Thermophilic inulin endonuclease as well as screening method, preparation method and application thereof

Abstract

The invention discloses a thermophilic endo-inulinase as well as a screening method, a preparation method and application thereof, and relates to the technical field of microbial engineering.The screening method of the thermophilic endo-inulinase comprises the following steps: collecting homologous sequences of known endo-inulinase by adopting a bioinformatics technology and forming a local protein sequence database; the thermophilic endo-inulinase I nuG with excellent stability is obtained by screening the thermophilic endo-inulinase according to the growth temperature of the strain from the homologous sequence and anchoring the homologous sequence corresponding to the strain with the highest growth temperature; according to the thermophilic inulin endonuclease as well as the screening method, the preparation method and the application thereof, the novel thermophilic inulin endonuclease I nuG is successfully obtained through a double screening strategy based on homologous sequence alignment and microbial growth temperature, and the high-temperature catalytic characteristic of the thermophilic inulin endonuclease I nuG not only reduces energy consumption and infectious microbe pollution risk, but also can improve the production efficiency of fructo-oligosaccharide, so that the novel thermophilic inulin endonuclease I nuG has broad application prospects. The problem that the substrate of the endo-inulinase is difficult to dissolve at normal temperature is solved.

Description

Technical Field

[0001] The present invention relates to microbial engineering technology, and in particular to a thermophilic inulin endonuclease and a screening method, a preparation method and an application thereof. Background Art

[0002] Fructooligosaccharides (FOS) are functional oligosaccharides with a degree of polymerization (DP) between 2 and 9. They are widely used in the food and pharmaceutical fields, such as improving intestinal health and promoting nutrient absorption. There are two main methods for their preparation: one uses sucrose as a substrate and utilizes β-fructosyltransferase or β-fructofuranosidase to catalyze the intermolecular fructosyl reaction; the other uses inulin as a substrate and utilizes endoinulinase to catalyze the hydrolysis reaction. The production of FOS using endoinulinase to hydrolyze inulin offers advantages such as a simple reaction, few byproducts, and a high yield, resulting in greater production value.

[0003] Endoinulinase is a glycoside hydrolase that randomly cleaves β-fructosidic bonds within fructan molecules. The main hydrolysis product is a mixture of oligofructose with varying degrees of polymerization (dp). However, current wild-type strains that produce endoinulinase suffer from low enzyme yields or specific activities, and secrete a complex enzyme system that makes enzyme isolation and purification difficult, leading to increased production costs and limiting its industrial application.

[0004] At the currently reported optimal reaction temperature for endoinulinase, the solubility of natural inulin is less than 30%. However, inulin solubility increases with increasing temperature, making raising the conversion temperature an effective measure to address the challenges of oligofructose production. However, existing endoinulinase enzymes are not stable enough at higher temperatures, making them difficult to meet the demands of industrial production. Therefore, screening for thermophilic endoinulinase suitable for industrial production is crucial to resolving the bottleneck in oligofructose production. Summary of the Invention

[0005] The purpose of the present invention is to provide a thermophilic inulinase and a screening method, a preparation method and an application thereof, so as to solve the above-mentioned deficiencies in the prior art.

[0006] In order to achieve the above object, the present invention provides the following technical solution: a method for screening thermophilic inulin endoinuclease, comprising the following steps:

[0007] S1. Use bioinformatics technology to collect homologous sequences of known inulin endonucleases and form a local protein sequence database;

[0008] S2. Screening thermophilic inulin endonuclease by the growth temperature of the strain from which the homologous sequence is derived, anchoring the homologous sequence of the strain with the highest growth temperature, and obtaining the thermophilic inulin endonuclease InuG.

[0009] Furthermore, the S1 includes the following steps:

[0010] S11, first select inulin endonuclease I nu1 from Arthrobacter sp.S37 strain as the probe sequence;

[0011] S12, then according to the principle of sequence similarity, using BLAST to compare in the NCB I database to find homologous sequences of the probe sequence Inu1, and setting the similarity condition of the homologous sequence to be greater than 20%;

[0012] S13. These homologous sequences were then constructed into a local protein sequence database.

[0013] Furthermore, the S2 includes the following steps:

[0014] S21, determining the growth temperature of the strain from which the homologous sequence in the protein sequence database obtained in S1 is derived;

[0015] S22, then selecting homologous sequences of strains with a growth temperature equal to or higher than 50°C, and reordering these homologous sequences according to the growth temperature of the strains;

[0016] S23. Finally, the homologous sequence of the strain with the highest growth temperature was anchored and named inuG, which is the thermophilic inulin endonuclease InuG.

[0017] A method for preparing thermophilic inulin endoinuclease comprises the following steps:

[0018] A1. After codon optimization of the inuG sequence screened out by the screening method for the thermophilic inulin endonuclease, an InuG coding gene suitable for the E. coli expression system is obtained by whole gene synthesis technology; the signal peptide fragment of the optimized InuG coding gene is removed by PCR, and then the gene is ligated with the cloning vector pLB to construct a recombinant plasmid pLB-inuG; the recombinant plasmid is transferred into the cloning host E. coli DH5α, and the E. coli DH5α is cultured. After the culture is completed, the recombinant plasmid is extracted, and then the extracted recombinant plasmid is double-digested with the pET-28a(+) expression vector, the target gene inuG obtained by the double-digestion and the linearized pET-28a(+) are recovered, and then the target gene inuG is ligated with the linearized pET-28a(+) to obtain the expression vector pET-28a-inuG;

[0019] A2. Transform the expression vector pET-28a-inuG into competent cells of the expression host E. coli Rosetta (DE3) and culture for 1 hour. After the culture is completed, centrifuge the culture, retain 100 μL of the supernatant and resuspend the bacterial solution, then spread it on the surface of LB solid medium containing kanamycin and culture overnight to obtain E. coli carrying the plasmid pET-28a-inuG;

[0020] A3. Inoculate E. coli carrying plasmid pET-28a-inuG into LB liquid medium for cultivation. 600 When the value reached 0.5, isopropyl-β-D-thiogalactopyranoside was added to the culture medium and cultured overnight. The cells were collected by centrifugation and ultrasonically disrupted. After disruption, the supernatant and precipitate were collected separately by centrifugation.

[0021] A4. Connect the Ni column to an AKTA protein purifier, and purify the supernatant described in A3 using the AKTA protein purifier to obtain the target protein InuG, i.e., thermophilic endonuclease.

[0022] Furthermore, the GenBank accession number of the inuG codon-optimized sequence of A1 is OP115877.

[0023] Furthermore, the culture conditions before adding isopropyl-β-D-thiogalactopyranoside to the culture medium in A3 are 37° C. and a rotation speed of 200 rpm; the culture conditions after adding isopropyl-β-D-thiogalactopyranoside to the culture medium in A3 are 16° C. and a rotation speed of 200 rpm.

[0024] Furthermore, the amount of isopropyl-β-D-thiogalactopyranoside added to the culture medium in A3 is such that its final concentration in the culture medium is 0.3 mmol / L.

[0025] A thermophilic inulinase is prepared by the preparation method. The coding sequence of the thermophilic inulinase is derived from Thermoanaerobacter italicus Ab9 and is thermostable.

[0026] Application of a thermophilic inulinase in the industrial production of oligofructose.

[0027] Compared with the existing technology, the present invention provides a thermophilic inulin endonuclease and its screening method, preparation method and application. Through a dual screening strategy based on homologous sequence alignment and microbial growth temperature, the thermophilic inulin endonuclease InuG is obtained, which solves the problem that traditional inulin endonucleases are difficult to dissolve substrates at room temperature.

[0028] InuG has an optimal reaction temperature of 70°C, a dissolution temperature of 78°C, and a long half-life of 82 hours. It stably catalyzes the hydrolysis of inulin (the main product is inulin trisaccharide) at high temperatures. Its wide pH adaptability (pH 4.0-8.0) is compatible with industrial fermentation environments. Its high-temperature catalytic properties eliminate the need for cooling steps, reducing energy consumption and the risk of bacterial contamination, thereby improving the efficiency of oligofructose production.

[0029] This enzyme and its screening strategy form a closed-loop innovation system of "basic research-industrial application", which has both the practical value of efficiently producing functional oligosaccharides and the theoretical value of guiding the rational design of enzymes, promoting the development of high-temperature biocatalysts. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments described in the present invention. For ordinary technicians in this field, other drawings can also be obtained based on these drawings.

[0031] Figure 1 A schematic diagram of the three-dimensional structure of InuG predicted by AlphaFold2 provided in an embodiment of the present invention;

[0032] Figure 2 Schematic diagram of protein sequence alignment analysis between InuG and the model sequences Inul and InuB provided in an embodiment of the present invention;

[0033] Figure 3 Schematic diagram of the results of removing the signal peptide from G provided in an embodiment of the present invention;

[0034] Figure 4 Double enzyme digestion gel image of plasmid pLB-inuG and pET-28a(+) vector provided in the embodiment of the present invention;

[0035] Figure 5 SDS-PAGE gel images at different stages provided by the embodiments of the present invention;

[0036] Figure 6 Schematic diagram of the main degradation products produced by the hydrolysis of inulin by InuG provided in an embodiment of the present invention;

[0037] Figure 7 Schematic diagram of the change of InuG catalytic activity at different reaction temperatures provided in an embodiment of the present invention;

[0038] Figure 8 Schematic diagram of thermal stability detection of InuG provided by an embodiment of the present invention;

[0039] Figure 9Schematic diagram of the effect of pH on InuG provided in an embodiment of the present invention;

[0040] Figure 10 Scoring of protein structure predictions for InuG, Inu1, and InuB using AlphaFold2 provided in the embodiments of the present invention;

[0041] Figure 11 A schematic diagram of the change of RMSD value with simulation time provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0042] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0043] Example 1:

[0044] See also Figures 1 to 2 , a method for screening thermophilic inulin endoinuclease, comprising the following steps:

[0045] S1. Use bioinformatics technology to collect homologous sequences of known inulin endonucleases and form a local protein sequence database;

[0046] S11, first select inulin endonuclease I nu1 from Arthrobacter sp.S37 strain as the probe sequence;

[0047] S12, then according to the principle of sequence similarity, using BLAST to compare in the NCB I database to find homologous sequences of the probe sequence Inu1, and setting the similarity condition of the homologous sequence to be greater than 20%;

[0048] S13. These homologous sequences were then constructed into a local protein sequence database.

[0049] S2. Screening thermophilic inulin endonuclease by the growth temperature of the strain from which the homologous sequence is derived, anchoring the homologous sequence of the strain with the highest growth temperature, and obtaining the thermophilic inulin endonuclease InuG.

[0050] S21, determining the growth temperature of the strain from which the homologous sequence in the protein sequence database obtained in S1 is derived;

[0051] S22, then selecting homologous sequences of strains with a growth temperature equal to or higher than 50°C, and reordering these selected homologous sequences according to the growth temperature of the strains;

[0052] S23. Finally, the homologous sequence of the strain with the highest growth temperature was anchored and named inuG, which is the thermophilic inulin endonuclease InuG.

[0053] In the first step, using the inulinase endonuclease I nu1 from Arthrobacter sp. S37 as a reference sequence, sequence alignment yielded 722 homologous sequences with greater than 20% sequence similarity. Inu1 was chosen as the reference sequence because it is the only bacterial inulinase with a published protein sequence and in-depth enzymatic properties. A literature search determined the growth temperatures of the strains from which the homologous sequences were derived within the protein sequence database. Using a strain growth temperature ≥ 50°C as a screening criterion, 27 homologous sequences were identified whose source strains met this screening criterion. Finally, the homologous sequences from this secondary screening were rearranged in descending order based on the source strain growth temperature. The homologous sequence inuG (WP_012996024) corresponding to the bacterium with the highest growth temperature was identified as a potential thermophilic inulinase and named InuG. The bacterium inuG, which originates from Thermoanaerobacter italicus Ab9, has a growth temperature of 70°C.

[0054] See also Figure 1 , AlphaFold2 was used to perform homology modeling on inulin endonuclease InuG, and the protein structure is as follows Figure 1 shown; see Figure 2 The amino acid residues marked with asterisks represent the catalytic active sites of GH32 activity. The CDD domain prediction analysis of NCB I revealed that InuG contains the N-terminal structural region (His274-Lle587) and the C-terminal domain (Glu592-Lys743) of GH32. The characteristic LamG-like jelly fold domain (Glu81-Glu232) of bacterial inulin endonuclease was detected at the N-terminus of InuG. Therefore, InuG belongs to GH32 and is speculated to be a bacterial inulin endonuclease.

[0055] Through the two-step thermophilic enzyme screening strategy developed in the laboratory, the thermophilic inulin endonuclease gene was successfully located. This method not only abandons the huge workload and lengthy working time of traditional transformation methods, but also avoids the contradiction between the growth environment temperature of natural inulin and the growth environment temperature of thermophilic microorganisms. It solves the problem of the difficulty in enriching extreme microorganisms in the plant growth environment from a new perspective, and provides ideas for the development of more extreme enzymes.

[0056] Example 2:

[0057] See also Figures 3 to 5 A method for preparing a thermophilic inulin endoinuclease comprises the following steps:

[0058] A1. After codon optimization of the inuG sequence screened out by the screening method for the thermophilic inulin endonuclease, an InuG coding gene suitable for the E. coli expression system is obtained by whole gene synthesis technology; the signal peptide fragment of the optimized InuG coding gene is removed by PCR, and then the gene is ligated with the cloning vector pLB to construct a recombinant plasmid pLB-inuG; the recombinant plasmid is transferred into the cloning host E. coli DH5α, and the E. coli DH5α is cultured. After the culture is completed, the recombinant plasmid is extracted, and then the extracted recombinant plasmid is double-digested with the pET-28a(+) expression vector, the target gene inuG obtained by the double-digestion and the linearized pET-28a(+) are recovered, and then the target gene inuG is ligated with the linearized pET-28a(+) to obtain the expression vector pET-28a-inuG;

[0059] PCR amplification was performed using the fully synthesized plasmid as a template to remove the signal peptide fragment on inuG and replace the restriction enzyme cleavage site to amplify the sample. Figure 3 In the gel electrophoresis results, M represents a DNA molecular weight standard; 1 represents the codon-optimized InuG gene with the signal peptide removed. The electrophoresis results show that the amplified InuG gene with the signal peptide removed is 2305 bp long and contains a single, clear band. The PCR amplification system is detailed in Table 1, and the specific PCR amplification conditions are listed in Table 2.

[0060] Table 1 PCR amplification system

[0061] Reagents Dosage (μL) template 50ng PrimeSTAR 0.25 Ab9(withoutSP)-BamHI-F 1 <![CDATA[Ab9-EcoRI -R ]]> 1 5xPrimeSTARBuffe 5

[0062] Table 2 PCR reaction conditions

[0063]

[0064] Use a gel extraction kit to recover the target band, then ligate the recovered target DNA fragment with the pLB vector to obtain the recombinant plasmid pLB-inuG. Finally, transform the constructed recombinant plasmid pLB-inuG into E. coli DH5α competent cells. The specific steps are as follows:

[0065] At room temperature, mix the gel recovery product and other components according to the reaction system in Table 3 and incubate in a 22°C incubator for 30 minutes to complete the ligation reaction. Mix the ligation product into E. coli DH5α competent cells at a volume ratio of 1:10 (ligation product: competent cells) and then incubate on ice for 20 minutes. Spread 100 μL of the bacterial solution on a solid LB plate containing ampicillin and incubate at 37°C for 10-12 hours. Pick a single colony and inoculate it into 1 mL of LB liquid medium containing ampicillin and incubate for 4-6 hours. After confirmation by PCR and electrophoresis, send it to a sequencing company for sequencing.

[0066] Table 3 Target gene and cloning vector connection system

[0067] Reagents Dosage (μL) Target gene gel recovery product 200ng pLB vector (35 ng / uL) 1 2*ReactionSolution 5 T4 DNA ligase 1 <![CDATA[ddH2O]]> Make up to 10uL

[0068] The strain corresponding to the correctly sequenced sample was cultured in LB liquid medium containing ampicillin and incubated at 37°C and 200 rpm for 16 hours. After the culture was completed, the bacterial suspension was collected and the recombinant plasmid was extracted, purified, and recovered using a plasmid extraction kit. The extracted plasmid and the prepared expression vector pET-28a were then treated with double enzyme digestion according to Table 4.

[0069] Table 4 Double enzyme digestion system

[0070] System Content Content (μL) Recombinant plasmid / pET-28a(+) 1 Q-cutEcoRI 1 Q-cutBamHI 1 Buffer 2 <![CDATA[ddH2O]]> 15

[0071] The enzyme digestion reaction was carried out at a constant temperature of 37°C for 30 minutes. The digestion products were then analyzed by agarose gel electrophoresis to verify the digestion effect. Figure 4 , M is a DNA molecular weight standard; 1 is a recombinant cloning vector plasmid digested with enzymes; 2 is an expression vector plasmid digested with enzymes. After successful enzyme digestion verification, the target gene band is immediately recovered and ligated with the expression vector pET-28a(+) digested with the same enzymes at 4°C overnight. The ligation system is shown in Table 5.

[0072] Table 5 Overnight connection system

[0073] System Content Content (μL) Target gene recovery product 5 Expression vector digestion product 3 Buffer 1 <![CDATA[ddH2O]]> 1

[0074] A2. Transform the expression vector pET-28a-inuG into competent cells of the expression host E. coli Rosetta (DE3).

[0075] E. coli Rosetta (DE3) was selected as the host bacteria for the expression vector. First, E. coli Rosetta (DE3) was prepared into competent cells so that the constructed expression vector could be transferred into the host bacteria. The following are the detailed steps for preparing and transforming the expression host competent cells:

[0076] Activate E. coli Rosetta (DE3) in LB liquid medium containing chloramphenicol; expand the activated two generations of seed liquid to 200 mL with LB medium and culture at 37°C and 200 rpm until OD 600 0.5; after placing on ice for 0.5 h, centrifuge at 4°C and 4000 rpm for 10 min, and then discard the supernatant; resuspend the cell pellet with pre-cooled 0.1 mol / L CaCl2, place on ice for 0.5 h, centrifuge at 4°C and 4000 rpm for 10 min, and then discard the supernatant; take out 60% glycerol and 0.1 mol / L CaCl2 previously placed in a 4°C refrigerator, add 600 μL glycerol and 1.4 mL CaCl2 to the bacterial pellet respectively, resuspend and distribute at 100 μL / tube, quickly freeze with liquid nitrogen and then quickly transfer to a -80°C refrigerator for storage. ; Take out the competent cells from the -80℃ freezer, place them on ice for 5 minutes to thaw, then add 10μL of the constructed expression vector pET-28a-inuG and ice bath for 20 minutes; heat shock at 42℃ for 90 seconds, and immediately place on ice for 5 minutes; then add 1mL of LB medium and recover at 37℃ and 200rpm for 1 hour; centrifuge at 4000rpm for 3 minutes, discard part of the supernatant medium, retain 100μL of liquid, resuspend the bacteria and spread them on LB solid medium containing kanamycin, culture at 37℃ for 14-16 hours, observe the transformation results and perform colony PCR identification.

[0077] A3. Inoculate E. coli Rosetta (DE3) carrying plasmid pET-28a-inuG into LB liquid medium (containing kanamycin) for cultivation. 600 When the pH reached 0.5, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to the culture medium and cultured overnight. The cells were collected by centrifugation and ultrasonically disrupted. After disruption, the cells were centrifuged and the supernatant and precipitate were collected separately.

[0078] The host strain E. coli Rosetta (DE3) carrying the plasmid pET-28a-inuG was inoculated into 200 mL of LB liquid medium (containing kanamycin) and cultured at 37°C and 200 rpm. Once the OD value of the host strain was observed, 600When the value reached approximately 0.5, isopropyl-β-D-thiogalactopyranoside was immediately added to the culture medium at a final concentration of 0.3 mmol / L to induce overexpression of InuG. Subsequently, the culture conditions were adjusted to 16°C and the rotation speed was maintained at 200 rpm, and the culture was continued overnight to ensure sufficient expression of the InuG protein.

[0079] The bacteria were collected by centrifugation (8000g, 5min), and the bacterial pellet was washed twice with PBS buffer. The entire centrifugation process was carried out at 4°C to ensure that the protein activity was not lost. After resuspending the bacteria in PBS buffer, ultrasonic disruption was performed. The bacterial solution was transferred to a 50mL beaker and disruption was started. The ultrasonic conditions were: 600W, ultrasonic on for 5s, off for 2s, and disruption for 20min. During disruption, it was necessary to ensure that the beaker containing the bacterial solution was fully in contact with the ice-water mixture below to eliminate the thermal effects of ultrasound. The disrupted bacterial solution was transferred to a centrifuge cup and centrifuged (10000g, 40min). The entire centrifugation process was carried out at 4°C. The supernatant (soluble protein) and precipitate (inclusion bodies) were collected separately. After the inclusion bodies were washed twice, they were dissolved with a buffer equal to the volume of protein and analyzed by SDS-PAGE gel electrophoresis for protein expression.

[0080] A4. Connect the Ni column to an AKTA protein purifier and purify the supernatant (soluble protein) using the AKTA protein purifier to obtain the target protein InuG, i.e., thermophilic endonuclease.

[0081] Connect the Ni column to the AKTA, flush ddH2O at a flow rate of 2mL / min to drain the ethanol sealing solution in the AKTA protein purifier and the Ni column, and end this step after a cycle of 10 column volumes; then flush PBS Buffer into the system at a flow rate of 2mL / min to drain the original ddH2O in the instrument and the Ni column, and end this step after a cycle of 10 column volumes to achieve the balance of the Ni column; the crude enzyme solution obtained after centrifugation was passed through the membrane and flushed into the Ni column at a flow rate of 0.8mL / min. After the sample is loaded, it was placed in a 4℃ refrigerator for binding for 1h; after the binding is completed, PBS Buffer was flushed into the column at a flow rate of 2mL / min to remove unbound impurities after loading and increase the purification efficiency. This step is ended after rinsing with 10 column volumes of PBS Buffer; Wash at a flow rate of 2mL / min. Buffer is flushed into the column to wash away non-specifically bound impurities, and this step is completed after 10 column volumes of flushing; Elution Buffer is flushed into the column at a flow rate of 2 mL / min to elute the specifically bound target protein, i.e., InuG, and this step is completed after 10 column volumes of flushing; after obtaining the target protein, it is stored at 4°C, and the purifier and Ni column are subsequently processed. First, ddH2O is flushed into the system at a flow rate of 2 mL / min to discharge the Elution Buffer in the instrument and Ni column, and the display is observed. After about 10 column volumes of the baseline is stable, 20% ethanol is flushed into the system at a flow rate of 2 mL / min to discharge the ddH2O in the instrument and Ni column, and this step is completed after 10 column volumes of flushing. The Ni column is sealed and stored at 4°C to obtain the target protein InuG, i.e., thermophilic inulinase.

[0082] See also Figure 5 , M is a molecular weight standard; 1 is an empty E. coli Rosetta-pET-28a vector; 2 is a crude E. coli Rosetta-pET-28a-InuG enzyme solution; and 3 is InuG purified by a Ni column. The thermophilic inulin endoenzyme prepared by this preparation method is thermostable.

[0083] Example 3:

[0084] See also Figure 6 This embodiment provides a technical solution based on the first or second embodiment: analysis of the main products of inulin hydrolysis by thermophilic endoinulinase.

[0085] First, according to the reaction system in the enzyme activity determination method, take 2% of the substrate inulin, add InuG, incubate in a 70°C water bath for 10 minutes, and terminate the reaction in a boiling water bath for 5 minutes. In order to determine the hydrolysis products of the reaction between InuG and inulin, the hydrolysis products of InuG were analyzed using the method developed by HPAEC-PAD (Dione x ICS-5000, Thermo Scientific, USA). The mixture after inulin hydrolysis was boiled for 10 minutes and then centrifuged at 13,000g for 40 minutes to remove residual enzyme protein. Then, the supernatant was filtered through a 0.22μm membrane and stored in a chromatographic vial for ion chromatography analysis. Then, 10μL of the appropriately diluted filtered solution was injected into the Dionex TM CarboPac TM A PA100 analytical column (250 mm × 4 mm) was used. The mobile phase consisted of water (A), 200 mmol / L sodium hydroxide solution (B), and 1000 mmol / L sodium acetate solution (C). Gradient elution conditions were: 0 to 10 min, 49% to 44.5% A, 50% B, and 1% to 5.5% C; 10 to 35 min, 44.5% to 22.5% A, 50% B, and 5.5% to 27.5% C; 35 to 60 min, 22.5% to 0% A, 50% B, and 27.5% to 50% C. During elution, the column temperature was maintained at 30°C, and the mobile phase flow rate was maintained at 1 mL / min.

[0086] See also Figure 6 In the figure, ① is inulin trisaccharide F3. The main product of inulin hydrolysis by InuG accounts for more than 90% of the total hydrolyzate. By comparing and analyzing with the standard, it is determined that the main product of inulin hydrolysis is inulin trisaccharide F3. According to the analysis of the hydrolyzed products, InuG is confirmed to be an inulin endo-enzyme.

[0087] Example 4:

[0088] See also Figures 7 to 9 This embodiment provides a technical solution based on the first or second embodiment: characterization of the enzymatic properties of thermophilic inulin endoinuclease.

[0089] 1. Optimal reaction temperature and temperature stability of InuG:

[0090] Enzyme activity assay: First, a 2% (w / v) inulin substrate solution was prepared using sodium hydrogen phosphate-citrate buffer (pH 7.0) as the solvent. 180 μL of substrate solution was added to 20 μL of purified InuG enzyme solution. The reaction was continued at 70°C for 10 minutes, followed by the addition of 3,5-dinitrosalicylic acid reagent to terminate the reaction. The mixture was then boiled at 100°C for 5 minutes to develop color. The DNS method was used to determine the amount of reducing sugars produced during inulin hydrolysis. One unit of enzyme activity was defined as the amount of enzyme required to produce 1 μmol / L of reducing sugar per minute. Fructose was oven-dried, weighed, and dissolved in deionized water at a concentration of 1 g / L as a high-concentration stock solution. The solution was then diluted according to a gradient of concentrations. Take 200 μL of dilutions of different concentrations and mix with an equal volume of DNS, boil in 100℃ water for 5 min to develop color, cool and add 1 mL of deionized water to make up to a total volume of 1.4 mL, and use a microplate reader to measure the OD 540 Measure the absorbance at 400 nm. Record and analyze the experimental data, set the fructose concentration on the horizontal axis and the OD value on the vertical axis, and draw the corresponding standard curve.

[0091] In order to determine the optimal reaction temperature, the enzyme activity of InuG was determined according to the above enzyme activity determination method; the melting temperature (T m ) and half-life (t 1 / 2 ) to evaluate the thermal stability parameters of InuG; T m Take measurements.

[0092] First, purified InuG dissolved in 50 mmol / L sodium hydrogen phosphate-citrate buffer (pH 7.0) was thoroughly mixed with SYPRO Orange dye (Invitrogen, CA, USA) at room temperature. The mixture was placed in a Light Cycler 480 Instrument II (Roche Diagnostics International Ltd) and gradually heated from 20°C to 90°C at a rate of 1°C / min. 470 nm and 570 nm were selected as the excitation and emission wavelengths, respectively. In addition, 1°C / min was selected as the sampling frequency. Experimental data were collected, and finally, T was determined based on the fitted curve. m The Bolzmann sigmoidal equation was used to process the experimental data and draw a fitting curve. 1 / 2To determine the activity of InuG, purified InuG in 50 mmol / L sodium hydrogen phosphate-citrate buffer (pH 7.0) is first incubated at 50, 60, and 70°C. Samples are then taken at defined intervals. The samples are then processed and tested for activity as described above. Relative activity is determined by the ratio of the residual activity in a sample extracted at a specific time point to the enzyme activity at time zero. 1 / 2 It refers to the incubation time when InuG loses 50% of its endoinulinase activity.

[0093] See also Figure 7 As the temperature increases from 30°C to 70°C, the relative activity of InuG increases by nearly 80%, reaching its peak activity at 70°C. However, between 70°C and 80°C, InuG activity decreases dramatically, with its relative activity dropping by 80% at 80°C. This result indicates that InuG has an optimal reaction temperature of 70°C and is a thermophilic enzyme. Among existing inulinase endoinulases, only two have an optimal temperature of 70°C. The optimal reaction temperature of other reported inulinase endoinulases does not exceed 60°C at most. InuG has a higher optimal reaction temperature than most reported inulinase endoinulases, and its optimal reaction temperature of 70°C significantly increases the solubility of natural inulin at this temperature. Its excellent optimal reaction temperature not only fills a gap in the research of thermophilic inulinase endoinulases but also opens up new possibilities for exploring their thermostability.

[0094] See also Figure 8 In the figure, (A) is the half-life of InuG at different temperatures; (B) is the T m Value; InuG at 70, 60 and 50 ℃ t 1 / 2 The values ​​were 82h, 146h, and 148h, respectively. Under incubation conditions at 70°C, the stability of InuG showed a three-phase change: from 0 to 12h, the residual activity of InuG decreased rapidly by 20%; from 12 to 72h, the residual activity of InuG remained above 70%; after 72h, the residual activity of InuG dropped sharply, and after 96h, the residual activity of InuG remained only 20% of the initial state. When incubated at 60°C and 50°C, the residual activity of the enzyme gradually decreased at a relatively slow rate. From 0 to 144h, it still maintained nearly 70% of the activity. After 144h, the activity of InuG decreased sharply, and at 145h, the activity of InuG had dropped to about 20% of the initial state.

[0095] SYPRO Orange fluorescent dye is a naturally quenched dye. After protein denaturation, it can efficiently bind to the exposed hydrophobic core of the protein, thereby enhancing the emitted fluorescent signal. That is, during the qRT-PCR process, the fluorescence signal intensity increases with increasing temperature. Therefore, according to the melting curve obtained from the qRT-PCR experiment, the T m The value was determined to be 78°C.

[0096] 2. Optimal reaction pH and pH stability of InuG:

[0097] To determine the optimal reaction pH value for InuG, different buffer systems were used: citric acid-sodium citrate buffer (50 mmol / L, pH 3.0-4.0), sodium acetate-acetic acid buffer (50 mmol / L, pH 4.0-6.0), disodium hydrogen phosphate-citric acid buffer (50 mmol / L, pH 6.0-8.0), tris-hydrochloric acid buffer (50 mmol / L, pH 8.0-10.0), and sodium carbonate-sodium hydroxide buffer (50 mmol / L, pH 10.0-13.0). The pH value was set in increments of 1.

[0098] Inulin was dissolved in buffer solutions of different pH values ​​to prepare a substrate with an inulin concentration of 2%. The purified InuG was evenly mixed with buffer solutions of different pH values ​​at a ratio of 1:9, and the mixture was incubated at 4°C for approximately 2 hours. Following the above-mentioned enzyme activity determination method, samples were taken after the incubation period for enzyme reaction and the residual enzyme activity was determined to determine the pH stability of InuG.

[0099] See also Figure 9 In the figure, (A) shows the optimal pH of InuG; (B) shows the pH stability of InuG. The results show that as the pH value increases from 3 to 13, the catalytic activity of InuG first increases and then decreases. During this process, when InuG reacts with 2% inulin dissolved in 50mmol / L sodium dihydrogen phosphate-citrate buffer at pH 7, the catalytic activity of InuG reaches its highest value, which is similar to the optimal pH of other reported bacterial inulin endonases. In addition, acidic (pH 3.0-4.0) or alkaline (pH 10.0-13.0) conditions have a significant effect on the catalytic activity of InuG, and its catalytic activity even drops to 0. The above results indicate that InuG is a neutral enzyme and its catalytic activity is very sensitive to changes in pH.

[0100] After incubation in the pH range of 3.0-13.0, InuG maintained over 60% relative enzyme activity. Within the pH range of 4.0-8.0, its stability was even better (maintaining over 90% relative activity). These results demonstrate that InuG maintains stability over a wide pH range. This broad pH tolerance makes InuG adaptable to the complex pH environments of industrial production.

[0101] 3. Effects of metal ions and some chemical reagents on InuG enzyme activity:

[0102] In order to explore the effects of various metal ions and some chemical reagents on InuG activity, different reagents were added to the above reaction mixture for enzyme reaction determination, with a final concentration of 5.0 mmol / L. All tested metal ions were provided in the form of chloride or sulfate, including Fe 3+ 、Fe 2+ 、Cu 2+ Mg 2+ 、Mn 2+ 、Zn 2+ , Ca 2+ , K + He Li + , where chemical reagents include SDS and EDTA.

[0103] Table 6 Effects of metal ions and chemical reagents on InuG enzyme activity

[0104]

[0105]

[0106] The experimental results are shown in Table 6. Mn 2+ Mg 2+ and K + After adding Mn, the activity of InuG was increased, but the degree of increase in enzyme activity was different. 2+ After addition of Mg, the activity of InuG increased to 158%, and the enzyme activity increased significantly. 2+ or K + After addition of Ca 2+ He Li + After treatment, InuG still maintained more than 80% of its activity. 2+ 、Cu 2+ 、Fe 2+ and Fe 3+After treatment, the enzyme activity of InuG was significantly inhibited. SDS almost completely inhibited the activity of InuG, while after EDTA treatment, the enzyme activity decreased by less than 25%, indicating that InuG does not belong to the metalloenzyme class.

[0107] 4. Determination of InuG substrate specificity and kinetic parameters:

[0108] To investigate the substrate specificity of InuG, several standard polysaccharides with different types of glycosidic bonds were selected as substrates for reaction with InuG. Enzyme activity was measured under optimal reaction conditions to investigate the specificity of InuG for substrates with different bond types. The experimental results are shown in Table 7.

[0109] Table 7 Specificity of InuG for different substrates

[0110]

[0111]

[0112] Substrate specificity results showed that InuG had no activity against a series of highly polymerized polysaccharides, including soluble starch, D-trehalose, carboxymethyl cellulose, xanthan gum, hemicellulose, glucomannan, and pullulan. InuG only exhibited catalytic activity against substrates with a β-(2-1) glycosidic bond with a degree of polymerization greater than 3. Unlike other reported inulin endo-nucleases, the smallest substrate that InuG could react with was GF3, which other reported inulin endo-nucleases could not react with.

[0113] To investigate the kinetic parameters of InuG, different concentrations of GF3 and inulin were prepared as substrates for the reaction with InuG. At the start of the experiment, 20 μL of purified InuG enzyme solution was added to 180 μL of the substrate solutions of varying concentrations to initiate the reaction. The reaction was maintained at the optimal temperature and pH for 3 minutes, and the amount of reducing sugars produced during the reaction was measured. The results are shown in Table 8.

[0114] Table 8 InuG kinetic parameters

[0115]

[0116] The experimental results show that the K m Value and K of GF3 m The values ​​are very close, while the V max It is almost 55 times that of GF3. In addition, the catalytic efficiency (k cat / K m), the catalytic efficiency of InuG for inulin was much higher than that for GF3. In summary, InuG has higher catalytic efficiency for substrates with higher DP, such as inulin and GF5, and lower catalytic efficiency for substrates with lower DP, such as GF3 and GF4. This indicates that InuG exhibits stronger catalytic activity for substrates with DP greater than 6, weaker activity for substrates with DP between 4 and 5, and no activity for substrates with DP less than 4.

[0117] Embodiment 5:

[0118] See also Figure 10 This embodiment provides a technical solution based on the first or second embodiment: protein structure prediction of InuG.

[0119] The protein structures of the inulinase endonuclease InuG, I nu1 from Arthrobacter sp. S37 (CAB63119), and InuB from Paenibacillus sp. LX16 (QAT77244) were predicted using the Alphafold2 program on a local GPU workstation. After model construction, the most confident model was selected based on the predicted local distance difference test (pLDDT) score. The pLDDT score, a key metric for assessing Alphafold2 model confidence, ranges from 0 to 100. Specifically, regions with scores exceeding 90 are considered highly accurate, while regions with scores between 70 and 90 demonstrate high structural quality. Regions with scores between 50 and 70 indicate relatively low model quality, while regions with scores below 50 indicate poor quality. The selected model was then used in subsequent molecular dynamics (MD) simulations to further explore its structural and dynamic properties.

[0120] By using full_dbsdata and default parameters, five relaxation models were obtained for each of the three endoinulinase enzymes. Figure 10 The predicted model is represented by the pLDDT score. In the figure, (A) is InuG; (B) is Inul; and (C) is InuB. The structure of the simulated protein with the best pLDDT value is selected for subsequent molecular dynamics (MD) simulation. After the predicted structure is imported into the PyMOL software, the structure is colored according to the previous pLDDT score. According to four different score ranges, namely greater than 90 points, 70-90 points, 50-70 points and less than 50 points, dark blue, light blue, yellow and orange are used to distinguish them respectively. Overall, the simulated structure is mainly blue, which indicates that the modeling quality is relatively high.

[0121] Example 6:

[0122] See also Figure 11 This embodiment provides a technical solution based on the first or second embodiment: molecular dynamics simulation (MD) experiment.

[0123] The first step is to set up the simulation system using the topology file generated by the CHARMM36 force field. The pre-processed initial structure is set as the center and the simulation box is set to determine the size of the system. In this step, the protein is acted on by the amber14SB force field and the water molecules are acted on by the TIP3P force field. The TIP3P water molecule model is filled into the box to perform protein solvation treatment, and the minimum distance between the protein structure and the water molecule cube boundary is set to Adding Na to the system + and Cl - Adjust the charge of the system to make it a neutral system.

[0124] The second step involves energy minimization to eliminate inappropriate atomic contacts. The first minimization constrains all components except the water molecules, allowing them to move freely to eliminate inappropriate contacts. The second minimization releases all constraints to fully optimize the structure. After minimization, NVT equilibration is performed for 500 ps. Each system is slowly heated from 0 K to the target temperature (303.15 K, 323.15 K, or 343.15 K) using a Langevin thermostat.

[0125] After the system was heated to the target temperature, a 1-ns density equilibration was performed in an NPT system (300 K, 1.0 atm) using a Berendsen barostat and a 2 fs collision frequency until the density stabilized. All restraints on the protein were then removed, followed by a 3-ns MD equilibration to reach the target temperature and pressure. Finally, a 100-ns MD simulation was performed in each system with a 2 fs integration step size to analyze the thermal stability mechanisms.

[0126] The root mean square deviation (RMSD) is generally used to evaluate the thermal fluctuation of protein conformation. The RMSD value is inversely proportional to the thermal stability of the protein, that is, proteins with higher thermal stability tend to have smaller RMSD values.

[0127] See also Figure 11 Analysis of the molecular dynamics simulation results shows that since the RMSD value of lnuG is lower than that of Inu1 and InuB during the simulation, and the curve fluctuation of InuG is the smallest during the simulation, it shows that lnuG has the best overall conformational stability compared with the other two inulin endonucleases.

[0128] The above description is merely illustrative of certain exemplary embodiments of the present invention. It goes without saying that those skilled in the art will be able to modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the above drawings and description are illustrative in nature and should not be construed as limiting the scope of protection of the claims.

Claims

1. A method for screening thermophilic inulinase, characterized in that: The steps include: S1. Use bioinformatics technology to collect homologous sequences of known inulin endonucleases and form a local protein sequence database; S2. Screening thermophilic inulin endonuclease by the growth temperature of the strain from which the homologous sequence is derived, anchoring the homologous sequence of the strain with the highest growth temperature, and obtaining the thermophilic inulin endonuclease InuG.

2. The method for screening a thermophilic inulinase according to claim 1, wherein The S1 comprises the following steps: S11, first select the inulin endonuclease Inu1 from Arthrobacter sp.S37 strain as the probe sequence; S12, then based on the principle of sequence similarity, using BLAST to search for homologous sequences of the probe sequence Inu1 in the NCBI database, and setting the similarity condition of the homologous sequences to be greater than 20%; S13. These homologous sequences were then constructed into a local protein sequence database.

3. The method for screening a thermophilic inulinase according to claim 1, wherein The S2 comprises the following steps: S21, determining the growth temperature of the strain from which the homologous sequence in the protein sequence database obtained in S1 is derived; S22, then selecting homologous sequences of strains with a growth temperature equal to or higher than 50°C, and reordering these homologous sequences according to the growth temperature of the strains; S23. Finally, the homologous sequence of the strain with the highest growth temperature was anchored and named inuG, which is the thermophilic inulin endonuclease InuG.

4. A method for preparing a thermophilic inulin endoinuclease, characterized in that: The steps include: A1. After codon optimization of the inuG sequence screened out by the screening method for thermophilic inulin endonuclease according to any one of claims 1 to 3, an InuG encoding gene suitable for the E. coli expression system is obtained by whole gene synthesis technology; The optimized InuG encoding gene was used to remove the signal peptide fragment by PCR, and then ligated with the cloning vector pLB to construct a recombinant plasmid pLB-inuG; the recombinant plasmid was transferred into the cloning host E. coli DH5α, and E. coli DH5α was cultured. After the culture was completed, the recombinant plasmid was extracted, and then the extracted recombinant plasmid was double-digested with the pET-28a(+) expression vector. The target gene inuG obtained by the double-digestion and the linearized pET-28a(+) were recovered, and then the target gene inuG was ligated with the linearized pET-28a(+) to obtain the expression vector pET-28a-inuG; A2. Transform the expression vector pET-28a-inuG into competent cells of the expression host E. coli Rosetta (DE3) and culture for 1 hour. After the culture is completed, centrifuge the culture, retain 100 μL of the supernatant and resuspend the bacterial solution, then spread it on the surface of LB solid medium containing kanamycin and culture overnight to obtain E. coli carrying the plasmid pET-28a-inuG; A3. Inoculate E. coli carrying plasmid pET-28a-inuG into LB liquid medium for cultivation. 600 When the value reached 0.5, isopropyl-β-D-thiogalactopyranoside was added to the culture medium and cultured overnight. The cells were collected by centrifugation and ultrasonically disrupted. After disruption, the supernatant and precipitate were collected separately by centrifugation. A4. Connect the Ni column to an AKTA protein purifier, and purify the supernatant described in A3 using the AKTA protein purifier to obtain the target protein InuG, i.e., thermophilic endonuclease.

5. The method for preparing a thermophilic inulin endoinuclease according to claim 4, wherein: The GenBank accession number of the inuG codon-optimized sequence of A1 is OP115877.

6. The method for preparing a thermophilic inulin endoinuclease according to claim 4, wherein: The culture conditions before adding isopropyl-β-D-thiogalactopyranoside to the culture medium in A3 are 37° C. and a rotation speed of 200 rpm; the culture conditions after adding isopropyl-β-D-thiogalactopyranoside to the culture medium in A3 are 16° C. and a rotation speed of 200 rpm.

7. The method for preparing a thermophilic inulin endoinuclease according to claim 4, characterized in that: The amount of isopropyl-β-D-thiogalactopyranoside added to the culture medium in A3 is such that the final concentration in the culture medium is 0.3 mmol / L.

8. A thermophilic inulinase, characterized in that The thermophilic inulinase is prepared by the preparation method according to claim 4. The coding sequence of the thermophilic inulinase is derived from Thermoanaerobacter italicus Ab9 and is thermostable.

9. Use of the thermophilic endoinulinase according to claim 8 in the industrial production of fructooligosaccharides.

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