Endo-inulinase ER-INUI as well as preparation and application thereof

Abstract

The invention relates to the technical field of enzyme gene engineering, in particular to endo-inulinase ER-INUI as well as preparation and application thereof, and the amino acid sequence of the endo-inulinase ER-INUI is as shown in SEQ ID NO. 2. The invention further discloses a method for hydrolyzing inulin. The endo-inulinase ER-INUI and the inulin are used for carrying out glycolysis reaction. The invention also discloses an application of the endo-inulinase ER-INUI in hydrolysis of inulin into fructo-oligosaccharide. The endo-inulinase ER-INUI provided by the invention can be widely applied to the fields of preparation of agriculture, food, feed additives, medicines, fructo-oligosaccharide and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme gene engineering, and particularly relates to an endoinulinase ER-INUI and a preparation and application thereof. Background Art

[0002] Inulin, also known as inulin, is a reserve polysaccharide that mainly comes from plants. It is rich in the roots of Jerusalem artichokes, chicory, dahlias and other Asteraceae plants, and is a rich renewable resource. However, inulin has problems such as low sweetness and poor water solubility. At present, my country's inulin industry in the food industry mostly stays at the primary processing level, and high-value processing technology is urgently needed. Converting inulin into high-value-added oligofructose is an important direction of the inulin deep processing industry.

[0003] Fructo oligosaccharide (FOS) refers to a mixture of functional oligosaccharides connected by β-(2→1) glycosidic bonds of fructose, with a degree of polymerization between 2-10. Fructo oligosaccharide is a strong beneficial bio-nutrient that can effectively improve the health level of the human body and plays an important role in disease prevention and treatment: FOS can selectively stimulate the growth and maintain colonization of probiotics in the gastrointestinal tract, especially bifidobacteria and lactic acid bacteria; prevent and treat diabetes, lower blood lipids and prevent cholesterol; promote mineral absorption: FOS can form complexes with minerals to promote the absorption and utilization of trace elements iron and calcium to prevent osteoporosis; in addition, FOS is a natural active substance that not only maintains the pure sweetness of sucrose, but is also more refreshing than sucrose, with a fat-like aroma and a refreshing and greasy feel. In recent years, oligofructose products have been popular in the health care product markets of Japan, Europe, and the United States, and are widely used in infant food, dairy products, condiments and other functional foods, with broad application prospects.

[0004] Inulinase, also known as β-fructanase, is a glycoside hydrolase that catalyzes the β-2,1-fructofuranose bonds in inulin. It is mainly derived from microorganisms, and mammals lack enzymes that can decompose inulin. According to the different modes of action of inulinase on substrates, it is further divided into endo-inulinase (Endo-inulinase, EC 3.2.1.7) and exo-inulinase (Exo-inulinase, EC3.2.1.6). Different types of inulinase can obtain different products after degrading inulin. Among them, endo-inulinase acts on the glycosidic bonds inside inulin, and the hydrolysis products are mainly a mixture of oligofructose with different degrees of polymerization (DP).

[0005] At present, domestic FOS is mainly produced by fructosyltransferase, which has low conversion efficiency and cumbersome product separation steps. In contrast, the production of oligofructose by hydrolyzing inulin with endo-inulinase (EC3.2.1.7) is not only simple in reaction, with few by-products and high yield, but also has greater production value. In addition, endo-inulinase is also the basis and key to the high-value utilization of inulin. Therefore, oligofructose produced by endo-inulinase has greater commercial application value.

[0006] However, the current wild-type strains of endoinulinase have low yields and complex enzyme systems, which make enzyme separation and purification difficult and costly. At the same time, the sources of these microorganisms are very limited and have low specific activities. For example, the specific enzyme activity of endoinulinase from Aspergillus niger DSM 2466 can only reach 4.91U / mg; the specific enzyme activity of endoinulinase from Arthrobacter sp.S37 can only reach 93.4U / mg, the specific activity of endoinulinase from Microbulbifer sp. is 38.2U / mg, and the specific enzyme activity of endoinulinase isolated from A. niger is 565.6U / mg.

[0007] These problems limit the industrial application of endoinulinase. Therefore, it is of great research significance and practical application value to find diverse microbial sources to obtain new endoinulinase that can efficiently and specifically catalyze inulin to produce oligofructose. Summary of the invention

[0008] In order to solve the above problems, the present application uses gene mining technology to discover an endoinulinase ER-INUI from Emericella rugulosa strain NRRL 11440, and then uses it to hydrolyze inulin to prepare oligofructose.

[0009] In a first aspect, the present application provides an endoinulinase ER-INUI, the amino acid sequence of the endoinulinase is shown in SEQ ID NO.2.

[0010] The second aspect of the present application provides an isolated polynucleotide, which encodes the above-mentioned endoinulinase.

[0011] The third aspect of the present application provides a construct, which contains the isolated polynucleotide mentioned above.

[0012] The fourth aspect of the present application provides a host cell, which comprises the above-mentioned construct, or the above-mentioned polynucleotide is integrated into the genome of the host cell.

[0013] The fifth aspect of the present application provides a method for preparing endoinulinase ER-INUI, which comprises culturing the above-mentioned cells to express endoinulinase.

[0014] A sixth aspect of the present application provides a method for hydrolyzing inulin, comprising using the above-mentioned endoinulinase ER-INUI to perform an enzymatic hydrolysis reaction on inulin.

[0015] The seventh aspect of the present application provides the use of the above-mentioned endoinulinase ER-INUI in hydrolyzing inulin into oligofructose.

[0016] The beneficial effects that the endoinulinase ER-INUI proposed in this specification may bring include but are not limited to:

[0017] 1. The ER-INUI coding sequence from Emericella rugulosa strain NRRL 11440 with high specific enzyme activity and catalytic efficiency was obtained by bioinformatics methods. The sequence had a similarity of 69% with the reference sequence. The sequence was recombined into E. coli engineering bacteria to obtain the recombinant E. coli engineering bacteria pET28a(+)-inui / BL21(DE3) that can efficiently express the gene, abandoning the traditional method of screening and isolating new enzymes from environmental samples such as soil, broadening the resources for the exploration and application of new enzymes, and saving time and energy for screening bacteria.

[0018] 2. The endoinulinase INUI provided by the present invention has an amino acid sequence as shown in SEQ ID No. 2 and has a high specific enzyme activity of up to 1161 U / mg and a maximum reaction rate (VMAX) of up to 2500 μmol·min -1 mg -1 The substrate affinity (Km) can reach 2.25mM, and the catalytic constant (Kcat) can reach 7252s -1 , kcat / Km can reach 3223s -1 mM -1 , which can meet the requirements of industrial production of endoinulinase and fructooligosaccharides (FOS) and has extremely high application prospects in the food field.

[0019] 3. The endoinulinase ER-INUI of the present invention, whose amino acid sequence is shown in SEQ ID No. 2, is used to degrade inulin into oligofructose (FOS) products, the degree of polymerization of which is mainly distributed in DP3-5 and has a high purity; and the endoinulinase has a strong specificity for inulin, providing a more convenient solution for the industrial processing of inulin. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The figure is the result of the amino acid sequence alignment between ER-INUI and known endoinulinase;

[0021] Figure 2 This is the electropherogram of PCR amplification of the ER-INUI encoding gene;

[0022] Figure 3 is a schematic diagram of the ER-INUI gene expression vector;

[0023] Figure 4 This is the SDS-PAGE electrophoresis of INUI after expression and purification. The arrow indicates the location of ER-INUI;

[0024] Figure 5 are the enzymatic properties of ER-INUI; A is the effect of temperature on ER-INUI; B is the effect of temperature on the stability of ER-INUI; the horizontal axis represents temperature and the vertical axis represents relative enzyme activity; C is the effect of pH on ER-INUI; D is the effect of pH on the stability of INUI; the horizontal axis represents pH and the vertical axis represents relative enzyme activity;

[0025] Figure 6 The results of thin layer chromatography of ER-INUI hydrolyzing GF, GF2, GF3, GF4, and GF5; A is GF as substrate, B is GF2 and GF3 as substrates, and C is GF4 and GF5 as substrates. ER- represents the control group without ER-INUI enzyme; ER+ represents the experimental group with ER-INUI added.

[0026] Figure 7 This is the thin layer chromatography spectrum of the product analysis when ER-INUI is converted with inulin as substrate. DETAILED DESCRIPTION

[0027] In order to more clearly explain the technical solutions of the embodiments of this specification, the following is a brief introduction to the drawings required for the embodiments. Obviously, the drawings described below are only some examples or embodiments of this specification. For ordinary technicians in this field, this specification can also be applied to other similar scenarios based on these drawings without creative work. Unless it is obvious from the language environment or otherwise explained, the same reference numerals in the figures represent the same structure or operation.

[0028] It should be understood that the terms used in the examples of the present invention are intended to describe specific embodiments rather than to limit the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated, the singular forms "a", "an" and "the" include plural forms. When the examples give numerical ranges, it should be understood that unless otherwise specified in the present invention, both endpoints of each numerical range and any value between the two endpoints can be selected.

[0029] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, any methods, equipment, and materials of the prior art that are similar or equivalent to the methods, equipment, and materials in the embodiments of the present invention may be used to implement the present invention, based on the prior art mastery of those skilled in the art and the description of the present invention.

[0030] The present application provides an endoinulinase ER-INUI, the amino acid sequence of the endoinulinase ER-INUI is shown in SEQ ID NO.2.

[0031] In one embodiment, the amino acid sequence of the endoinulinase ER-INUI is derived from Emericella rugulosa strain NRRL 11440.

[0032] In one embodiment, the working pH range of the endoinulinase ER-INUI can be 4.5-6.5; preferably, the working pH range of the endoinulinase can be 5.0-6.0; more preferably, the working pH range of the endoinulinase can be 5.5.

[0033] In one embodiment, the operating temperature range of the endoinulinase is 45°C to 65°C; preferably, the operating temperature range of the endoinulinase is 50°C to 60°C; more preferably, the operating temperature range of the endoinulinase is 55°C.

[0034] The present application also provides an isolated polynucleotide, which encodes the above-mentioned endoinulinase ER-INUI.

[0035] The polynucleotide of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.

[0036] In one embodiment, the isolated polynucleotide has a nucleotide sequence as shown in SEQ ID No.1.

[0037] The present application also provides a construct, wherein the construct contains the isolated polynucleotide described above.

[0038] In certain embodiments of the present invention, the construct is constructed by inserting the isolated polynucleotide into the multiple cloning site of an expression vector. The expression vector in the present invention generally refers to various commercially available expression vectors well known to those skilled in the art, such as bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors.

[0039] The present application also provides a host cell, wherein the host cell comprises the above construct, or the above polynucleotide is integrated into the host cell genome.

[0040] In some embodiments, the host cell may be an Escherichia coli host cell Escherichia coli BL21 (DE3), Escherichia coli Rosetta (DE3), Escherichia coli JM109 or Escherichia coli DH5α; in some embodiments, the cell may be a Bacillus subtilis host cell Bacillus subtilis R25, Bacillus subtilis SCK22, Bacillus subtilis 168 or Bacillus subtilis 9920; in some embodiments, the cell may be a yeast host cell Saccharomyces cerevisiae, Pichiapastoris or Kluyveromyces lactis; in some embodiments, the cell may be a lactic acid bacteria host cell Lactiplantibacillus plantarum or Lactic acid bacteria COCC101; in some embodiments, the cell may be an actinomycete host cell Streptomyces spp; in some embodiments, the cell may be a filamentous fungus host cell Trichoderma viride, Trichoderma reesei, Aspergillus niger or Aspergillus nidulans; in some embodiments, the cell may be an insect cell Bombyx mori or Antharaea eucalypti; in some embodiments, the cell may be a mammalian cell Chinese hamster ovary cell CHO or Chinese hamster lung cell CHL.

[0041] The present application also provides a method for preparing endoinulinase ER-INUI, which comprises inoculating the above-mentioned cells into a culture medium, culturing and inducing the expression of endoinulinase ER-INUI.

[0042] In one embodiment, the culture medium includes but is not limited to LB medium or TB medium.

[0043] In one embodiment, the inducing agent used to induce expression is selected from IPTG.

[0044] In one embodiment, the method further comprises purifying the expressed endoinulinase ER-INUI.

[0045] Specifically, the purification includes: collecting fermentation supernatant, purifying the supernatant, and collecting endoinulinase. More specifically, the purification includes but is not limited to affinity chromatography and / or ion exchange chromatography.

[0046] The present application also provides a method for hydrolyzing inulin, using the above-mentioned endoinulinase ER-INUI to carry out enzymatic hydrolysis reaction with inulin.

[0047] Specifically, the endoinulinase ER-INUI is added into a buffer solution containing inulin for conversion to obtain oligofructose with high activity and purity.

[0048] In one embodiment, the inulin is dissolved in a buffer, and the buffer is an ammonium acetate buffer.

[0049] In the enzymatic hydrolysis system, the mass concentration of the inulin is 2% to 10%; preferably, the mass concentration of the inulin may be 4% to 6%, and more preferably, the mass concentration of the inulin may be 5%.

[0050] In one embodiment, the pH range of the reaction may be 4.5 to 6.5; preferably, the pH range of the reaction may be 5.0 to 6.0; more preferably, the pH of the reaction may be 5.5.

[0051] In one embodiment, the reaction temperature may be 45°C to 65°C; preferably, the reaction temperature may be 50°C to 60°C; more preferably, the reaction temperature may be 55°C.

[0052] In one embodiment, the reaction time may be 10 min to 2 h; preferably, the reaction time may be 10 min to 30 min; more preferably, the reaction time may be 10 min.

[0053] The present application also provides the use of the above-mentioned endoinulinase in hydrolyzing inulin into oligofructose.

[0054] In one embodiment, the application is the application of one or more of the endoinulinase, isolated polynucleotide, and construct as a catalyst in the preparation of oligofructose. Further, the application is the application of inulin in the preparation of oligofructose.

[0055] Preferably, the oligofructose may include any one or more of kestose, kestose, and pentose.

[0056] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent companies unless otherwise specified. The quantitative experiments in the following examples are repeated three times, and the results are averaged.

[0057] Example 1 - Screening and bioinformatics analysis of endoinulinase encoding genes

[0058] The endoinulinase gene was selected by bioinformatics method, and the amino acid sequence from Lipomyces staryi reported was searched by tblastn in NCBI database, and the sequences with homology between 30-80% were retained. In order to further narrow the scope, microorganisms were used as important indicators for screening. Oligofructose is mainly used in the food industry, so the endoinulinase from pathogenic microorganisms was excluded, and the amino acid sequence of Emericella rugulosastrain NRRL 11440 (NCBI number AFT91384.1) was found to have a homology of 69% with the probe, so it was considered that this amino acid sequence might be the amino acid sequence of endoinulinase, and was named the target amino acid sequence (amino acid sequence SEQ ID No. 2).

[0059] To further determine whether the target sequence is an endoinulinase sequence, the target sequence was then compared with the amino acid sequences of other known endoinulinase sequences (NCBI numbers are ODQ71402.1, O94220.1, and BAA12321.1) for conserved regions. Figure 1 According to existing reports, endoinulinase generally contains 6 conserved regions: WMNEPNGL, Q, FT, RDP, EVP and SVEVF, respectively. Figure 1 It can be seen that the target amino acid sequence contains 6 conserved region sequences of endoinulinase. Therefore, the target amino acid sequence is further confirmed to be the amino acid sequence of endoinulinase, and the corresponding protein is named ER-INUI (amino acid sequence is SEQ ID NO.2).

[0060] Example 2 - Cloning, expression and purification of the endoinulinase gene described in this example

[0061] Construction of recombinant bacteria with ER-INUI encoding gene

[0062] According to the amino acid sequence of the target endoinulinase found from NCBI, the codon composition of the target inulinase encoding gene was optimized. The codon-optimized gene sequence encoding endoinulinase was obtained through whole gene synthesis technology at GENEWIZ Biotechnology Co., Ltd. The corresponding nucleotide sequence is named ER-inui (nucleotide sequence is SEQ ID NO.1). The full length of the gene (from the start codon to the stop codon) is 1527bp, encoding 508 amino acids, and its amino acid sequence is SEQ ID NO.2.

[0063] The nucleotide sequence encoding endoinulinase is shown in SEQ ID NO.1.

[0064] Information of SEQ ID NO.1

[0065] (a) Sequence characteristics

[0066] Length: 1527 nucleotides

[0067] Type: Nucleotide

[0068] Chain type: Single chain

[0069] (b) Molecule type: DNA

[0070] Sequence description: SEQ ID NO.1

[0071]

[0072] The amino acid sequence of endoinulinase is shown in SEQ ID NO.2.

[0073] Information of SEQ ID NO.1

[0074] (a) Sequence characteristics

[0075] Length: 508 amino acids

[0076] Type: Amino Acid

[0077] Chain type: Single chain

[0078] (b) Molecular type: protein

[0079] Sequence description: SEQ ID NO.2

[0080] MFLHILCLLAGQALADDYRPVFHFVPEKNWMNEPNGLIKIGSTWHLFYQHNPTANVWGNLNWGHATSSDLVHWTHEPLAITSENGIEAFTGTSYYDAENTSGLGTSDNPPYLAWYTGYFPSNGTQDQ RLAFSIDAGETWTKFEGNPVISAAQEAPHDATGGLETRDPKVFFHADSGKWIMVLAHGGQNKMSFWTSVDAKRWSWASDLTSTQVPGLPSAVKGWEVPDMFEVPVHGTDKTTWVLIFTPAEGSPAGG NGVLALTGSFDGTVFHPNPVNVSTLWLDYGRDFDGALSWENLPDSDGHRILAAISNSYGANPPTNTWKGMLSFPRTLSLHQSQTGQYFLQQPVSNLDTVSTPLVSVKNQTIAPGQVLLSSVRGTALD IRIAFSANAGTVLSLAVRKAGSQETVIQYRQSDATLSVDRTTSGLTSYDPAAGGVHTAPLRPDASGVVQIRALVDTCSVEVFGGQGEVVISDLIFPDETSDGLALQVIGGTAVLRSLEVREISLDLE

[0081] Obtain a recombinant bacterium that efficiently expresses ER-INUI intracellularly, i.e., a genetically engineered bacterium: clone ER-inui into the Nco I and Xho I sites of the expression vector pET28a(+), fuse the ER-inui coding sequence with the 6 His tag gene sequences present in the pET28a(+) expression system (located at the C-terminus), and obtain the corresponding recombinant expression plasmid to construct the recombinant plasmid pET28a(+)-inui( Figure 3 The recombinant plasmid was transformed into Escherichia coli BL21 (DE3), spread on LB solid medium plates containing 50 μg / mL kanamycin, and colony PCR was performed using F (SEQ ID NO.3) and R (SEQ ID NO.4) to select positive bacteria ( Figure 2 ) to obtain the engineered strain pET28a(+)-inui / BL21(DE3).

[0082] F:GTTATTGCTCAGCGGTGGCAG(SEQ ID NO.3)

[0083] R:ATTAATACGACTCACTATAG(SEQ ID NO.4)

[0084] Expression of recombinant ER-INUI

[0085] Activation of strains: The genetically engineered bacteria pET28a(+)-inui / BL21(DE3) obtained by screening were inoculated into 2 mL LB liquid culture medium containing 50 μg / mL kanamycin, and cultured at 37°C and 200 rpm for 12 h.

[0086] Fermentation of strains: The activated genetically engineered bacteria pET28a(+)-inui / BL21(DE3) bacterial culture fluid was inoculated at 1% inoculation into 50 mL of TB liquid culture medium containing 50 μg / mL kanamycin, and fermented at 200 rpm and 20°C. When the OD600 of the bacterial fluid increased to 0.5, 1 mM IPTG was added to induce expression for 20 hours. After stopping the fermentation, the fermentation fluid was centrifuged at 10,000 rpm for 30 minutes, and the precipitate was collected. The precipitate was crushed with an ultrasonic crusher and centrifuged again to obtain the intracellular supernatant and precipitate, and the crude enzyme in the cell was obtained.

[0087] Among them, LB medium: tryptone (trypticase): 10g / L; yeast extract: 5g / L; NaCl: 10g / L; agar powder (add to solid LB medium): 1g / L-2g / L; finally, add distilled water to 1L, adjust pH to 7.0, and sterilize at 121℃ for 20min. TB liquid medium: peptone 12g / L, yeast powder 24g / L, potassium dihydrogen phosphate 2.31g / L, potassium dihydrogen phosphate 12.54g / L, glycerol 4mL / L.

[0088] Purification of recombinant INUI

[0089] The cell supernatant was purified by nickel column affinity chromatography to obtain ER-INUI. Based on the results of SDS-PAGE electrophoresis, it was found that ER-INUI mainly appeared in the 100 mM imidazole eluate ( Figure 4 ); collect the purified samples, desalt them, and measure the specific enzyme activity of the desalted samples. The test results show that the specific enzyme activity of ER-INUI is 1161U / mg. The specific enzyme activity of endoinulinase from A. niger is 565.6U / mg, while the specific enzyme activity level of endoinulinase from the source bacteria is much lower, such as Microbulbifer sp. only 38.2U / mg, Arthrobacter sp.S37 only 93.4U / mg, indicating that the specific enzyme activity of ER-INUI is at a higher level than that of endoinulinase reported in the literature.

[0090] Detection of endoinulinase ER-INUI activity: prepare 1 mg / mL fructose solution, dilute it into fructose solutions of different concentrations (0, 0.2, 0.4, 0.6, 0.8, 1 mg / mL), add 1 mL of DNS, react in boiling water for 10 min, measure the absorbance at a wavelength of 540 nm, and draw a standard curve with the absorbance as the ordinate and the corresponding fructose concentration as the abscissa.

[0091] Take 800uL of 2.0% (w / v) inulin solution, add 200μL enzyme solution, react for 10min at 55℃ and pH 5.5, add 1mL DNS reagent to terminate the reaction, heat at 100℃ for 10min, cool, and measure the absorbance of the reaction solution at 540nm. According to the standard curve, the enzyme activity of endoinulinase ER-INUI can be calculated.

[0092] The definition of endoinulinase activity is: under the conditions of pH 5.5 and temperature 55°C, the amount of enzyme (mL) required to hydrolyze inulin to produce 1 umoL reducing sugar per minute is 1 enzyme activity unit (U);

[0093] The specific activity of endoinulinase is defined as the number of enzyme activity units (U) per unit weight (mg) of endoinulinase at a pH of 5.5 and a temperature of 55°C.

[0094] Example 3 - Study on the Enzymatic Properties of ER-INUI

[0095] Effect of temperature on enzyme activity

[0096] Determination of the optimal reaction temperature: The activity of the purified ER-INUI obtained in Example 2 was measured at different temperatures (30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C) and pH 5.5. The highest enzyme activity was set to 100%. Each experiment was performed in triplicate and the results were averaged. Figure 5 A shows that the optimal reaction temperature of recombinant ER-INUI is 55°C. The optimal temperatures of endoinulinases from Arthrobacter sp. (CAB63119.1) and Microbulbifer sp. (AB669414) are reported to be 45-50°C and 35°C, respectively. It can be seen that the optimal temperature of ER-INUI is higher than these endoinulinases, indicating that ER-INUI has great industrial potential.

[0097] Determination of thermal stability: The ER-INUI obtained in Example 2 was incubated at 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, and 80°C for 1 h, quickly cooled on ice, and the residual enzyme activity was measured for each reaction, with the unincubated enzyme activity being taken as 100%. Figure 5 B shows that the ER-INUI was determined to be relatively stable between 30℃-55℃, and the endoinulinase from Microbulbifersp. and A.niger remained stable below 35℃ and 45℃, respectively. ER-INUI showed good thermal stability and could maintain high enzyme activity within the temperature range of 55℃.

[0098] Effect of pH on enzyme activity

[0099] Determination of the optimal reaction pH: The activity of ER-INUI was measured at different pH values ​​(2.5, 3.0, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 10.5, 11.0, 11.5, 12.0) at 55°C, and the highest activity was set as 100%. Figure 5C showed that the optimal pH of recombinant ER-INUI was 5.0-6.0, which was consistent with the optimal pH of Lipomyces starkeyiNRRL Y-11557 (ODQ71402.1) (5.0-6.0).

[0100] Determination of pH stability: The ER-INUI obtained in Example 2 was kept at pH (2.5, 3.0, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 10.5, 11.0, 11.5, 12.0) at 4°C for 12 h and the residual activity was measured respectively, with the unincubated enzyme activity being 100%. Figure 5 D shows that the activity of recombinant ER-INUI remains above 80% in the pH range of 3.0-10.0, and has a good pH stability range.

[0101] Effects of metal ions on the activity of recombinant ER-INUI

[0102] Prepare 50mM stock solutions of CaC12, MnC12, KC1, MgC12, LiCl, NaCl, A1Cl3, SnC12, Fe2(SO4)3, FeSO4, CoSO4, ZnSO4, and CuSO4, and add the prepared stock solutions to the appropriately diluted enzyme solution to a final concentration of 5mM. After keeping at room temperature for 1 hour, determine the residual enzyme activity according to the enzyme activity determination method, with the enzyme activity without adding metal ions as 100%. The residual enzyme activity was determined according to the enzyme activity determination method. The results are shown in Table 1. Compared with the blank control group without adding any metal ions, Ca2+ has a significant promoting effect, with the increase ratio reaching 104%; according to current reports, Ca2 has no activating effect on endoinulinase from most other sources, such as Arthrobacter sp. (GenBank No. CAB63119.1), L. starkeyi NRRL Y-11557 (ODQ71402.1); while Al3+, Fe3+, Fe2+, Zn2+ and Cu2+ have a significant inhibitory effect on ER-INUI enzyme activity.

[0103] Table 1 Effects of metal ions on ER-INUI enzyme activity

[0104]

[0105] Example 4—Kinetic parameter analysis of ER-INUI

[0106] In the study of enzyme catalytic reaction kinetics, Km and kcat are the most important characteristic constants of enzyme catalytic reactions, which reflect the affinity between the enzyme and the substrate and the reaction rate of the enzyme, respectively. This example determines the kinetic parameters of ER-INUI.

[0107] Inulin with a concentration of 0.25 to 20 mg / ml was used as a substrate, 200 uL of the purified ER-INUI obtained in Example 2 was added, and the reaction was carried out at a temperature of 55°C and a pH of 5.5 for 10 minutes. The initial velocity V of the ER-INUI enzyme reaction and the substrate concentration [S] were nonlinearly fitted. The maximum reaction rate (Vmax), the Michaelis constant (Km), the catalytic constant (kcat) of the enzyme, and the catalytic efficiency (Km / kcat) were determined by fitting, as shown in Table 2.

[0108] According to the molecular weight of ER-INUI of 55.7KDa, the V of ER-INUI was calculated. MAX The value, kcat value and kcat / Km value were 2500 μmol·min -1 mg -1 、7252s -1 and 3223s -1 mM -1 The catalytic efficiency is at a relatively high level reported so far. Endoinulinase V from Aspergillus fumigatus CL1 MAX The value is 1590 μmol·min -1 mg -1 The endoinulinase from A. niger was 666.7 μmol·min -1 mg -1 , Endoinulinase V from A.arachidicola MAX The value is only 23.2 μmol·min -1 mg -1 It can be seen that ER-INUI has high catalytic efficiency and meets the requirements of industrial applications.

[0109] Table 2 Kinetic parameters of recombinant ER-INUI

[0110]

[0111] Example 5 - Analysis of the conversion products of inulin hydrolyzed by recombinant ER-INUI

[0112] Analysis of hydrolysis of different substrates by recombinant ER-INUI

[0113] 5% GF (sucrose), GF2 (tetrose), GF3 (tetrose), GF4 (pentaose), GF5 (hexaose) were used as substrates of the obtained ER-INUI, respectively, and reacted at 55°C for 1 hour to obtain a reaction solution. At the same time, the reducing sugar content was determined by the DNS method. The results showed that there was no increase in reducing sugar when GF (sucrose), GF2 (tetrose), GF3 (tetrose), GF4 (pentaose), and GF5 (hexaose) were used as substrates. The composition of these products was analyzed by TLC thin layer chromatography, and the results are as follows: Figure 6 As shown in AC, no hydrolysis products were generated, indicating that the recombinant ER-INUI had no hydrolysis activity on low-polymerization degree inulin with DP≤6.

[0114] Thin layer chromatography (TLC) method: Take the reaction solution and spot it on the TLC plate, place the TLC plate in the chromatography cylinder, develop it with the developing agent, blow it dry, spray the color developer and then develop it at 105℃ for 5 minutes until the color is clear.

[0115] Analysis of the conversion of inulin into oligofructose products by recombinant ER-INUI

[0116] ER-INUI was treated with 5% commercial inulin as substrate at 55°C for 10 min, 1 h, and 2 h to obtain a reaction solution. The hydrolysis product was analyzed by TLC thin layer chromatography. The TLC results showed that ( Figure 7 ), after ER-INUI reacted with commercial inulin for 10 minutes, the inulin with high polymerization degree was almost completely degraded, and the main products were GF2-GF4. It can be determined that ER-INUI is an endo-inulinase, and its inulin hydrolysis products are mainly inulin trisaccharide-inulin pentasaccharide. At the same time, it can be seen that the inulin substrate contains a small amount of monosaccharides and sucrose, and the monosaccharides and sucrose produced by ER-INUI hydrolysis do not increase significantly. It can be seen that the oligofructose (FOS) produced by hydrolyzing inulin with ER-INUI has high purity, is easy to separate and purify, and has strong specificity, which is of great value for the production of oligofructose.

[0117] The basic concepts have been described above. Obviously, for those skilled in the art, the above detailed disclosure is only for example and does not constitute a limitation of this specification. Although not explicitly stated here, those skilled in the art may make various modifications, improvements and corrections to this specification. Such modifications, improvements and corrections are suggested in this specification, so such modifications, improvements and corrections still belong to the spirit and scope of the exemplary embodiments of this specification.

[0118] At the same time, this specification uses specific words to describe the embodiments of this specification. For example, "one embodiment", "an embodiment", and / or "some embodiments" refer to a certain feature, structure or characteristic related to at least one embodiment of this specification. Therefore, it should be emphasized and noted that "one embodiment" or "an embodiment" or "an alternative embodiment" mentioned twice or more in different positions in this specification does not necessarily refer to the same embodiment. In addition, certain features, structures or characteristics in one or more embodiments of this specification can be appropriately combined.

[0119] In some embodiments, numbers describing the number of components and attributes are used. It should be understood that such numbers used in the description of the embodiments are modified by the modifiers "about", "approximately" or "substantially" in some examples. Unless otherwise specified, "about", "approximately" or "substantially" indicate that the numbers are allowed to vary by ±20%. Accordingly, in some embodiments, the numerical parameters used in the specification and claims are approximate values, which may change according to the required features of individual embodiments. In some embodiments, the numerical parameters should take into account the specified significant digits and adopt the general method of retaining digits. Although the numerical domains and parameters used to confirm the breadth of their range in some embodiments of this specification are approximate values, in specific embodiments, the setting of such numerical values ​​is as accurate as possible within the feasible range.

[0120] Finally, it should be understood that the embodiments described in this specification are only used to illustrate the principles of the embodiments of this specification. Other variations may also fall within the scope of this specification. Therefore, as an example and not a limitation, alternative configurations of the embodiments of this specification may be considered consistent with the teachings of this specification. Accordingly, the embodiments of this specification are not limited to the embodiments explicitly introduced and described in this specification.

Claims

1. An endoinulinase ER-INUI, characterized in that The amino acid sequence of the endoinulinase ER-INUI is shown in SEQ ID NO.

2.

2. The endoinulinase ER-INUI according to claim 1, characterized in that The amino acid sequence of the endoinulinase ER-INUI is derived from Emericella rugulosa strain NRRL 11440.

3. An isolated polynucleotide, characterized in that The isolated polynucleotide encodes the endoinulinase ER-INUI according to claim 1 or 2.

4. A construct, characterized in that The construct contains the isolated polynucleotide of claim 3.

5. A host cell, characterized in that The host cell comprises the construct according to claim 4, or the host cell genome is integrated with the polynucleotide according to claim 3.

6. A method for preparing endoinulinase ER-INUI, characterized in that: The method comprises inoculating the host cell according to claim 5 into a culture medium, culturing the culture medium and inducing the expression of endoinulinase ER-INUI.

7. A method for hydrolyzing inulin, characterized in that: The endoinulinase ER-INUI as claimed in claim 1 or 2 is used to carry out enzymatic hydrolysis reaction with inulin.

8. The method according to claim 7, characterized in that The inulin is dissolved in a buffer, and the buffer is an ammonium acetate buffer; And / or, in the enzymatic hydrolysis system, the mass concentration of inulin is 2% to 10%; preferably, the mass concentration of inulin is 4% to 6%, and more preferably, the mass concentration of inulin is 5%; And / or, the pH range of the reaction is 4.5 to 6.5; preferably, the pH range of the reaction is 5.0 to 6.0; more preferably, the pH of the reaction is 5.5; And / or, the reaction temperature is 45°C to 65°C; preferably, the reaction temperature is 50°C to 60°C; more preferably, the reaction temperature is 55°C; And / or, the reaction time is 10 min to 2 h; preferably, the reaction time is 10 min to 30 min; more preferably, the reaction time is 10 min.

9. Use of the endoinulinase ER-INUI according to claim 1 or 2 in hydrolyzing inulin into oligofructose.

10. The use according to claim 9, characterized in that: The oligofructose includes any one or more of kestose, kestose, and pentose.

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