Glucose isomerase mutant and application thereof

Abstract

The invention discloses a glucose isomerase mutant and application thereof. Belongs to the technical field of enzyme engineering. The amino acid sequence of the glucose isomerase mutant is as shown in SEQ ID NO. 2. Compared with the prior art, the glucose isomerase mutant has the advantages that the glucose isomerase mutant resistant to high temperature and low pH value is provided, the optimum pH value of the mutant is reduced to 6.0 from 7.0, the optimum temperature is increased to 100 DEG C from 95 DEG C, and the problem that existing glucose isomerase is poor in acid resistance and high temperature resistance is solved.

Description

Technical Field

[0001] The present invention relates to the technical field of enzyme engineering, and more particularly to a glucose isomerase mutant and application thereof. Background Art

[0002] Glucose isomerase (EC 5.3.1.5) is a monosaccharide enzyme that converts D-glucose into D-fructose. It is used industrially to produce high fructose corn syrup (HFCS). HFCS has good solubility, high sweetness and cost-effectiveness, and is widely used as a sweetener substitute and food additive in beverages, candies and baked goods.

[0003] HFCS is a liquid mixture of glucose and fructose, which is usually obtained by acid or enzyme conversion in industrial production. Enzyme conversion is far better than acid conversion because it is safer, has milder reaction conditions and obtains higher fructose yield.

[0004] In the production process of HFCS, high temperature shifts the enzyme reaction balance in the positive direction, increasing fructose production, accelerating the reaction rate, and reducing substrates. Acid-resistant glucose isomerase can reduce the browning and by-product formation of HFCS. High-temperature and acid-resistant glucose isomerase is more suitable for the industrial production of high fructose corn syrup. However, most glucose isomerases in the industry have poor acid and high-temperature resistance, and the HFCS produced is of poor quality and high cost.

[0005] In summary, how to provide an acid-resistant and high-temperature-resistant glucose isomerase is a problem that those skilled in the art need to solve urgently. Summary of the invention

[0006] In view of this, the present invention provides a glucose isomerase mutant and application thereof.

[0007] In view of the fact that most glucose isomerases show low activity or inactivation at high temperature and low pH, the present invention selects the thermostable Thermotoga mariitma glucose isomerase MSB8 gene, mutates the three amino acid residues E237H / N298K / N337R near the enzyme active site to introduce positive charge, uses Swiss-model to use the crystal structure of glucose isomerase of Thermotoganeapolitana (PDB ID 1A0E) as a template for homology modeling, uses Pymol to view the three-dimensional structure of its visual model, and analyzes the interaction pattern between the mutation site, active center and substrate. The obtained mutant gene is optimized by Pichia codon preference, commissioned to be synthesized by a gene synthesis company, and the synthesized target gene is connected to the Pichia expression vector pPinkα-HC to obtain the pPinkα-HC-TMGI-MSB8C recombinant plasmid, and after linearizing the recombinant plasmid, it is electrotransferred to PichiaPink TM Strain4 Pichia pastoris competent cells were cultured on PAD plates to obtain positive clones, named PP-pPinkα-HC-TMGI-MSB8C. The positive recombinant strain was then induced with methanol to express the target protein, and the fermentation and induction conditions were explored and optimized to obtain a higher expression level. The enzymatic activity of glucose isomerase was then determined using the improved cysteine-carbazole method. The experiment showed that the optimal pH value of the glucose isomerase obtained after mutation was reduced from 7.0 to 6.0, and the optimal temperature was increased from 95°C to 100°C. The crude enzyme activity obtained by fermentation in a 5L fermenter could reach 84.6U / ml. The enzymatic properties and kinetic parameters of the expressed enzyme were determined, the pKa value of its catalytic residues was analyzed, the expressed enzyme was subjected to molecular dynamics simulation, and finally the modified sepiolite was used to immobilize glucose isomerase.

[0008] In order to achieve the above object, the present invention adopts the following technical solution:

[0009] A glucose isomerase mutant is obtained by site-directed mutation of the amino acids in the sequence shown in SEQ ID NO.1, wherein the mutation sites are the 237th, 298th and 337th positions.

[0010] Furthermore, the amino acids at positions 237, 298 and 337 of the sequence as shown in SEQ ID NO. 1 are mutated to histidine, lysine and arginine, respectively.

[0011] Furthermore, its amino acid sequence is shown in SEQ ID NO.2.

[0012] Furthermore, its nucleotide sequence is shown in SEQ ID NO.3.

[0013] Application of the above-mentioned glucose isomerase mutant in catalytic production of D-fructose.

[0014] It can be seen from the above technical solution that, compared with the prior art, the beneficial effects achieved by the present invention are:

[0015] The invention provides a glucose isomerase mutant which is resistant to high temperature and low pH value. The optimum pH value of the mutant is reduced from 7.0 to 6.0, and the optimum temperature is increased from 95 DEG C to 100 DEG C, thus solving the problem that the existing glucose isomerase has poor acid and high temperature resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0017] Figure 1 The amino acid residues selected for site-directed mutagenesis in Example 1 of the present invention (the mutated residues are indicated in purple);

[0018] Figure 2 The SDS-PAGE electrophoresis diagram of the target protein in Example 2 of the present invention, wherein lane 1 represents the TMGI-MSB8 result, and lane 2 represents the E237H / N298K / N337R mutant result;

[0019] Figure 3 The enzymatic activity characteristics of TMGI-MSB8 and E237H / N298K / N337R mutants in Example 4 of the present invention, wherein a is the determination result of the optimum pH value; b is the pH tolerance result; c is the determination result of the optimum temperature; d is the thermal stability result;

[0020] Figure 4 is the pKa value of the catalytic residues in TMGI-MSB8 and the E237H / N298K / N337R mutant in Example 6 of the present invention;

[0021] Figure 5 It is a molecular docking diagram of TMGI-MSB8 and E237H / N298K / N337R mutant in Example 7 of the present invention, wherein a is the molecular docking of TMGI-MSB8; b is the molecular docking of E237H / N298K / N337R mutant;

[0022] Figure 6 RMSD results of TMGI-MSB8 and E237H / N298K / N337R mutant in Example 7 of the present invention;

[0023] Figure 7 This is the RMSF result of TMGI-MSB8 and E237H / N298K / N337R mutant in Example 7 of the present invention. DETAILED DESCRIPTION

[0024] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] The drugs required for the present invention are conventional experimental drugs, which are purchased from commercial channels; the experimental methods not mentioned are conventional experimental methods and will not be described in detail here.

[0026] Example 1

[0027] Mutation of three amino acid residues near the active site (E237H / N298K / N337R) introduces positive charge

[0028] The protein sequence of glucose isomerase (TMGI-MSB8) from Thermotoga marimita MSB8 was obtained from the NCBI database (ID: AKE29424.1), and its amino acid sequence is shown in SEQ ID NO. 1. Swiss-model (https: / / swissmodel.expasy.org) was used to perform homology modeling using the reported crystal structure of glucose isomerase from Thermotoga neapolitana (PDB ID 1A0E) as a template (homology of 95.03% and sequence coverage of 100%), and the PDB file was obtained.

[0029] The original amino acid sequence of Thermotoga maritima glucose isomerase:

[0030] MAEFFPEIPKIQFEGKESTNPLAFRFYDPNEVIDGKPLKDHLKFSVAFWHTFVNEGRDPFGDPTAERPWNRFSDPMDKAFARVDALFEFCEKLNIEYFCFHDRDIAPEGKTL RETNKILDKVVERIKERMKDSNVKLLWGTANLFSHPRYMHGAATTCSADVFAYAAAQVKKALEITKELGGEGYVFWGGREGYETLLNTDLGLELENLARFLRMAVEYAKKIGF TGQFLIEPKPKEPTKHQYDFDVATAYAFLKNHGLDEYFKFNIEANHATLAGHTFQHELRMARILGKLGSIDANQGDLLLGWDTDQFPTNIYDTTLAMYEVIKAGGFTKGGLN FDAKVRRASYKVEDLFIGHIAGMDTFALGFKIAYKLAKDGVFDKFIEEKYRSFKEGIGKEIVEGKTDFEKLEEYIIDKEDIELPSGKQEYLESLLNSYIVKTIAELR, SEQID NO.1.

[0031] Screening of TMGI-MSB8 active sites The non-conservative and non-basic amino acids in the range are mutated into basic positively charged amino acids (mutating acidic amino acids into basic amino acids makes the residues near the active site more active, which may increase the chance of contact with the substrate, thereby improving the specific activity of the enzyme; at the same time, an alkaline region is formed to tolerate higher concentrations of H + , thereby lowering the optimal pH value), the amino acid residues selected by site-directed mutagenesis are as follows Figure 1 As shown (the mutated residues are indicated in purple). Pymol software was used to view the three-dimensional structure of the model and analyze the action mode of the mutation site in the protein structure.

[0032] ΔΔG values ​​were determined using FoldX (https: / / tango.crg.es / ) and the Deep DDG website (http: / / protein.org.cn / ddg.html), and energy calculations were performed based on the homology model three-dimensional structure. ΔΔG < 0 kcal·mol -1 The amino acids in the protein sequence were identified as potential mutation sites. This revealed the relative free energy change (ΔΔG) of all amino acids in the protein sequence except cysteine. The calculation formula is as follows:

[0033]

[0034] The results are shown in Table 1.

[0035] Table 1 ΔΔG values ​​of mutants calculated by FoldX

[0036]

[0037]

[0038] According to the results in Table 1, the mutation sites selected were E237H / N298K / N337R (mutating three sites simultaneously can more easily form an alkaline environment, making it easier to lower the optimal pH value of the enzyme).

[0039] The amino acid sequence of the mutant Thermotoga maritime glucose isomerase:

[0040]

[0041] in,

[0042] Mutation sites (bold font size): E237H / N298K / N337R;

[0043] Catalytic site (bold italics): H101 D104;

[0044] Binding sites (bold and underlined): E232 E268 H271 D296 D307 D309 D339.

[0045] Example 2

[0046] PichiaPink Pichia yeast expression system to express target protein

[0047] The original gene sequence of Thermotoga maritime glucose isomerase:

[0048]

[0049] The mutant gene was optimized by Pichia pastoris codon preference, and the optimized Thermotoga maritime glucose isomerase gene sequence was as follows:

[0050] ATGGCTGAATTTTTCCCTGAAATTCCTAAAATTCAATTTGAAGGTA

[0051] AAGAATCTACTAATCCATTGGCTTTTAGATTCTACGATCCAAATGAAGT

[0052] TATTGATGGTAAACATTGAAGGATCATCTGAAATTTTCTGTTGCCTTTT

[0053] GGCATACCTTTGTTAACGAAGGTAGAGATCCATTTGGTGACCCAACTGC

[0054] TGAAAGACCATGGAATAGATTTTCTGATCCAATGGATAAGGCTTTTGCT

[0055] AGAGTTGATGCTTTGTTTGAATTCTGTGAAAAGTTGAATATTGAATACT

[0056] TTTGTTTTCATGACAGAGACATTGCTCCCAGAAGGTAAAACTTTGAGAGA

[0057] AACTAACAAGATTTTGGATAAGGTCGTGAAAGAATTAAGGAAAGAAT

[0058] GAAAGACTCTAACGTTAAATTGTTGTGGGGTACTGCTAACTTGTTCTCT

[0059] CACCCAAGATATATGCACGGAGCTGCAACTACCTGTAGTGCTGATGTTT

[0060] TTGCTTACGCTGCCGCCCAAGTTAAAAAGGCTTTGGAAATAACTAAGG

[0061] AATTGGGTGGTGAAGGTTACGTTTTCTGGGGTGGTAGAGAAGGTTATG

[0062] AAACTTTGTTGAACACTGATTTGGGTTTGGAATTGGAAAACTTGGCTAG

[0063] ATTTTTGAGAATGGCTGTTGAGTACGCTAAAAGATTGGTTTTACTGGA

[0064] CAGTTTTTGATTGAGCCTAAACCTAAGCACCCAACTAAACATCAATATG

[0065] ATTTTGATGTTGCCACTGCTTACGCTTTTCTGAAGAATCATGGACTTGAT

[0066] GAATATTTTAAATTCAACATTGAGGCTAACCATGCTACCCTTGCAGGAC

[0067] ATACTTTCCAACATGAATTGAGAATGGCTAGAATTTTGGGTAAATTGGG

[0068] TTCTATTGATGCTAAGCAAGGAGATTTGTTGTTAGGTTGGGATACTGAT

[0069] CAATTCCCTACTAACATCTACGATACTACCTTAGCTATGTATGAAGTTA

[0070] TTAAGGCTGGTGGTTTTACTAAAGGAGGTTTAAGATTCGATGCTAAGGT

[0071] TAGAAGAGCTTCTTTACAAGGTTGAAGATCTGTTTATTGGTCATATTGCT

[0072] GGTATGGATACTTTCGCTTTGGGTTTTAAGATTGCCTACAAGTTGGCTA

[0073] AGGACGGTGTTTTTGATAAATTCATTGAAGAAAAATAGATCCTTTAA

[0074] GGAAGGAATTGGAAAGGAAATTGTTGAAGGTAAAACTGATTTCGAAAA

[0075] GTTGGAAGAATACATTATTGATAAGGAGGATATTGAATTGCCATCTGG

[0076] AAAGCAAGAGTACTTGGAATCTTTGCTGAATTCTTATATTGTTAAGACT

[0077] ATTGCTGAATTGAGATGA, SEQ ID NO.3.

[0078] The original gene and optimized mutant gene sequences were synthesized by a biological company. The PichiaPink expression system was used for the first time to connect the target gene to the pPinkα-HC vector to obtain the pPinkα-HC-TMGI-MSB8C recombinant plasmid. The target gene was then cut with Spe I enzyme and the linearized recombinant plasmid was electroporated into the PichiaPink using a Bio-rad electroporator. TM Strain4 Pichia pastoris competent cells, then spread the mixture on PAD medium, culture at 30℃ for 24 hours, pick the white colonies on the plate, extract their chromosomes for PCR and sequencing identification, and obtain the positive clone, named PP-pPinkα-HC-TMGI-MSB8C. Then ferment the positive recombinant bacteria for expression, the steps are as follows:

[0079] The selected positive recombinant bacteria were cultured in YPD (1% (w / v) yeast extract, 2% (w / v) peptone, 2% (w / v) glucose) at 200 rpm and 30°C for 24 h, and then inoculated into BMGY (1% (w / v) yeast extract, 2% (w / v) peptone, 100 mM phosphate buffer, 1.34% (w / v) YNB, 4×10 -5 D-biotin, 1% (w / v) glycerol) medium, 25, 28 or 31 ° C, 200 rpm culture for 24 hours, glycerol as the carbon source, methanol was used to induce the expression of the target protein after the glycerol was consumed, and the concentration was added every day at 1-3%, and the induction was continuous for 72-144 hours. The fermentation broth was centrifuged to obtain the supernatant for SDS-PAGE and protein separation and expression determination. The optimal induction expression conditions were explored according to different temperatures, pH values, fermentation time, and carbon sources. The results are shown in Table 2.

[0080] Table 2 Experimental design and expression results

[0081]

[0082] After exploration, under 2% methanol induction, the pH value of the culture medium was 6.0, and the induction temperature was 25°C. The expression level was the highest at 144h, reaching 466mg / L. The optimal fermentation conditions were used for fermentation in a fermenter. The SDS-PAGE electrophoresis of the target protein was as follows: Figure 2 shown.

[0083] Example 3

[0084] Optimization of the cysteine-carbazole method for enzyme activity determination

[0085] With reference to GB / T 23533-2009, the method for determining the enzyme activity of Streptomyces and Actinomycetes in immobilized glucose isomerase preparations was improved according to the characteristics of glucose isomerase from Thermotoga maritima. The improved steps are as follows:

[0086] Add 50 μl of 3 mol / L glucose solution and 400 μl of 100 mmol / L pH 7.0 potassium hydrogen phthalate-imidazole buffer (containing 5 mmol / L Mg 2+ , 1mmol / L Co 2+ ), 50 μl of diluted enzyme solution, react at 95℃ for 10 min, and then terminate the reaction with 500 μl of 50% trichloroacetic acid. Control group: Glucose and potassium hydrogen phthalate-imidazole buffer were added to the test tube, trichloroacetic acid was added to terminate the reaction, and then placed at 95℃ for 10 min before adding enzyme solution.

[0087] Take an appropriate amount of reaction solution (50 μl), and then add distilled water to 500 μl, then add 0.1 mL of cysteine ​​hydrochloride solution and 3 mL of sulfuric acid solution to each tube, shake well, and immediately add 0.1 mL of carbazole alcohol solution, shake well, and keep warm in a 60°C water bath for 10 min. Take out and cool with water to turn it reddish purple. Use a 10 mm optical path cuvette to compare the color at a wavelength of 560 nm. The A560 value obtained can be referred to the fructose standard curve to obtain the fructose content in the liquid to be tested.

[0088] Definition of enzyme unit (U): the amount of enzyme required to convert 1 umol of fructose within 1 minute under the reaction conditions.

[0089] GI enzyme activity (U / mL) = Q×V1 / (180×T×V2)×N, where:

[0090] Q: generated fructose concentration; N: initial dilution factor of enzyme solution; V1: reaction volume (mL); V2: amount of enzyme added during reaction (mL or g); T: reaction time (min); 180: molecular weight of fructose.

[0091] The mutant reduced the optimal pH of glucose isomerase from 7.0 to 6, and the maximum specific enzyme activity was 55.92 U / mg, which was 1.35 times that of the original sequence (41.38 U / mg). At pH 6, the specific enzyme activity of the mutant was 55.92 U / mg, which was 1.62 times that of the original sequence (34.51 U / mg).

[0092] Example 4

[0093] Determination of enzyme properties

[0094] In order to determine the optimal reaction pH value of TMGI-MSB8 and its mutants, the activity of glucose isomerase was measured in the pH range of 4.5 to 8.5. Figure 3 a. The results showed that the enzyme activity of the mutant was higher than that of the wild type, the optimal pH of the enzyme dropped from 7.0 to 6.0, and the maximum activity of the mutant was 55.92 U / mg, which was 1.35 times that of TMGI-MSB8 (41.38 U / mg). At pH 6.0, the specific enzyme activity of E237H / N298K / N337R was 1.62 times higher than that of TMGI-MSB8, indicating that the mutant had better acid resistance than TMGI-MSB8.

[0095] In order to determine the optimal reaction temperature of TMGI-MSB8 and its mutants, the activity of glucose isomerase was measured in the range of 65-100°C. Figure 3 c. The results showed that compared with TMGI-MSB8, the optimum temperature of the mutant E237H / N298K / N337R was determined to be 100°C, which was 5°C higher than that of TMGI-MSB8.

[0096] The purified glucose isomerase was incubated in 50 mM potassium hydrogen phthalate-sodium imidazole buffer (pH 4.5-8.5) at room temperature for 4 hours, and the residual activity of the enzyme was measured. The results were as follows: Figure 3 As shown in b.

[0097] The purified glucose isomerase was incubated in 50 mM potassium hydrogen phthalate-sodium imidazole buffer at room temperature for 2, 4, 6, 8, 10 and 12 h at 95 °C, and the residual activity of the enzyme was determined. The results are as follows: Figure 3 The results showed that the specific enzyme activity of the mutant E237H / N298K / N337R after incubation at 95°C for 4 h was 45.37 U / mg, which was 1.58 times higher than that of TMGI-MSB8.

[0098] In summary, modification near the active site of the enzyme has the potential to improve acid resistance and thermal stability.

[0099] Example 5

[0100] Determination of enzyme kinetic parameters

[0101] The enzyme kinetic parameters of glucose isomerase before and after mutation were measured, and the results are shown in the following table.

[0102] Table 3 Enzyme kinetic parameters determination results

[0103]

[0104] Note: Data are presented as mean ± standard deviation (n = 3). Asterisks indicate significant differences between TMGI-MSB8 and E237H / N298K / N337R mutants (P < 0.01).

[0105] K of TMGI-MSB8 m The value is 1.09 times higher than that of E237H / N298K / N337R, indicating that the mutant has a higher affinity for the substrate than the wild type. cat The value is 1.53 times that of the wild type, indicating that the catalytic efficiency of the mutant is significantly improved compared with the wild type. cat / Km The value is 1.66 times that of the wild type, indicating that the affinity and enzymatic reaction rate of the mutant are significantly improved.

[0106] Example 6

[0107] Determination of pKa values ​​of catalytic residues

[0108] The pKa values ​​of H101 and D104 of glucose isomerase before and after mutation were measured. The results are as follows: Figure 4 As shown. The p values ​​of H101 and D104 of E237H / N298K / N337R mutants K The a values ​​changed from 4.89 and 2.33 to 4.77 and 2.57, respectively. The lower pKa value of H101 in the E237H / N298K / N337R mutant may be the main reason for the acid shift of the optimal pH of the enzyme.

[0109] Example 7

[0110] Molecular docking and molecular dynamics simulation

[0111] (1) Molecular docking

[0112] In this study, AutoDockVinav1.2.3 software was used for molecular docking analysis. After checking that the spatial structure of glucose was correct, the AutoDock Toolv1.5.6 software was used to add atomic charges, assign atom types, and change all flexible bonds to default rotatable and save them in "pdbqt" format as docking ligands. MSB8, MSB8-pka, and MSB8-sueface were treated by adding hydrogen atoms and deleting all water molecules. Then the docking box (Girdbox) was set to X: 30, Y: 30, Z: 30, and finally the optimized protein was saved in "pdbqt" format as a docking receptor. Subsequently, AutoDock Vina v1.2.3 software was used for docking and the binding affinity (kJ / mol) was calculated. Molecular visualization is a key aspect of modeling research analysis and communication. Therefore, the Molecular Operating Environment (MOE) v2022.02 software was used to analyze the details of the interaction between the ligand and the receptor and draw a 2D diagram of the interaction.

[0113] The results are as follows Figure 5 As shown, glucose forms hydrogen bonds with Asp234, Asp339 and His50 amino acid residues of TMGI-MSB8 and E237H / N298K / N337R mutants, forms a metal covalent bond with the cobalt atom (Co5), forms hydrophobic interactions with Phe60, Trp188, Phe145 and Trp49, and forms cationic bonds with His271 and Asp309.

[0114] (2) Affinity value

[0115] The affinity values ​​of glucose isomerase before and after mutation were measured, and the results were -5.817 kcal / mol and -5.819 kcal / mol, respectively.

[0116] (3)RMSD

[0117] The RMSD results are as follows Figure 6 As shown, the average RMSD is 0.2 nm (wild type) and 0.18 nm (E237H / N298K / N337R mutant). Compared with the wild-type TMGI-MSB8 protein, it seems that the complex formed by the E237H / N298K / N337R mutant and glucose is more stable.

[0118] (4)RMSF

[0119] The RMSF results are as follows Figure 7As shown, RMSF analysis showed that the RMSF of TMGI-MSB8 and E237H / N298K / N337R complexes did not change significantly, indicating that glucose had no obvious effect on the flexibility of the mutated protein.

[0120] (5) Binding free energy

[0121] The binding free energy of glucose isomerase before and after mutation was measured, and the results are as follows.

[0122] Table 4 Binding free energy of the complex at 300K (kcal / mol)

[0123]

[0124]

[0125] The results showed that the total binding free energy of the TMGI-MSB8 complex was -21.06 kcal / mol, of which van der Waals force (-13.35 kcal / mol), electrostatic force (-56.03 kcal / mol) and gas phase energy (-69.38 kcal / mol) were conducive to the stability of the TMGI-MSB8 complex system. The total binding free energy of the E237H / N298K / N337R complex was -26.08 kcal / mol, of which van der Waals force (-20.02 kcal / mol), electrostatic force (-45.79 kcal / mol) and gas phase energy (-65.81 kcal / mol) were conducive to the stability of the E237H / N298K / N337R complex system. Compared with the wild-type TMGI-MSB8 protein, the mutant E237H / N298K / N337R can form a more stable complex system with glucose.

[0126] Example 8

[0127] Immobilization of Glucose Isomerase on Modified Sepiolite

[0128] Sepiolite is treated with 2mol / L capric acid at 60℃ for 110min, separated, washed to neutrality, vacuum dried, crushed, sieved through 200 mesh, and dried for use. Take 20g of the above acid-treated sepiolite, add 50ml of 5% KH550 aqueous solution by volume, adjust pH to 5-6 with capric acid, stir at room temperature overnight, centrifuge, wash repeatedly, vacuum dry, sieve through 200 mesh, and obtain silane coupling agent KH550 modified sepiolite. The anti-swelling effect of the modified sepiolite is greatly enhanced, and a rigid ordered structure with large cavities is formed between the layers.

[0129] Use the bifunctional reagent glutaraldehyde and adopt the cross-linking method for fixation. Add KH550 modified sepiolite to 10ml glutaraldehyde solution, stir for 0.5h, centrifuge, wash repeatedly to remove free glutaraldehyde, and dry in vacuum at room temperature. Take 0.3g of glutaraldehyde-treated carrier, add 30ml of 1mg / mL glucose isomerase solution (0.1mol / L phosphate buffer solution, pH 6.0), mix well, and react on a 200r / min water bath constant temperature oscillator for 6 hours. Then centrifuge, determine the enzyme content of the supernatant, and calculate the immobilization rate; wash the lower precipitate repeatedly to obtain immobilized glucose isomerase, dry it in vacuum at room temperature, and determine its enzyme activity. Through experiments, it is known that the immobilization rate of this method can reach 63%, the product enzyme activity can reach 5329.8U / g, and the enzyme activity recovery rate can reach 52%.

[0130] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0131] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A glucose isomerase mutant, It is characterized in that The amino acid in the sequence shown in SEQ ID NO.1 is subjected to site-directed mutations, and the sites of the mutation are position 237, position 298 and position 337; MAEFFPEIPKIQFEGKESTNPLAFRFYDPNEVIDGKPLKDHLKFSVAFWHTFVNEGRDPFGDPTAERPWNRFSDPMDKAFARVDALFEFCEKLNIEYFCFHDRDIAPEGKTLRETNKILDKVVERIKERMKDSNVKLLWGTANLFSHPRYMHGAATTCSADVFAYAAAQVKKALEITKELGGEGYVFWGGREGYETLLNTDLGLELENLARFLRMAVEYAKKIG FTGQFLIEPKPKEPTKHQYDFDVATAYAFLKNHGLDEYFKFNIEANHATLAGHTFQHELRMARILGKLGSIDANQGDLLLGWDTDQFPTNIYDTTLAMYEVIKAGGFTKGGLNFDAKVRRASYKVEDLFIGHIAGMDTFALGFKIAYKLAKDGVFDKFIEEKYRSFKEGIGKEIVEGKTDFEKLEEYIIDKEDIELPSGKQEYLESLLNSYIVKTIAELR, SEQ IDNO.

1.

2. The glucose isomerase mutant of claim 1, It is characterized in that The amino acids shown in the sequences such as SEQ ID NO.1 are mutated to histidine, lysine and arginine, respectively.

3. The glucose isomerase mutant of claim 1, It is characterized in that Its amino acid sequence is shown in SEQ IDNO.2; MAEFFPEIPKIQFEGKESTNPLAFRFYDPNEVIDGKPLKDHLKFSVAFWHTFVNEGRDPFGDPTAERPWNRFSDPMDKAFARVDALFEFCEKLNIEYFCFHDRDIAPEGKTLRETNKILDKVVERIKERMKDSNVKLLWGTANLFSHPRYMHGAATTCSADVFAYAAAQVKKALEITKELGGEGYVFWGGREGYETLLNTDLGLELENLARFLRMAVEYAKKIG FTGQFLIEPKPKHPTKHQYDFDVATAYAFLKNHGLDEYFKFNIEANHATLAGHTFQHELRMARILGKLGSIDAKQGDLLLGWDTDQFPTNIYDTTLAMYEVIKAGGFTKGGLRFDAKVRRASYKVEDLFIGHIAGMDTFALGFKIAYKLAKDGVFDKFIEEKYRSFKEGIGKEIVEGKTDFEKLEEYIIDKEDIELPSGKQEYLESLLNSYIVKTIAELR, SEQ IDNO.

2.

4. The glucose isomerase mutant of claim 1, It is characterized in that Its nucleotide sequence is shown in SEQ IDNO.3; ATGGCTGAATTTTTCCCTGAAATTCCTAAAATTCAATTTGAAGGTAAAGAATCTACTAATCCATTGGCTTTTAGATTCTACGATCCAAATGAAGTTATTGATGGTAAACCATTGAAGGATCATCTGAAATTTTCTGTTGCCTTTGGCATACCTTTGTTAACGAAGG TAGAGATCCATTTGGTGACCCAACTGCTGAAAGACCATGGAATAGATTTTTCTGATCCAATGGATAAGGCTTTTGCTAGAGTTGATGCTTTGTTGAATTCTGTGAAAAGTTGAATTGAATATTGAATACTTTGTTTTCATGACAGAGACATTGCTCCCAGAAGGTAAAACTT TGAGAGAAACTAACAAGATTTTGGATAAGGTCGTTGAAAGAATTAAGGAAAGAATGAAAGAATGAAAGACTCTAACGTTAAATTGTTGGGGTACTGCTAACTTGTTCTCTCACCCAAGATATATGCACGGAGCTGCAACTACCTGTAGTGCTGATGTTTTTGCTTACGCTGCC GCCCAAGTTAAAAAGGCTTTGGAAATAACTAAGGAATTGGGTGGTGAAGGTTACGTTTTCTGGGTGGTAGAGAAGGTTATGAAACTTTGTTGAACACTGATTTGGGTTGGAATTGGAAAACTTGGCTGTTGAGTAAAAAGATT GGTTTTACTGGACAGTTTTTGATTGAGCCTAAACCTAAACCTAAACATCAATATGATTTTGATGTTGCCACTGCTTTCTGAAGAATCATGGACTTGATGAATATTTTAAATTCAACATTGAGGCTAACCATGCTTGCAGGACATACTTTTTTT CCAACATGAATTGAGAATGGCTAGAATTTTGGGTAAATTGGGTTCTATTGATGCTAAGCAAGGAGATTTGTTGTTAGGTTGGGATACTGATCAATTCCCTACTAACATCTACGATACTACCTTAGCTATGTATGAAGTTATTAAGGCTGGTGGTTTTACTAAAGGAGG TTTAAGATTCGATGCTAAGGTTAGAAGAGCTTCTTACAAGGTTGAAGATCTGTTTATTGGTCATATTGCTGGTATGGATACTTTCGCTTTGGGTTTTAAGATTGCCTACAAGTTGGCTAAGGACGGTGTTTTTGATAAATTCATTGAAGAAAATATATCCCTTTA AGGAAGGAATTGGAAAGGAAATTGTTGAAGGTAAAACTGATTTCGAAAAGTTGGAAGAATACATTATTGATAAGGAGGATTGAATTGAATTGCCATCTGGAAAGCAAGAGTACTTGGAATCTTTGCTGAATTTGAATTGAATGA, SEQ ID NO.

3.

5. The use of glucose isomerase mutant as claimed in any one of claims 1-4 in catalytic production of D-fructose.

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