35 °C, and further,
[0025] the temperature is 30 °C.
[0026] The present invention further analyzes and identifies the product.
[0027] The beneficial effect of the present invention is that the present invention uses endo-inulinase Inu-B to degrade inulin into prebiotic inulin trisaccharide with high activity and high purity. The purity of the inulin trisaccharide is as high as 92.3%, overcoming the disadvantage of low yield of inulin trisaccharide generated by the enzymatic method, reducing and simplifying the operation difficulty of separation and purification, and providing a more convenient scheme for the industrial production of inulin trisaccharide. Description of the Drawings
[0028] Figure 1 is the SDS-PAGE electrophoresis pattern of the purified endo-inulinase Inu-B of the present invention; among them, lane 1 is the pET28a(+) empty vector; lane 2 is the crude enzyme solution induced by IPTG; lane 3 is the protein purified by Ni-NTA.
[0029] Figure 2 is the ion chromatogram of the degradation of inulin fructan by the endo-inulinase Inu-B of the present invention to generate inulin trisaccharide.
[0030] Figure 3 is the secondary mass spectrum of the main product of the degradation of inulin fructan by Inu-B of the present invention and the mass spectrum comparison diagram with the inulin trisaccharide standard.
[0031] Figure 4 is the optimal reaction temperature and temperature stability range of the endo-inulinase Inu-B of the present invention; among them, a is the optimal reaction temperature of the Inu-B enzyme activity; b is the temperature stability range of the Inu-B enzyme activity. Detailed Embodiments
[0032] The following non-limiting examples can enable those of ordinary skill in the art to understand the present invention more comprehensively, but do not limit the present invention in any way.
[0033] Example 1
[0034] The method for cloning, expressing and purifying the endo-inulinase Inu-B gene described in this example is as follows:
[0035] A. The inulin fructan-degrading bacterium Paenibacillus sp. LX16, as the source bacterium of endo-inulinase Inu-B, is from the previous screening in this experiment;
[0036] The inulin fructan-degrading bacterium Paenibacillus sp. LX16 described above was isolated from the soil in this laboratory and preserved in the China Center for Type Culture Collection (CCTCC NO: M2015644); the preservation information is as follows: the name of the preservation unit: China Center for Type Culture Collection, the address of the preservation unit: Wuhan University, China, the preservation number: CCTCC NO: M2015644, the preservation date: October 26, 2015, the taxonomic name of the biological preservation: Paenibacillus sp. LX16).
[0037] B. Using the genome of Paenibacillus sp. LX16 as a template by PCR technology, the target fragment, the coding gene of endo-inulinase Inu-B, that is, the nucleotide sequence of SEQ ID NO.2, was amplified;
[0038] Forward primer: SEQ ID NO.3:
[0039] 5’-GC GAATTC ATGAGCCATGCAACCGATAA-3’ (the horizontal line is the EcoRI restriction site)
[0040] Reverse primer: SEQ ID NO.4:
[0041] 5’-GC GCGGCCGC TTACCAAATGGACTGCAT-3’ (the horizontal line is the NotI restriction site)
[0042] Among them, the PCR reaction system is: 20 μL Premix Prime STAR, 2 μL forward primer, 2 μL reverse primer, 100 ng genome, and ddH2O was added to make up to 40 μL
[0043] The PCR reaction amplification conditions are: pre-denaturation at 98 °C for 5 min; then successively 98 °C for 30 s, 60.5 °C for 30 s, 72 °C for 2.5 min, and the reaction was carried out for 30 cycles.
[0044] C. The obtained target fragment and the pLB cloning vector are ligated using T4 ligase;
[0045] D. The ligation product is transferred into the cloning strain E. coli DH5α by heat transformation;
[0046] The above-mentioned E. coli DH5α was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.
[0047] E. The transformation solution is plated on an LB solid medium containing ampicillin (100 mg / L), and incubated upside down in a 37°C incubator for 12 h. The obtained single colonies are used for plasmid extraction. The plasmid is digested and identified by reacting with EcoRI and NotI restriction endonucleases at 30°C for 20 min. The plasmid with correct digestion identification is sent to BGI (Beijing) for sequencing;
[0048] LB medium contains 10 g / L tryptone, 5.0 g / L yeast extract, 10 g / L sodium chloride, (20 g / L agar is added to the solid medium), pH 7.0
[0049] F. For the plasmid with correct sequencing, the target fragment and the pET28a(+) plasmid are simultaneously digested with EcoRI and NotI. The digestion products are ligated using T4 ligase; transferred into E. coli Rosetta(DE3) competent cells. The transformation solution is plated on an LB solid medium containing kanamycin (final concentration 50 mg / L), and incubated upside down in a 37°C incubator for 12 h. The obtained single colonies are subjected to colony verification. The colonies with correct verification are cultured in a shaker at 37°C and 200 rpm for 12 h, and then inoculated into an LB low-salt medium at a ratio of 1:50 and cultured until OD 600 reaches 0.5, and 0.1 mM IPTG is added at 20°C to induce the expression of the target gene. The cells are collected, ultrasonically disrupted, and the supernatant is collected by centrifugation at 10,000 rpm for 30 min as the crude enzyme solution;
[0050] LB low-salt medium contains 10.0 g / L tryptone, 5.0 g / L yeast extract, 5.0 g / L sodium chloride, pH 7.0
[0051] The above-mentioned E. coli Rosetta(DE3) was purchased from Merck (China) Co., Ltd.
[0052] G. The crude enzyme solution is purified through a Ni-NTA column (Shanghai Sangon) to obtain the purified endoinulinase Inu-B. The results are as Figure 1As shown, the size of Inu-B is approximately 89 kD. It can be seen from the electrophoresis diagram that after purification by the Ni-NTA column, a single band of the target protein was obtained.
[0053] Example 2
[0054] The inulin triose was obtained by incubating 0.5 mg / mL of endo-inulinase (prepared in this experiment) with 20 g / L of inulin fructan at 30 °C for 20 min.
[0055] As described above, the inulin fructan was reacted with the endo-inulinase Inu-B of the present invention at 30 °C for 1 min, 5 min, 10 min, and 20 min. During the process, the products were analyzed by ion chromatography. The results are as Figure 2 shown (ion chromatograms at 0 min and 20 min of the reaction). The entire degradation of inulin fructan is a dynamic process. When the endo-inulinase Inu-B acts on inulin fructan for 1 min, obvious products are generated. After 20 min, the substrate is almost completely consumed, a large amount of inulin triose is produced, the oligosaccharide peaks above DP3 are hardly visible, and the peak areas of oligosaccharides with smaller molecular weights increase. The area of inulin triose increases significantly, and finally the purity of inulin triose reaches as high as 92.3%.
[0056] Compared with the reported endo-inulinases from fungi or other bacteria sources, Inu-B has distinct characteristics of its own. Through the analysis of the ion chromatography results, it was found that there are no oligosaccharides above trisaccharide in the products of Inu-B enzymatic hydrolysis of inulin fructan, and the oligosaccharide products are single, which is very different from the reported endo-inulinase products that are mostly mixtures of oligosaccharides (GF2, GF3, GF4, GF5, F3, and F4). This makes it possible to produce prebiotic inulin triose with high activity and high purity by applying Inu-B.
[0057] During the process of fermenting and producing inulin triose using endo-inulinase, the closer the enzymatic hydrolysis temperature is to room temperature, the greater the benefits an enterprise can obtain under the premise of low energy consumption. The reported endo-inulinases applied in industrial production mostly come from fungi, and the optimal enzymatic activity temperature is generally 55 - 65 °C. However, the optimal temperature of the endo-inulinase Inu-B in the present invention for the action on inulin fructan is 30 °C, and more than 95% of the enzyme activity can be retained within the temperature range of 20 - 35 °C for 24 h. Therefore, in industrial production, the enzymatic hydrolysis using the endo-inulinase Inu-B of this study can be carried out at room temperature, greatly reducing the production cost and energy input of enterprises.
Claims
1. Use of endoinulinase Inu-B in degrading inulin fructan and producing prebiotic inulotriose with high activity and high purity, characterized in that, The endo-inulinase Inu-B is a protein composed of the amino acid sequence shown in SEQ ID NO.
1.
2. Use of an endoinulinase Inu-B as claimed in claim 1 in degrading inulin fructan and producing highly active and highly pure prebiotic inulotriose, characterized in that, The gene encoding the endo-inulinase Inu-B has a nucleotide sequence shown in SEQ ID NO.
2.
3. Use of an endo-inulinase Inu-B as claimed in claim 2 for degrading inulin fructan and producing prebiotic inulotriose with high activity and high purity, characterized in that, The recombinant plasmid carries the gene.
4. Use of an endoinulinase Inu-B as claimed in claim 3 in degrading inulin fructan and producing highly active and highly pure prebiotic inulotriose, characterized in that, The vector of the recombinant plasmid is the pET28a(+) vector.
5. Use of an endoinulinase Inu-B as claimed in claim 2 in degrading inulin fructan and producing highly active and highly pure prebiotic inulotriose, characterized in that, The host cell carries the gene.
6. Use of an endoinulinase Inu - B as described in claim 3 or 4 for degrading inulin fructan and producing highly active and highly pure prebiotic inulotriose, characterized in that, The host cell carries the recombinant plasmid.
7. Use of an endo-inulinase Inu-B as claimed in claim 5 for degrading inulin fructan and producing prebiotic inulotriose with high activity and high purity, characterized in that, The host cell is Escherichia coli.
8. Use of an endo-inulinase Inu-B for degrading inulin fructan and producing highly active and highly pure prebiotic inulin triose as claimed in claim 1, characterized in that, The preparation method of the endo-inulinase Inu-B comprises adding the host cell described in claim 5 or 7 into a culture medium and culturing until OD 600 reaches 0.5 - 0.6, then adding IPTG into the fermentation broth and continuing the induction culture at 20 °C for 16 h to obtain the endo-inulinase Inu-B.
9. A method for degrading inulin fructan and producing highly active and highly pure prebiotic inulotriose, characterized in that, The endo-inulinase Inu-B in the application described in claim 1 is added to a buffer containing inulin for reaction to obtain prebiotic inulotriose with high activity and high purity.
10. A method for degrading inulin fructan and producing highly active and highly pure prebiotic inulotriose as claimed in claim 9, characterized in that, The temperature of the endo-inulinase Inu-B during the degradation of inulin fructan is 25 - 35 °C, and the pH is 7.0 - 8.0.