Low-temperature endo-inulinase mutant Y94V and application thereof

Abstract

The invention relates to the technical field of enzyme gene engineering, in particular to an endo-inulinase mutant and application thereof. The invention provides an endo-inulinase mutant, compared with wild type endo-inulinase, the endo-inulinase mutant comprises Y94V mutation, and the amino acid sequence of the wild type endo-inulinase is as shown in SEQ ID NO. 1. The amino acid sequence of the endo-inulinase mutant is as shown in SEQ ID NO. 2. Compared with wild type endo-inulinase, the endo-inulinase mutant provided by the invention has higher inulin hydrolytic activity, the optimum temperature is reduced from 55 DEG C to 45 DEG C, the specific activity at 45 DEG C is 1.3 times that of the wild type endo-inulinase, the industrial energy consumption is remarkably reduced, high-temperature side reaction is avoided, and the endo-inulinase mutant has a wider market prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of enzyme genetic engineering, in particular to a low-temperature endo-inulinase mutant Y94V and application thereof. BACKGROUND

[0002] Inulin is a natural fructan widely existing in plants such as Jerusalem artichoke and chicory, and is an important raw material for producing high fructose syrup, oligofructose (FOS) and other high value-added products and bioethanol. Endo-inulinase (EC 3.2.1.7) can randomly hydrolyze the internal β-2,1-glycosidic bond of inulin to produce fructooligosaccharides with different degrees of polymerization, and is one of the key enzymes for efficient utilization of inulin.

[0003] The wild type of the currently known endo-inulinase from microorganisms generally has a relatively high optimal reaction temperature, usually between 50℃ and 60℃, for example, the optimal temperature of the endo-inulinase from common Aspergillus niger is about 55℃. Although a higher reaction temperature can sometimes improve the reaction rate, in many practical application scenarios, high-temperature operation has significant disadvantages: (1) huge energy consumption, especially in large-scale production; (2) may cause undesirable side reactions, such as the Maillard reaction to produce pigments and odors, affecting the color and flavor of the final product (especially in the food field); (3) may destroy the simultaneously existing heat-sensitive raw material components or products; (4) limits the coupling efficiency with low-temperature fermentation processes (such as some yeast fermentation) (in the field of biofuels). Therefore, developing an endo-inulinase with a lower optimal temperature while maintaining high catalytic activity and good stability is of great significance for reducing production costs, improving product quality and expanding application range. However, there is no report on lowering the optimal temperature of endo-inulinase to 45℃ and improving low-temperature activity by single-point mutation. SUMMARY

[0004] In view of the above-mentioned disadvantages of the prior art, the purpose of the present application is to provide an endo-inulinase mutant, a preparation method and application thereof, to solve the problems in the prior art.

[0005] To achieve the above-mentioned objects and other related objects, the first aspect of the present application provides an endo-inulinase mutant, wherein the endo-inulinase mutant comprises a Y94V mutation compared to a wild type endo-inulinase mutant, and the amino acid sequence of the wild type endo-inulinase is shown in SEQ ID NO. 1.

[0006] The second aspect of the present application provides an isolated polynucleotide encoding the aforementioned endo-inulinase mutant.

[0007] The third aspect of the present application provides a construct comprising the isolated polynucleotide.

[0008] The fourth aspect of the present application provides a host cell containing the construct or the isolated polynucleotide integrated into the genome of the host cell.

[0009] The fifth aspect of the present application provides a method for preparing the endoxylanase, comprising the following steps: introducing the aforementioned polynucleotide or the aforementioned construct into a host cell, inducing the expression of the exogenous gene, and isolating and purifying to obtain the endoxylanase mutant.

[0010] The sixth aspect of the present application provides the use of the endoxylanase, the isolated polynucleotide, the nucleic acid construct or the engineered cell in the preparation of a hydrolyzed inulin product.

[0011] The eighth aspect of the present application provides a method for hydrolyzing inulin using the aforementioned endoxylanase mutant, wherein the temperature for hydrolysis is selected from 30-40℃, and the pH is selected from 5.0-6.0.

[0012] Compared with the prior art, the beneficial effects of the present application include:

[0013] The present application obtains the endoxylanase mutant Y94V by site-directed mutagenesis of the wild-type enzyme in Emericella rugulosa NRRL 11440. Compared with the wild-type endoxylanase ER-INUI, the catalytic properties of the mutant Y94V are significantly changed, and the low-temperature catalytic efficiency and industrial applicability are breakthroughly improved. The low-temperature activity is significantly enhanced: the optimal temperature of the mutant is reduced from 55℃ to 45℃, and the activity at 40℃ is 1.6 times that of the wild-type enzyme at the same temperature. The relative enzyme activity at 40℃ can be maintained at ≥95% (the wild-type is ≤80%). The low-temperature stability and activity are synergistically optimized: after 1 hour of treatment at 40℃, the enzyme activity retention rate of the mutant is >99% (equivalent to the wild-type under the same conditions); although the stability at 45℃ is reduced (70% activity is retained for 1 hour), this defect can be avoided by controlling the reaction temperature ≤40℃, so that high activity and high stability coexist. The mutant Y94V has high activity and stability when catalyzing inulin hydrolysis under mild conditions ≤40℃ (40℃ for 1 hour retains >95% activity). The industrial energy consumption is significantly reduced and the high-temperature side reactions are avoided, which is suitable for the industrialized production of oligofructose and has outstanding advantages in industrial applications. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 The SDS-PAGE electropherogram of ER-INUI and mutant Y94V after expression and purification is shown. The arrow indicates the position of the endoxylanase protein band.

[0015] Figure 2The effect of pH on ER-INUI and mutant Y94V is shown. In which, the abscissa represents pH, and the ordinate represents relative enzyme activity.

[0016] Figure 3 The effect of pH on the stability of ER-INUI and mutant Y94V is shown. In which, the abscissa represents pH, and the ordinate represents relative enzyme activity.

[0017] Figure 4 The effect of temperature on ER-INUI and mutant Y94V is shown. In which, the abscissa represents temperature, and the ordinate represents relative enzyme activity.

[0018] Figure 5 The effect of temperature on the stability of ER-INUI and mutant Y94V is shown. In which, the abscissa represents temperature, and the ordinate represents relative enzyme activity.

[0019] Figure 6 The product analysis thin layer chromatogram of the hydrolysis of wild type ER-INUI and mutant Y94V is shown when the inulin is used as the substrate for the conversion reaction of ER-INUI. DETAILED DESCRIPTION

[0020] The embodiments of the present application will be described in detail below with specific reference being made to certain embodiments. As would be obvious to one skilled in the art, other advantages and benefits of the present application can be readily ascertained from the disclosure. The present application can be practiced in other embodiments and by using different methods than those described herein without departing from the spirit of the present application.

[0021] In the present application, by mutating the endo-inulinase, an endo-inulinase mutant is obtained. Compared with the wild type endo-inulinase, the catalytic properties of the mutant are significantly changed, the low temperature catalytic efficiency and industrial applicability are breakthroughly improved, the low temperature activity is significantly enhanced, and high activity and high stability can coexist.

[0022] The present application first provides an endo-inulinase mutant, which contains a Y94V mutation compared with the wild type endo-inulinase mutant, and the amino acid sequence of the wild type endo-inulinase is shown in SEQ ID NO. 1.

[0023] In the present application, the amino acid sequence of the endo-inulinase mutant is shown in SEQ ID NO. 2.

[0024] SEQ ID NO. 1:

[0025] MFLHILCLLAGQALADDYRPVFHFVPEKNWMNEPNGLIKIGSTWHLFYQHNPTANVWGNLNWGHATSSDLVHWTHEPLAITSENGIEAFTGTSYYDAENTSGLGTSDNPPYLAWYTGYFPSNGTQDQRLAFSIDAGETWTKFEGNPVISAAQEAPHDATGGLETRDPKVFFHADSGKWIMVLAHGGQNKMSFWTSVDAKRWSWASDLTSTQVPGLPSAVKGWEVPDMFEVPVHGTDKTTWVLIFTPAEGSPAGGNGVLALTGSFDGTVFHPNPVNVSTLWLDYGRDFDGALSWENLPDSDGHRILAAISNSYGANPPTNTWKGMLSFPRTLSLHQSQTGQYFLQQPVSNLDTVSTPLVSVKNQTIAPGQVLLSSVRGTALDIRIAFSANAGTVLSLAVRKAGSQETVIQYRQSDATLSVDRTTSGLTSYDPAAGGVHTAPLRPDASGVVQIRALVDTCSVEVFGGQGEVVISDLIFPDETSDGLALQVIGGTAVLRSLEVREISLDLE.

[0026] SEQ ID NO. 2:

[0027] MFLHILCLLAGQALADDYRPVFHFVPEKNWMNEPNGLIKIGSTWHLFYQHNPTANVWGNLNWGHATSSDLVHWTHEPLAITSENGIEAFTGTSVYDAENTSGLGTSDNPPYLAWYTGYFPSNGTQDQRLAFSIDAGETWTKFEGNPVISAAQEAPHDATGGLETRDPKVFFHADSGKWIMVLAHGGQNKMSFWTSVDAKRWSWASDLTSTQVPGLPSAVKGWEVPDMFEVPVHGTDKTTWVLIFTPAEGSPAGGNGVLALTGSFDGTVFHPNPVNVSTLWLDYGRDFDGALSWENLPDSDGHRILAAISNSYGANPPTNTWKGMLSFPRTLSLHQSQTGQYFLQQPVSNLDTVSTPLVSVKNQTIAPGQVLLSSVRGTALDIRIAFSANAGTVLSLAVRKAGSQETVIQYRQSDATLSVDRTTSGLTSYDPAAGGVHTAPLRPDASGVVQIRALVDTCSVEVFGGQGEVVISDLIFPDETSDGLALQVIGGTAVLRSLEVREISLDLE.

[0028] In the endoxylanase mutant of the present application, the low-temperature activity of the endoxylanase mutant is significantly enhanced compared with the wild-type endoxylanase.

[0029] The present application also provides an isolated polynucleotide encoding the above-mentioned endoxylanase mutant.

[0030] The polynucleotide of the present application 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.

[0031] In one embodiment, the isolated polynucleotide has a nucleotide sequence as shown in SEQ ID No. 3.

[0032] SEQ ID No. 3:

[0033]

[0034] The present application also provides a construct comprising the isolated polynucleotide as described above.

[0035] In some embodiments of the present application, the construct is constructed by inserting the isolated polynucleotide into a multiple cloning site of an expression vector. The expression vector in the present application generally refers to various commercially available expression vectors well known in the art, such as bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus or other vectors. Preferably, the expression vector is pET28a.

[0036] The present application also provides a host cell comprising the construct as described above, or the host cell genome integrated with the polynucleotide as described above.

[0037] In some embodiments, the host cell comprises a prokaryotic cell or a eukaryotic cell. Specifically, the prokaryotic cell can be selected from Escherichia coli, Salmonella typhimurium, Klebsiella pneumoniae, Bacteroides ovatus, Campylobacter jejuni, Staphylococcus saprophyticus, Enterococcus faecalis, Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bacteroides uniformis, Lactobacillus casei, Bacteroides fragilis, Acinetobacter lwoffi, Fusobacterium nucleatum, Bacteroides joine, Bacteroides arnini, Lactobacillus rhamnosus, Bacteroides massiliosus, Bacteroides caccae, Fusobacterium mortiferum, Bifidobacterium breve or Listeria; or the eukaryotic cell can be selected from yeast cell, Streptomyces, Drosophila S2 or Sf9 insect cell, CHO, COS.293 cell or Bowes melanoma cell. Preferably, the host cell is Escherichia coli BL21 (DE3). The above-mentioned Escherichia coli is required to be able to express the above-mentioned endoxylanase mutant, so as to provide conditions for the presence of endoxylanase. Suitable methods for constructing the above-mentioned Escherichia coli should be known to those skilled in the art.

[0038] The present application also provides a method for preparing the endoxylanase mutant, which comprises: introducing the above-mentioned polynucleotide or the above-mentioned construct into a host cell, inducing the expression of the exogenous gene, and isolating and purifying to obtain the endoxylanase mutant.

[0039] In some embodiments, the host cell is selected from a prokaryotic cell or a eukaryotic cell. Specifically, the prokaryotic cell can be selected from E. coli, Salmonella typhimurium, Klebsiella pneumoniae, Bacteroides ovatus, Campylobacter jejuni, Staphylococcus saprophyticus, Enterococcus faecalis, Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bacteroides uniformis, Lactobacillus casei, Bacteroides fragilis, Acinetobacter lwoffi, Fusobacterium nucleatum, Bacteroides joine, Bacteroides arnini, Lactobacillus rhamnosus, Bacteroides massiliosus, Bacteroides caccae, Fusobacterium mortiferum, Bifidobacterium breve, or Listeria monocytogenes; or the eukaryotic cell can be selected from a yeast cell, Streptomyces, Drosophila S2 or Sf9 insect cell, CHO, COS.293 cell, or Bowes melanoma cell.

[0040] In some embodiments, the method of introduction can be selected from one or more of DEAE-dextran mediated transfection, liposome-mediated transfection, viral or bacteriophage infection, lipofection, transfection, conjugation, protoplast fusion, polyethylenimine-mediated transfection, electroporation, calcium phosphate precipitation, gene gun, calcium phosphate precipitation, microinjection, nanoparticle-mediated nucleic acid delivery.

[0041] In one embodiment, the inducer for inducing expression is selected from IPTG. IPTG induction is a conventional induction method for engineering bacteria to express a protein of interest, which is mature and reliable in the art.

[0042] The present application also provides use of one or more of the aforementioned endo-inulinase mutants, isolated polynucleotides, constructs or host cells in preparing a hydrolyzed inulin product.

[0043] The present application also provides a method of hydrolyzing inulin using the aforementioned endo-inulinase mutants, wherein the temperature of hydrolysis is selected from 30-40°C, and the pH is selected from 5.0-6.0.

[0044] In one embodiment, the temperature of hydrolysis is selected from 40°C, and the hydrolysis time is less than 2 hours. Further, the mass concentration of inulin is selected from 5-10%, preferably 5%. Still further, the mass ratio of the endo-inulinase mutant to inulin is 1:800-1000, preferably 1:1000.

[0045] Before further description of the application, it is understood that the application is not limited in scope to the specific embodiments described herein; and that the term "comprises" as used in the specification and claims is not intended to exclude the presence of other elements or steps. Furthermore, the use of the term "including" as well as other forms such as "include", "includes" for describing compositions containing certain elements or steps is not meant to be construed as limitations on the specific compositions or methods.

[0046] When the embodiments give numerical ranges, it is understood that unless the present invention indicates otherwise, every numerical range's two endpoints and any number between the two endpoints are optional. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices and materials similar or equivalent to those described in the embodiments of the present invention can be used according to the mastery of the prior art by those skilled in the art and the description of the present invention to implement the present invention.

[0047] Unless otherwise specified, the experimental methods, detection methods, preparation methods disclosed in the present invention all use conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and related fields.

[0048] Example 1: Construction of mutant Y94V encoding gene and preparation of recombinant vector

[0049] Using the published Emericella rugulosa NRRL 11440 wild-type endo-inulinase (the amino acid sequence of the wild-type endo-inulinase is shown as SEQ ID NO: 1, and the nucleotide sequence is shown as SEQ ID NO: 4), primers were designed according to the nucleotide sequence of the wild-type endo-inulinase. Forward primer Y94V-F: 5'-GTTTACCGGCACCTCTGTGTATGATGCGGAAAACAC-3', reverse primer Y94V-R: 5'-GTGTTTTCCGCATCATACACAGAGGTGCCGGTAAAC-3'. PCR amplification was carried out with plasmid pET28a(+)-inui as template. The codon was changed from TAT (tyrosine) to GTG (valine), and the mutant site corresponds to amino acid position Y94V, the mutant nucleotide sequence (SEQ ID NO: 3), with His tag at the C-terminal.

[0050] SEQ ID NO: 1:

[0051] MFLHILCLLAGQALADDYRPVFHFVPEKNWMNEPNGLIKIGSTWHLFYQHNPTANVWGNLNWGHATSSDLVHWTHEPLAITSENGIEAFTGTSYYDAENTSGLGTSDNPPYLAWYTGYFPSNGTQDQRLAFSIDAGETWTKFEGNPVISAAQEAPHDATGGLETRDPKVFFHADSGKWIMVLAHGGQNKMSFWTSVDAKRWSWASDLTSTQVPGLPSAVKGWEVPDMFEVPVHGTDKTTWVLIFTPAEGSPAGGNGVLALTGSFDGTVFHPNPVNVSTLWLDYGRDFDGALSWENLPDSDGHRILAAISNSYGANPPTNTWKGMLSFPRTLSLHQSQTGQYFLQQPVSNLDTVSTPLVSVKNQTIAPGQVLLSSVRGTALDIRIAFSANAGTVLSLAVRKAGSQETVIQYRQSDATLSVDRTTSGLTSYDPAAGGVHTAPLRPDASGVVQIRALVDTCSVEVFGGQGEVVISDLIFPDETSDGLALQVIGGTAVLRSLEVREISLDLE.

[0052] SEQ ID NO: 4:

[0053]

[0054] The obtained PCR product was added with 1 μL Dpn I and digested at 37 °C for 3 h (to degrade the methylated template plasmid completely). The recombinant plasmid was then transformed into E. coli BL21 (DE3) and plated on LB solid medium containing 50 μg / mL kanamycin. Single colonies were selected and sent to Jiweizhi Biotechnology Co., Ltd. for sequencing to confirm that the codon was changed from TAT to GTG (Y94V mutation), i.e., the recombinant plasmid pET28a-Y94V was obtained.

[0055] Example 2: Expression and purification of wild-type ER-INUI and mutant Y94V

[0056] E. coli BL21 (DE3) chemically competent cells (full type Jinbiotechnology) were used; the cells were stored in a glycerol tube (20% glycerol, L / L) at -80 °C and thawed in an ice bath before use. The recombinant plasmid pET28a-Y94V was transformed into E. coli BL21 (DE3) and plated on LB-Kan plates (composition: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, agar 15 g / L, kanamycin 50 μg / mL, pH 7.0) and incubated at 37 °C for 16 h.

[0057] Activation of the strain: a single colony was inoculated into 5 mL of TB-Kan liquid medium (peptone 12 g / L, yeast extract 24 g / L, potassium dihydrogen phosphate 2.31 g / L, dipotassium hydrogen phosphate 12.54 g / L, glycerol 4 mL / L) and incubated at 37 °C and 200 rpm for 12 h for standby.

[0058] Fermentation of the strain: the activated genetic engineering bacteria pET28a(+)-Y94V / BL21 (DE3) culture solution was inoculated into 30 mL of TB liquid medium containing 50 μg / mL kanamycin at an inoculation amount of 1%, and fermented at 180 rpm and 25 °C. When the OD 600 of the bacterial solution reached 0.5-0.8, 1 mM IPTG was added to induce expression for 18 h. After stopping the fermentation, the fermentation broth was centrifuged at 10,000 rpm for 30 min, and the precipitate was collected. The precipitate was crushed with an ultrasonic crusher and centrifuged again to obtain the intracellular supernatant, i.e., the crude enzyme in the intracellular.

[0059] The cell crushing supernatant was subjected to nickel column affinity chromatography to obtain purified Y94V. Based on the SDS-PAGE electrophoresis results, Y94V had a single band Figure 1 ); the purified sample was collected and desalted.

[0060] Example 3: Enzymatic property determination

[0061] (1) Activity analysis of purified wild-type ER-INUI and mutant Y94V:

[0062] The endo-inulinase activity was determined as follows: a standard curve was prepared by plotting the absorbance at 540 nm against the concentration of fructose in the range of 0-1 mg / mL. 800 μL of inulin solution (2.0% w / L) was incubated with 200 μL of enzyme solution at the reaction temperature and pH for 10 min, and then 1 mL of DNS reagent was added to stop the reaction. The mixture was heated at 100°C for 10 min to inactivate the enzyme, and then cooled, diluted 5-fold and the absorbance at 540 nm was measured. The endo-inulinase activity was calculated according to the standard curve. The enzyme activity was defined as 1 unit (U) of enzyme activity per mL of enzyme required to hydrolyze inulin to produce 1 μmol of reducing sugar per minute. The specific enzyme activity was defined as the number of units (U) of enzyme activity per mg of endo-inulinase.

[0063] The results are shown in Table 1. Figure 2 As shown in Table 1, the specific activity of mutant Y94V was 1.26 times that of wild-type ER-INUI at 40°C.

[0064] (2) pH activity and pH stability determination of purified wild-type ER-INUI and mutant Y94V

[0065] The pH activity of the enzyme was determined by incubating the enzyme solution at 37°C in a buffer having a pH of 3.0-12.0. The pH stability of the enzyme was determined by incubating the enzyme solution in a buffer having a pH of 3.0-12.0 at room temperature for 1 h, and then performing an enzymatic reaction at pH 6.0 and 55°C. The enzyme activity of wild-type ER-INUI and mutant Y94V was determined by using inulin as a substrate and incubating for 10 min.

[0066] The results show that the optimal pH of wild-type ER-INUI and mutant Y94V was 5.5, respectively. Figure 2 After incubation in a buffer having a pH of 3.0-10.0 for 1 h, the enzyme activity of ER-INUI and mutant Y94V was more than 60%. Figure 3

[0067] (3) Thermal activity and thermal stability determination of purified wild-type ER-INUI and mutant Y94V

[0068] The optimal reaction temperature was determined by measuring the activity of purified ER-INUI obtained in Example 2 at different temperatures (20-80°C) and pH 5.5. The highest enzyme activity was set as 100%, and each experiment was performed in triplicate and the results were averaged.​

[0069] The results show that the optimum temperature of wild enzyme ER-INUI is 55°C, and it has 10%, 36%, 75% and 84% of enzyme activity at 20°C, 25°C, 40°C and 45°C, respectively. The optimum temperature of mutant enzyme Y94V is 45°C, and it has 39%, 63%, 96% of enzyme activity at 20°C, 25°C and 40°C, respectively. Figure 4 The results show that the enzyme activity of mutant Y94V at 40°C is 1.62 times of that of wild type ER-INUI.

[0070] Determination of thermal stability: ER-INUI obtained in Example 2 was incubated at 30°C-80°C for 1 h, and then rapidly cooled on ice. The residual enzyme activity was determined, and the enzyme activity of the unincubated enzyme was taken as 100%. The results are shown in Table 2. Figure 5 The results show that the residual enzyme activity of wild enzyme ER-INUI and mutant Y94V is 99.9% and 99.8% respectively after treatment at 40°C for 1 h, indicating that both of them can remain stable at 40°C.

[0071] Example 4: Analysis of the products of inulin hydrolysis by purified wild enzyme ER-INUI and mutant enzyme Y94V

[0072] ER-INUI and mutant enzyme Y94V obtained in Example 2 were used as the enzyme, and 5% commercial inulin was used as the substrate. The mass ratio of ER-INUI or mutant enzyme Y94V to inulin was 1:1000, and the reaction was carried out at the optimum reaction temperature and the optimum pH value for 30 min to obtain the reaction solution. TLC thin layer chromatography was used to analyze the hydrolysis products. The results of TLC show that the main products of both wild type and mutant are inulin trisaccharide-inulin pentasaccharide (GF2-GF4). Figure 6

[0073] Although the present application has been disclosed with reference to the preferred embodiments as described above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, and therefore the protection scope of the present application should be defined by the claims.

[0074] The above examples are intended to illustrate the embodiments disclosed in the present application, and should not be construed as limiting the present application. In addition, various modifications and changes of the methods and compositions in the present application listed herein are obvious to those skilled in the art without departing from the scope and spirit of the present application. Although the present application has been specifically described in conjunction with various specific preferred embodiments of the present application, it should be understood that the present application should not be limited to these specific embodiments. In fact, various modifications as described above to obtain the present application are obvious to those skilled in the art and should be included in the scope of the present application.​

Claims

1. An endoxylanase mutant, characterized in that, The endoxylanase mutant comprises a Y94V mutation, and the amino acid sequence of the wild-type endoxylanase is shown in SEQ ID NO.

1.

2. The inulinase mutant according to claim 1, characterized in that, The amino acid sequence of the endoxylanase mutant is shown in SEQ ID NO. 2; and / or the endoxylanase mutant has higher low-temperature activity than the wild-type endoxylanase.

3. An isolated polynucleotide, comprising, The isolated polynucleotide encodes the endoxylanase mutant of claim 1 or 2.

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

5. The construct of claim 4, wherein, The construct is constructed by inserting the isolated polynucleotide into a multiple cloning site of an expression vector, preferably the expression vector is pET28a.

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

7. The host cell of claim 6, wherein The host cell comprises a prokaryotic cell or a eukaryotic cell, preferably the host cell is Escherichia coli BL21 (DE3).

8. A method for producing an endoxylanase mutant, said method comprising: After introducing the polynucleotide of claim 3, or the construct of claim 4 or 5 into an engineering cell, the expression of the exogenous gene is induced, and the endoxylanase mutant is obtained by separation and purification.

9. Use of the endoxylanase mutant of claim 1 or 2, the isolated polynucleotide of claim 3, the construct of claim 4 or 5, or the host cell of claim 6 or 7 in preparing a hydrolyzed inulin product.

10. A method for hydrolyzing inulin, wherein the inulin is hydrolyzed by the endoxylanase mutant of claim 1 or 2, the temperature of the hydrolysis is selected from 30-40℃, the pH is selected from 5.0-6.0, and the mass ratio of the endoxylanase to inulin is 1:800-1000.

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