Glucose isomerase mutant and application thereof

Abstract

The invention discloses a glucose isomerase mutant, a coding gene thereof, a recombinant vector constructed by the coding gene, a recombinant genetically engineered bacterium obtained by converting the recombinant vector, and application of the glucose isomerase mutant in catalyzing glucose to generate fructose. The glucose isomerase mutant with good thermal stability, good pH stability and high fructose conversion rate is obtained by performing site-specific mutagenesis on the wild glucose isomerase XAA and is successfully used for catalyzing glucose to generate fructose, and the conversion rate and reaction speed of the glucose isomerase mutant are both superior to those of the wild glucose isomerase XAA. The glucose isomerase mutant shows excellent application prospects and can be used for directly preparing 55% high fructose corn syrup.

Description

Technical Field

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

[0002] Fructose is an important functional sweetener widely used in the food, beverage, and pharmaceutical industries. Compared to sucrose, fructose offers advantages such as increased sweetness, lower calories, and resistance to crystallization. It is particularly suitable for the development of low-calorie foods and foods for diabetic patients, and has significant application value in the functional food sector.

[0003] Fructose production methods primarily include chemical synthesis and enzymatic methods. Chemical synthesis involves the isomerization of glucose under strong acid, strong base, and high temperature conditions. While the process is relatively simple, it suffers from drawbacks such as high energy consumption, low product purity, numerous byproducts, and severe environmental pollution. Furthermore, the product quality often fails to meet food-grade standards. Enzymatic methods, on the other hand, offer advantages such as mild reaction conditions, strong specificity, high product purity, and environmental friendliness, meeting current requirements for green chemistry and sustainable development.

[0004] Glucose isomerase is one of the most important industrial enzymes in fructose production, catalyzing the isomerization of glucose to fructose. However, the currently used wild-type glucose isomerase suffers from poor thermal and pH stability, as well as low fructose conversion efficiency, limiting its application in industrial production. Summary of the Invention

[0005] The present invention aims to provide a glucose isomerase mutant and its application to solve the deficiencies in the prior art.

[0006] The present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a glucose isomerase mutant, wherein the glucose isomerase mutant is a mutant of glucose isomerase XAA, the amino acid sequence of glucose isomerase XAA is shown in SEQ ID NO: 2, and the gene sequence is shown in SEQ ID NO: 1, and the glucose isomerase mutant is selected from any one of the following:

[0008] a) Glucose isomerase mutant XAA (H109Q / S154T / V194S): the mutation sites are: histidine at position 109 is mutated to glutamine (H109Q), serine at position 154 is mutated to threonine (S154T), and valine at position 194 is mutated to serine (V194S); the amino acid sequence is shown in SEQ ID NO: 3;

[0009] b) Glucose isomerase mutant XAA (H109Q / S154T / V194A): its mutation sites are: histidine at position 109 is mutated to glutamine (H109Q), serine at position 154 is mutated to threonine (S154T), and valine at position 194 is mutated to alanine (V194A); its amino acid sequence is SEQ ID NO: 4;

[0010] c) Glucose isomerase mutant XAA (H109Q / S154T / A277G): its mutation sites are: histidine at position 109 is mutated to glutamine (H109Q), serine at position 154 is mutated to threonine (S154T), and alanine at position 277 is mutated to glycine (A277G); its amino acid sequence is SEQ ID NO: 5;

[0011] d) Glucose isomerase mutant XAA (F108K / H109Q / S154T / V194S / A277D): the mutation sites are: phenylalanine at position 108 is mutated to lysine, i.e., F108K; histidine at position 109 is mutated to glutamine, i.e., H109Q; serine at position 154 is mutated to threonine, i.e., S154T; valine at position 194 is mutated to serine, i.e., V194S; and alanine at position 277 is mutated to aspartic acid, i.e., A277D; its amino acid sequence is set forth in SEQ ID NO: 6.

[0012] The second aspect of the present invention provides a gene encoding the above-mentioned glucose isomerase mutant.

[0013] The third aspect of the present invention provides a recombinant vector constructed with the gene encoding the glucose isomerase mutant.

[0014] The fourth aspect of the present invention provides a recombinant genetically engineered bacterium obtained by transformation of the above-mentioned recombinant vector.

[0015] The fifth aspect of the present invention provides the use of the above-mentioned glucose isomerase mutant in catalyzing the production of fructose from glucose.

[0016] Furthermore, an enzyme-catalyzed reaction is carried out in a reaction medium at a certain temperature and stirring speed using a glucose isomerase mutant as a catalyst, glucose as a substrate, and magnesium ions as a cofactor, wherein the source of the magnesium ions includes magnesium sulfate. After the reaction is completed, fructose is separated and purified to obtain fructose.

[0017] Furthermore, the dosage of the glucose isomerase mutant is 100-800 mg / L, the glucose concentration is 100-500 g / L, and the magnesium sulfate concentration is 1-3 g / L; the reaction medium includes Tris-HCl, with a pH of 7.0-8.5; the temperature of the enzyme-catalyzed reaction is 50-75°C, the stirring speed is 70-300 rpm, and the time is 3-8 hours.

[0018] Furthermore, the glucose is D-glucose, and the fructose is D-fructose.

[0019] Beneficial effects of the present invention:

[0020] The present invention, through site-directed mutagenesis of the wild-type glucose isomerase XAA, has generated a glucose isomerase mutant with excellent thermal and pH stability and a high fructose conversion rate. The mutant has been successfully used to catalyze glucose to fructose, achieving a conversion rate and reaction rate superior to those of the wild-type glucose isomerase XAA. The glucose isomerase mutant has shown excellent application prospects and can be used to directly prepare 55% fructose syrup. DETAILED DESCRIPTION

[0021] The present invention will be further explained below in conjunction with the examples. The following examples are only used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0022] Example 1: Amplification of the glucose isomerase gene XAA

[0023] Based on the information of the glucose isomerase gene XAA from Xanthomonas axonopodis included in Genebank (the gene sequence is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2), the total genomic DNA of the bacteria was extracted using a rapid nucleic acid extractor. Using the genomic DNA as a template, PCR amplification was performed under the action of primer 1 (shown in SEQ ID NO: 33, 5'-gggtttcatatgagcaacacc-3') and primer 2 (shown in SEQ ID NO: 34, 5'-ccgctcgagtcaacgcgtcag-3'). The PCR reaction system (total volume 50 μL) consisted of 5 μL of 10× Pfu DNA Polymerase Buffer, 1 μL of 10 mM dNTP mixture (2.5 mM each of dATP, dCTP, dGTP, and dTTP), 1 μL each of Primer 1 and Primer 2 (both at a concentration of 50 μM), 1 μL of genomic DNA (50 ng / μL), 1 μL of Pfu DNA Polymerase (2 U / μL), and 40 μL of ddH₂O. Using a BioRad PCR instrument, the PCR reaction conditions were: initial denaturation at 95°C for 5 min; 30 cycles of denaturation at 95°C for 30 s, annealing at 65°C for 45 s, and extension at 72°C for 1 min; and a final extension at 72°C for 10 min. The PCR reaction was analyzed by electrophoresis on a 0.9 m / v% agarose gel, and the fragment was purified and recovered to obtain the glucose isomerase gene XAA.

[0024] Example 2: Construction of recombinant Escherichia coli expressing glucose isomerase gene XAA

[0025] 1. Obtaining the recombinant plasmid pET28a-XAA

[0026] The XAA gene sequence was obtained. After accurate sequencing, the amplified fragment was treated with Nde I and Xho I restriction endonucleases (TaKaRa), and the fragment was ligated with the commercial vector pET28a treated with the same restriction endonucleases using T4 DNA ligase (TaKaRa) to construct the expression vector pET28a-XAA, i.e., the recombinant plasmid pET28a-XAA.

[0027] 2. Transformation of recombinant plasmid pET28a-XAA into E. coli BL21 (DE3)

[0028] In each tube, 100 μL of E. coli BL21 (DE3) competent cell suspension (OD 600 =0.4-0.6), add 10 μL of recombinant plasmid (i.e., recombinant plasmid pET28a-XAA, concentration 10 ng / μL), mix gently, and let it stand in an ice bath for 30 minutes. Transfer to a 42°C water bath and heat shock for 90 seconds. Quickly transfer to an ice bath and cool for 3 minutes. Add 700 μL of LB liquid medium (LB liquid medium formula: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.0; the same below) to each tube and incubate at 37°C, 100 rpm on a shaker for 1 hour. After incubation, centrifuge the bacterial solution at 3000 rpm for 2 minutes, discard 700 μL of the supernatant, mix the remaining bacterial solution, and spread it onto LB solid medium (LB solid medium formula: 1.5 m / v% agar powder added to the LB liquid medium formula; the same below) containing 50 μg / mL kanamycin sulfate, and incubate inverted at 37°C overnight.

[0029] Selection of positive clones: Four single colonies were selected from the transformation plate and transferred to 5 mL of LB liquid medium containing 50 μg / mL kanamycin sulfate. The cells were cultured at 37°C and 200 rpm for 8 hours. The plasmid was extracted using the Mini-Plasmid Rapid Isolation Kit (Beijing Broadtech Bio-Gene Technology Co., Ltd.). 20 μL of the plasmid was sequenced at Sangon Biotech (Shanghai) Co., Ltd. to verify the correct E. coli BL21(DE3) / pET28a-XAA clone.

[0030] Example 3: Amplification of Glucose Isomerase Mutant Genes XAA(H109Q / S154T / V194S), XAA(H109Q / S154T / V194A), XAA(H109Q / S154T / A277G), and XAA(F108K / H109Q / S154T / V194S / A277D)

[0031] The primers at both ends were synthesized. The primer sequences corresponding to XAA (H109Q / S154T / V194S) were shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively. The primer sequences corresponding to XAA (H109Q / S154T / V194A) were shown in SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively. The primer sequences corresponding to XAA (H109Q / S154T / A277G) were shown in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24. NO: 24, and the corresponding sequences of XAA (F108K / H109Q / S154T / V194S / A277D) are shown in SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32, respectively (108 and 109 can be spliced ​​using overlapping primers, so only four pairs of primers are needed to complete the amplification).

[0032] The mutation was introduced using the PCR method. The specific method is as follows: PCR reaction system: ddH2O 22μL, Premix PrimeSTAR 25μL, upstream primer (20μM) 1μL, downstream primer (20μM) 1μL, template plasmid 1μL (the template plasmid is the recombinant plasmid pET28a-XAA prepared in Example 2, with a concentration of 10ng / μL). PCR reaction: 98℃ pre-denaturation for 30s; 98℃ denaturation for 30s, 55℃ annealing for 30s, 72℃ extension for 7min, 30 cycles; 72℃ extension for 10min. Using the glucose isomerase gene XAA as a template, PCR reaction was performed with primers to obtain the mutated full-length gene. DNA fragments were purified using the 3S Spin Agarose Gel DNA Purification Kit (Shanghai Shenneng Biotechnology Co., Ltd.).

[0033] The purified gene PCR product was digested with Dpn I enzyme to form the template plasmid:

[0034] Digestion system: 30 μL of purified gene PCR product (concentration 10 ng / μL), 5 μL of 10× Buffer, 1.5 μL of Dpn I (concentration 2 U / μL), and ddH2O to make up to 50 μL. After 2 h of digestion, DNA fragments were purified using a 3S Spin Agarose Gel DNA Purification Kit (Shanghai Shenneng Biotechnology Co., Ltd.).

[0035] The digested and purified PCR reaction solution was detected by 0.9 m / v% agarose gel electrophoresis and the gel was cut to recover and purify the fragments to obtain the glucose isomerase mutant genes XAA(H109Q / S154T / V194S), XAA(H109Q / S154T / V194A), XAA(H109Q / S154T / A277G), and XAA(F108K / H109Q / S154T / V194S / A277D).

[0036] Example 4: Construction of recombinant Escherichia coli expressing the glucose isomerase mutant genes XAA(H109Q / S154T / V194S), XAA(H109Q / S154T / V194A), XAA(H109Q / S154T / A277G), and XAA(F108K / H109Q / S154T / V194S / A277D)

[0037] 1. Obtaining recombinant plasmids pET28a-XAA(H109Q / S154T / V194S), pET28a-XAA(H109Q / S154T / V194A), pET28a-XAA(H109Q / S154T / A277G), and pET28a-XAA(F108K / H109Q / S154T / V194S / A277D)

[0038] The gene sequences of each mutant were obtained. After accurate sequencing, the amplified fragments were treated with Nde I and Xho I restriction endonucleases (TaKaRa), and the fragments were ligated with the commercial vector pET28a treated with the same restriction endonucleases using T4 DNA ligase (TaKaRa) to construct expression vectors pET28a-XAA (H109Q / S154T / V194S), pET28a-XAA (H109Q / S154T / V194A), pET28a-XAA (H109Q / S154T / A277G), and pE T28a-XAA(F108K / H109Q / S154T / V194S / A277D) is the recombinant plasmid pET28a-XAA(H109Q / S154T / V194S), pET28a-XAA(H109Q / S154T / V194A), pET28a-XAA(H109Q / S154T / A277G), and pET28a-XAA(F108K / H109Q / S154T / V194S / A277D).

[0039] 2. Recombinant plasmids pET28a-XAA(H109Q / S154T / V194S), pET28a-XAA(H109Q / S154T / V194A), pET28a-XAA(H109Q / S154T / A277G), and pET28a-XAA(F108K / H109Q / S154T / V194S / A277D) were transformed into E. coli BL21(DE3)

[0040] In each tube, 100 μL of E. coli BL21 (DE3) competent cell suspension (OD 600 =0.4-0.6) was added with 10 μL of recombinant plasmid (recombinant plasmid pET28a-XAA(H109Q / S154T / V194S), pET28a-XAA(H109Q / S154T / V194A), pET28a-XAA(H109Q / S154T / A277G), pET28a-XAA(F108K / H109Q / S154T / V194S / A277D)) at a concentration of 50 ng / μL), gently mixed, and allowed to stand on ice for 30 min. Transfer to a 42°C water bath and heat shock for 90 s. Quickly transfer to an ice bath and cool for 3 min. Add 700 μL of LB liquid medium to each tube and incubate at 37°C with a shaker at 100 rpm for 1 h. After culture, the bacterial solution was centrifuged at 3000 rpm for 2 min, 700 μL of the supernatant was discarded, and the remaining bacterial solution was mixed and spread on an LB solid culture medium plate containing 50 μg / mL kanamycin sulfate, and cultured in an inverted manner at 37°C overnight.

[0041] Selection of positive clones: Four single colonies were selected from the transformation plate and transferred to 5 mL of LB liquid medium containing 50 μg / mL kanamycin sulfate. The cells were cultured at 37°C and 200 rpm for 8 h. Plasmids were then extracted using the Mini-Plasmid Rapid Isolation Kit (Beijing Broadtech Bio-Gene Technology Co., Ltd.). 20 μL of the plasmid was then sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. Verify the correct E. coli BL21(DE3) / pET28a-XAA(H109Q / S154T / V194S), E. coli BL21(DE3) / pET28a-XAA(H109Q / S154T / V194A), E. coli BL21(DE3) / pET28a-XAA(H109Q / S154T / A277G), and E. coli BL21(DE3) / pET28a-XAA(F108K / H109Q / S154T / V194S / A277D).

[0042] Sequencing confirmed that the amino acid sequences of XAA (H109Q / S154T / V194S), XAA (H109Q / S154T / V194A), XAA (H109Q / S154T / A277G), and XAA (F108K / H109Q / S154T / V194S / A277D) had the following mutations relative to the XAA amino acid sequence (as shown in SEQ ID NO. 2):

[0043] For XAA (H109Q / S154T / V194S), its amino acid sequence is shown in SEQ ID NO. 3: the histidine at position 109 is mutated to glutamine (H109Q), the serine at position 154 is mutated to threonine (S154T), and the valine at position 194 is mutated to serine (V194S).

[0044] For XAA (H109Q / S154T / V194A), its amino acid sequence is shown in SEQ ID NO. 4: the histidine at position 109 is mutated to glutamine (H109Q), the serine at position 154 is mutated to threonine (S154T), and the valine at position 194 is mutated to alanine (V194A).

[0045] For XAA (H109Q / S154T / A277G), its amino acid sequence is shown in SEQ ID NO. 5: the histidine at position 109 is mutated to glutamine (H109Q), the serine at position 154 is mutated to threonine (S154T), and the alanine at position 277 is mutated to glycine (A277G).

[0046] For XAA (F108K / H109Q / S154T / V194S / A277D), its amino acid sequence is shown in SEQ ID NO. 6: the phenylalanine at position 108 is mutated to lysine (F108K); the histidine at position 109 is mutated to glutamine (H109Q); the serine at position 154 is mutated to threonine (S154T); the valine at position 194 is mutated to serine (V194S); and the alanine at position 277 is mutated to aspartic acid (A277D).

[0047] Example 5: Construction of recombinant Bacillus subtilis expressing the glucose isomerase mutant genes XAA(H109Q / S154T / V194S), XAA(H109Q / S154T / V194A), XAA(H109Q / S154T / A277G), and XAA(F108K / H109Q / S154T / V194S / A277D)

[0048] I. Obtaining the recombinant plasmids pHCMC04-XAA(H109Q / S154T / V194S), pHCMC04-XAA(H109Q / S154T / V194A), pHCMC04-XAA(H109Q / S154T / A277G), and pHCMC04-XAA(F108K / H109Q / S154T / V194S / A277D)

[0049] The gene sequences of each mutant were obtained. After accurate sequencing, the amplified fragments were treated with Kpn I and Sma I restriction endonucleases (TaKaRa) and ligated with the commercial vector pHCMC04 treated with the same restriction endonucleases using T4 DNA ligase (TaKaRa) to construct expression vectors pHCMC04-XAA (H109Q / S154T / V194S), pHCMC04-XAA (H109Q / S154T / V194A), pHCMC04-XAA (H109Q / S154T / A277G), and pHCMC04-XAA (F108K / H109Q / S154T / V194S / A277D).

[0050] 2. Transformation of recombinant plasmids pHCMC04-XAA(H109Q / S154T / V194S), pHCMC04-XAA(H109Q / S154T / V194A), pHCMC04-XAA(H109Q / S154T / A277G), and pHCMC04-XAA(F108K / H109Q / S154T / V194S / A277D) into Bacillus subtilis SCK6

[0051] Preparation of SCK6 competent cells: Inoculate a single colony from a plate containing Bacillus subtilis SCK6 in LB solid medium supplemented with 100 μg / mL ampicillin into 5 ml of LB liquid medium. Cultivate overnight with shaking at 37°C, 200 rpm. Transfer 2.5 ml of the culture to 10 ml of LB liquid medium and dilute to an OD600 of approximately 1.0. Add xylose to a final concentration of 1 w / v%, incubate at 37°C, 200 rpm, and shake for 2 hours. Add glycerol to a final concentration of 10 v / v%, aliquot 500 μl per tube, and freeze at -70°C until ready for use.

[0052] 20 μL of recombinant plasmid (recombinant plasmid pHCMC04-XAA (H109Q / S154T / V194S), pHCMC04-XAA (H109Q / S154T / V194A), pHCMC04-XAA (H109Q / S154T / A277G), pHCMC04-XAA (F108K / H109Q / S154T / V194S / A277D)) was added per 500 μL of SCK6 competent cells, with a concentration of 50 ng / μL), and cultured at 37 ° C and 200 rpm with shaking for 1.5 h. The culture medium was spread on an LB solid medium plate containing 100 μg / mL ampicillin and cultured inverted at 37 ° C overnight.

[0053] Selection of positive clones: Four single clones were picked from the transformation plate and inoculated into 5 ml of LB liquid medium. The culture was shaken at 37°C and 200 rpm overnight. 3 ml of the culture was centrifuged (12,000 rpm, 1 min), the cells were collected, and resuspended in 150 μL of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0; the same below). Lysozyme was added to a final concentration of 1 mg / mL and incubated at 37°C for 1 h. Sodium dodecyl sulfate (SDS) was added to a final concentration of 0.5 w / v% and proteinase K was added to a final concentration of 50 μg / mL. The mixture was inverted and incubated at 37°C until it became clear. 300 μL of TE buffer was added, the mixture was inverted and centrifuged (12,000 rpm, 1 min). The supernatant was collected and the plasmid was extracted using a small-scale plasmid extraction kit (Omega, cat. no. D6943-02). The supernatant was purified by adsorption on a silica-based column and 50 μL of PBS was added. Plasmid was eluted with TE buffer. 20 μL of the plasmid was removed and sequenced at Sangon Biotech (Shanghai) Co., Ltd. The correct plasmids were SCK6 / pHCMC04-XAA(H109Q / S154T / V194S), SCK6 / pHCMC04--XAA(H109Q / S154T / V194A), SCK6 / pHCMC04--XAA(H109Q / S154T / A277G), and SCK6 / pHCMC04--XAA(F108K / H109Q / S154T / V194S / A277D).

[0054] Sequencing confirmed that the amino acid sequences of XAA (H109Q / S154T / V194S), XAA (H109Q / S154T / V194A), XAA (H109Q / S154T / A277G), and XAA (F108K / H109Q / S154T / V194S / A277D) had the following mutations relative to the XAA amino acid sequence (as shown in SEQ ID NO. 2):

[0055] For XAA (H109Q / S154T / V194S), its amino acid sequence is shown in SEQ ID NO. 3: the histidine at position 109 is mutated to glutamine (H109Q), the serine at position 154 is mutated to threonine (S154T), and the valine at position 194 is mutated to serine (V194S).

[0056] For XAA (H109Q / S154T / V194A), its amino acid sequence is shown in SEQ ID NO. 4: the histidine at position 109 is mutated to glutamine (H109Q), the serine at position 154 is mutated to threonine (S154T), and the valine at position 194 is mutated to alanine (V194A).

[0057] For XAA (H109Q / S154T / A277G), its amino acid sequence is shown in SEQ ID NO. 5: the histidine at position 109 is mutated to glutamine (H109Q), the serine at position 154 is mutated to threonine (S154T), and the alanine at position 277 is mutated to glycine (A277G).

[0058] For XAA (F108K / H109Q / S154T / V194S / A277D), its amino acid sequence is shown in SEQ ID NO. 6: the phenylalanine at position 108 is mutated to lysine (F108K); the histidine at position 109 is mutated to glutamine (H109Q); the serine at position 154 is mutated to threonine (S154T); the valine at position 194 is mutated to serine (V194S); and the alanine at position 277 is mutated to aspartic acid (A277D).

[0059] Example 6: Induced expression culture of recombinant Escherichia coli of glucose isomerase mutant genes XAA(H109Q / S154T / V194S), XAA(H109Q / S154T / V194)A, XAA(H109Q / S154T / A277)G, and XAA(F108K / H109Q / S154T / V194S / A277D)

[0060] In order to verify the protein expression of recombinant E. coli, the constructed E. coli BL21 (DE3) / pET28a-XAA (H109Q / S154T / V194S), E. coli BL21 (DE3) / pET28a Induction expression was performed on recombinant E. coli BL21(DE3) / pET28a-XAA(H109Q / S154T / V194A), E. coli BL21(DE3) / pET28a-XAA(H109Q / S154T / A277G), E. coli BL21(DE3) / pET28a-XAA(F108K / H109Q / S154T / V194S / A277D), and wild-type E. coli BL21(DE3) / pET28a-XAA. An empty vector (E. coli BL21(DE3) / pET28a, lacking the target gene) was used as a negative control. Recombinant E. coli cells from each treatment were inoculated into 5 mL of LB liquid medium supplemented with 50 μg / mL kanamycin sulfate and cultured overnight at 37°C with shaking at 200 rpm. Transfer 10 mL of culture medium to 1 L of LB liquid medium containing 50 μg / mL kanamycin sulfate and culture at 37°C, 200 rpm, shaking until the OD600 is approximately 0.6-0.8. Add isopropyl-β-D-thiogalactoside to a final concentration of 0.5 mM to the culture medium and induce the culture at 25°C, 200 rpm, shaking for 12 hours. After the induction culture is completed, centrifuge at 12,000 rpm for 10 minutes, discard the supernatant, and collect the bacterial pellet. Dissolve the collected bacterial pellet in PBS buffer (20 mM, pH 7.0) and ultrasonically disrupt for 30 minutes (ultrasonic power of 150 W, ultrasonic frequency of 20 kHz, working time of 2 seconds, rest time of 3 seconds). The disrupted bacteria were centrifuged at 12000 rpm for 40 min, and the supernatant and precipitate were collected separately. Protein expression was detected by SDS-PAGE as follows: 20 μl of supernatant was added to 5 μl of 4×SDS-PAGE loading buffer, mixed and then treated in a boiling water bath for 10 min, with a sample volume of 20 μl; the precipitate was evenly dispersed with PBS buffer (20 mM, pH 7.0) equal to the volume of the supernatant, 20 μl was added to 5 μl of 4×SDS-PAGE loading buffer, mixed and then treated in a boiling water bath for 10 min, with a sample volume of 20 μl. Electrophoresis was performed using a 5 m / v% stacking gel and a 12 m / v% separating gel. The electrophoresis conditions were as follows: the inner tank was a negative electrode buffer (25 mM Tris, 192 mM glycine, 0.1 w / v% SDS), the outer tank was a positive electrode buffer (25 mM Tris, 192 mM glycine, 0.1 w / v% SDS), and the voltage was set to 80 V (stacking gel) and 120 V (separating gel). After electrophoresis, the gel was stained (Coomassie Brilliant Blue R-250) and decolorized to observe protein bands. Clear bands were observed at the corresponding molecular weight for each treatment, indicating that the protein was correctly expressed.

[0061] Example 7: Induced expression culture of recombinant Bacillus subtilis of glucose isomerase mutant genes XAA(H109Q / S154T / V194S), XAA(H109Q / S154T / V194)A, XAA(H109Q / S154T / A277)G, and XAA(F108K / H109Q / S154T / V194S / A277D)

[0062] To verify protein expression in recombinant Bacillus subtilis, the constructed SCK6 / pHCMC04-XAA(H109Q / S154T / V194S), SCK6 / pHCMC04--XAA(H109Q / S154T / V194A), SCK6 / pHCMC04-XAA(H109Q / S154T / A277G), and SCK6 / pHCMC04-XAA(F108K / H109Q / S154T / V194S / A277D) strains were induced for expression. An empty vector (SCK6 / pHCMC04, without the target gene inserted) was used as a negative control. Each recombinant Bacillus subtilis strain was inoculated into 5 ml of LB liquid medium containing 25 μg / ml chloramphenicol and cultured overnight at 37°C with shaking at 200 rpm. 2 ml of culture medium was transferred to 50 ml of 5YC liquid medium (5YC liquid medium formula: 5 g / L yeast extract, 10 g / L peptone, 5 g / L NaCl, 10 g / L glucose, pH 7.0) containing 25 μg / ml chloramphenicol. Culture was shaken at 33°C and 200 rpm for 8 h. Isopropyl-β-D-thiogalactopyranoside was added to the culture medium to a final concentration of 1 mM, and the culture was shaken for another 24 h. After completion of the culture, the cells were centrifuged at 4000 rpm for 10 min, and the supernatant was collected. Protein expression was analyzed by SDS-PAGE as follows: 20 μl of supernatant was added to 5 μl of 4× SDS-PAGE loading buffer, mixed, and incubated in a boiling water bath for 10 min. A 20 μl sample volume was loaded. Electrophoresis was performed using a 5 m / v% stacking gel and a 17 m / v% separating gel. The electrophoresis conditions were as follows: the inner tank was a negative electrode buffer (25 mM Tris, 192 mM glycine, 0.1 w / v% SDS), the outer tank was a positive electrode buffer (25 mM Tris, 192 mM glycine, 0.1 w / v% SDS), and the voltage was set to 80 V (stacking gel) and 120 V (separating gel). After electrophoresis, the gel was stained (Coomassie Brilliant Blue R-250) and decolorized to observe protein bands. Clear bands were observed at the corresponding molecular weight for each treatment, indicating that the protein was correctly expressed.

[0063] Example 8: Purification of Recombinant Proteins XAA(H109Q / S154T / V194S), XAA(H109Q / S154T / V194A), XAA(H109Q / S154T / A277G), XAA(F108K / H109Q / S154T / V194S / A277D), and Wild-Type XAA (Expression in Recombinant E. coli BL21(DE3) System)

[0064] Recombinant proteins XAA (H109Q / S154T / V194S), XAA (H109Q / S154T / V194A), XAA (H109Q / S154T / A277G), XAA (F108K / H109Q / S154T / V194S / A277D), and wild-type XAA were purified using His-Trap HP affinity chromatography columns:

[0065] Pick the positive monoclonal clones (prepared in Example 4 and Example 2) and inoculate them into 5 mL of LB liquid culture medium containing 50 μg / mL kanamycin sulfate, and culture them at 37°C, 200 rpm and shaking overnight. Take 10 mL of culture solution and transfer it to 1 L of LB liquid culture medium containing 50 μg / mL kanamycin sulfate, and culture them at 37°C, 200 rpm and shaking until the OD600 is about 0.6-0.8. Add isopropyl-β-D-thiogalactoside at a final concentration of 0.5 mM to the culture solution, and induce the culture at 25°C, 200 rpm and shaking for 12 hours. After the induction culture is completed, centrifuge at 12000 rpm for 10 minutes, discard the supernatant, and collect the bacterial precipitate. Wash the collected bacterial precipitate 3 times with pre-cooled physiological saline (0.9w / v% NaCl), centrifuge at 12000 rpm for 5 minutes each time, and discard the supernatant. Finally, collect the bacterial precipitate (wet bacteria). Weigh 2 g of wet cells and add 20 mL of 20 mM Tris buffer (pH 8.0) containing 150 mM NaCl. Ultrasonicate in an ice bath for 30 min (ultrasonic power 150 W, ultrasonic frequency 20 kHz, working time 2 s, rest time 3 s). Centrifuge at 12,000 rpm for 30 min at 4°C, and collect the supernatant as the crude enzyme solution. The crude enzyme solution was purified using a His-Trap HP affinity chromatography column manufactured by GE. After ultrafiltration and desalination, the pure enzyme solution can be directly used for the specific conversion of D-glucose or freeze-dried and stored for future use (in the following examples, mutant XAA (H109Q / S154T / V194S), mutant XAA (H109Q / S154T / V194A), mutant XAA (H109Q / S154T / A277G), mutant XAA (F108K / H109Q / S154T / V194S / A277D) and wild-type XAA are freeze-dried products).

[0066] Example 9: Wild-type XAA catalyzes D-glucose to produce D-fructose

[0067] The reaction system consisted of 2 ml Tris-HCl (pH 7.5), 0.6 g glucose, 0.005 g magnesium sulfate, and 1 mg enzyme (prepared in Example 8, wild-type XAA). The reaction conditions were 65°C, 150 rpm, and 5 h. The reaction was terminated by adding 1 M sulfuric acid to adjust the pH to 2.

[0068] HPLC analysis of D-fructose conversion was performed using an Agilent 1260 high-performance liquid chromatograph with a differential refractive index detector. The HPLC conditions included a Carbomix Ca-NP column (4.6 x 300 mm), mobile phase: pure water, flow rate: 0.6 ml / min, run time: 25 min, column temperature: 65°C, detector temperature: 50°C. The reaction solution was analyzed by HPLC, and the D-fructose conversion was 46%.

[0069] Example 10: Mutant XAA (H109Q / S154T / V194S) catalyzes D-glucose to produce D-fructose

[0070] The reaction system consisted of 2 ml of Tris-HCl (pH 7.5), 0.6 g of glucose, 0.005 g of magnesium sulfate, and 1 mg of enzyme (prepared in Example 8, mutant XAA (H109Q / S154T / V194S)). The reaction was carried out at 65°C and 150 rpm for 5 h. The reaction was terminated by adding 1 M sulfuric acid to adjust the pH to 2. The reaction solution was analyzed by HPLC (HPLC conditions were the same as in Example 9), revealing a D-fructose conversion rate of 52%.

[0071] Example 11: Mutant XAA (H109Q / S154T / V194A) catalyzes D-glucose to produce D-fructose

[0072] The reaction system consisted of 2 ml of Tris-HCl (pH 7.5), 0.6 g of glucose, 0.005 g of magnesium sulfate, and 1 mg of enzyme (prepared in Example 8, mutant XAA (H109Q / S154T / V194A)). Reaction conditions were 65°C, 150 rpm, and 5 h. The reaction was terminated by adding 1 M sulfuric acid to adjust the pH to 2. The reaction solution was analyzed by HPLC (HPLC conditions were the same as in Example 9), revealing a D-fructose conversion rate of 58%.

[0073] Example 12: Mutant XAA (H109Q / S154T / A277G) catalyzes D-glucose to produce D-fructose

[0074] The reaction system consisted of 2 ml of Tris-HCl (pH 7.5), 0.6 g of glucose, 0.005 g of magnesium sulfate, and 1 mg of enzyme (prepared in Example 8, mutant XAA (H109Q / S154T / A277G)). The reaction was carried out at 65°C and 150 rpm for 5 h. The reaction was terminated by adding 1 M sulfuric acid to adjust the pH to 2. The reaction solution was analyzed by HPLC (HPLC conditions were the same as in Example 9), revealing a D-fructose conversion rate of 61%.

[0075] Example 13: Mutant XAA (F108K / H109Q / S154T / V194S / A277D) catalyzes D-glucose to produce D-fructose

[0076] The reaction system consisted of 2 ml of Tris-HCl (pH 7.5), 0.6 g of glucose, 0.005 g of magnesium sulfate, and 1 mg of enzyme (prepared in Example 8, mutant XAA (F108K / H109Q / S154T / V194S / A277D)). The reaction was carried out at 65°C and 150 rpm for 5 h. The reaction was terminated by adding 1 M sulfuric acid to adjust the pH to 2. The reaction solution was analyzed by HPLC (HPLC conditions were the same as in Example 9), revealing a D-fructose conversion rate of 56%.

[0077] Example 14: Wild-type XAA catalyzes D-glucose to produce D-fructose

[0078] The reaction system consisted of 2 ml of Tris-HCl (pH 7.5), 0.6 g of glucose, 0.005 g of magnesium sulfate, and 1 mg of enzyme (prepared in Example 8, wild-type XAA). The reaction conditions were 75°C and 150 rpm for 5 h. The reaction was terminated by adding 1 M sulfuric acid to adjust the pH to 2. The reaction solution was analyzed by HPLC (HPLC conditions were the same as in Example 9), yielding a D-fructose conversion rate of 31%.

[0079] Example 15: Mutant XAA (H109Q / S154T / V194S) catalyzes D-glucose to produce D-fructose

[0080] The reaction system consisted of 2 ml of Tris-HCl (pH 7.5), 0.6 g of glucose, 0.005 g of magnesium sulfate, and 1 mg of enzyme (prepared in Example 8, mutant XAA (H109Q / S154T / V194S)). The reaction was carried out at 75°C and 150 rpm for 5 h. The reaction was terminated by adding 1 M sulfuric acid to adjust the pH to 2. The reaction solution was analyzed by HPLC (HPLC conditions were the same as in Example 9), yielding a D-fructose conversion rate of 55%.

[0081] Example 16: Mutant XAA (H109Q / S154T / A277G) catalyzes D-glucose to produce D-fructose

[0082] The reaction system consisted of 2 ml of Tris-HCl (pH 7.5), 0.6 g of glucose, 0.005 g of magnesium sulfate, and 1 mg of enzyme (prepared in Example 8, mutant XAA (H109Q / S154T / A277G)). The reaction conditions were 75°C and 150 rpm for 5 h. The reaction was terminated by adding 1 M sulfuric acid to adjust the pH to 2. The reaction solution was analyzed by HPLC (HPLC conditions were the same as in Example 9), yielding a D-fructose conversion rate of 63%.

[0083] Example 17: Wild-type XAA catalyzes D-glucose to produce D-fructose

[0084] The reaction system consisted of 2 ml of Tris-HCl (pH 7.5), 0.6 g of glucose, 0.005 g of magnesium sulfate, and 1 mg of enzyme (prepared in Example 8, wild-type XAA). The reaction was carried out at 50°C and 150 rpm for 5 h. The reaction was terminated by adding 1 M sulfuric acid to adjust the pH to 2. The reaction solution was analyzed by HPLC (HPLC conditions were the same as in Example 9), yielding a D-fructose conversion rate of 37%.

[0085] Example 18: Mutant XAA (H109Q / S154T / A277G) catalyzes D-glucose to produce D-fructose

[0086] The reaction system consisted of 2 ml of Tris-HCl (pH 7.5), 0.6 g of glucose, 0.005 g of magnesium sulfate, and 1 mg of enzyme (prepared in Example 8, mutant XAA (H109Q / S154T / A277G)). The reaction was carried out at 50°C and 150 rpm for 5 h. The reaction was terminated by adding 1 M sulfuric acid to adjust the pH to 2. The reaction solution was analyzed by HPLC (HPLC conditions were the same as in Example 9), yielding a D-fructose conversion rate of 40%.

[0087] Example 19: Mutant XAA (H109Q / S154T / A277G) catalyzes D-glucose to produce D-fructose

[0088] The reaction system consisted of 2 ml of Tris-HCl (pH 7.0), 0.6 g of glucose, 0.005 g of magnesium sulfate, and 1 mg of enzyme (prepared in Example 8, mutant XAA (H109Q / S154T / A277G)). The reaction was carried out at 65°C and 150 rpm for 5 h. The reaction was terminated by adding 1 M sulfuric acid to adjust the pH to 2. The reaction solution was analyzed by HPLC (HPLC conditions were the same as in Example 9), yielding a D-fructose conversion rate of 61%.

[0089] Example 20: Mutant XAA (H109Q / S154T / A277G) catalyzes D-glucose to produce D-fructose

[0090] The reaction system consisted of 2 ml of Tris-HCl (pH 8.0), 0.6 g of glucose, 0.005 g of magnesium sulfate, and 1 mg of enzyme (prepared in Example 8, mutant XAA (H109Q / S154T / A277G)). The reaction was carried out at 65°C and 150 rpm for 5 h. The reaction was terminated by adding 1 M sulfuric acid to adjust the pH to 2. The reaction solution was analyzed by HPLC (HPLC conditions were the same as in Example 9), yielding a D-fructose conversion rate of 60%.

[0091] Example 21: Mutant XAA (H109Q / S154T / A277G) catalyzes D-glucose to produce D-fructose

[0092] The reaction system consisted of 2 ml of Tris-HCl (pH 8.5), 0.6 g of glucose, 0.005 g of magnesium sulfate, and 1 mg of enzyme (prepared in Example 8, mutant XAA (H109Q / S154T / A277G)). The reaction was carried out at 65°C and 150 rpm for 5 h. The reaction was terminated by adding 1 M sulfuric acid to adjust the pH to 2. The reaction solution was analyzed by HPLC (HPLC conditions were the same as in Example 9), yielding a D-fructose conversion rate of 58%.

Claims

1. A glucose isomerase mutant, characterized in that The glucose isomerase mutant is a mutant of glucose isomerase XAA, the amino acid sequence of glucose isomerase XAA is shown in SEQ ID NO: 2, and the gene sequence is shown in SEQ ID NO:

1. The glucose isomerase mutant is selected from any one of the following: a) Glucose isomerase mutant XAA (H109Q / S154T / V194S): the mutation sites are: histidine at position 109 is mutated to glutamine (H109Q), serine at position 154 is mutated to threonine (S154T), and valine at position 194 is mutated to serine (V194S); the amino acid sequence is shown in SEQ ID NO: 3; b) Glucose isomerase mutant XAA (H109Q / S154T / V194A): its mutation sites are: histidine at position 109 is mutated to glutamine (H109Q), serine at position 154 is mutated to threonine (S154T), and valine at position 194 is mutated to alanine (V194A); its amino acid sequence is SEQ ID NO: 4; c) Glucose isomerase mutant XAA (H109Q / S154T / A277G): its mutation sites are: histidine at position 109 is mutated to glutamine (H109Q), serine at position 154 is mutated to threonine (S154T), and alanine at position 277 is mutated to glycine (A277G); its amino acid sequence is SEQ ID NO: 5; d) Glucose isomerase mutant XAA (F108K / H109Q / S154T / V194S / A277D): the mutation sites are: phenylalanine at position 108 is mutated to lysine, i.e., F108K; histidine at position 109 is mutated to glutamine, i.e., H109Q; serine at position 154 is mutated to threonine, i.e., S154T; valine at position 194 is mutated to serine, i.e., V194S; and alanine at position 277 is mutated to aspartic acid, i.e., A277D; its amino acid sequence is set forth in SEQ ID NO:

6.

2. The gene encoding the glucose isomerase mutant according to claim 1.

3. A recombinant vector constructed from the gene encoding the glucose isomerase mutant according to claim 2.

4. The recombinant genetically engineered bacteria obtained by transformation with the recombinant vector according to claim 3.

5. Use of the glucose isomerase mutant according to claim 1 in catalyzing the production of fructose from glucose.

6. The use according to claim 5, characterized in that The method uses a glucose isomerase mutant as a catalyst, glucose as a substrate, and magnesium ions as a cofactor. The source of the magnesium ions includes magnesium sulfate. An enzyme-catalyzed reaction is carried out in a reaction medium at a certain temperature and stirring speed. After the reaction is completed, fructose is separated and purified to obtain fructose.

7. The use according to claim 6, characterized in that The dosage of the glucose isomerase mutant is 100-800 mg / L, the glucose concentration is 100-500 g / L, and the magnesium sulfate concentration is 1-3 g / L; the reaction medium includes Tris-HCl with a pH of 7.0-8.5; the temperature of the enzyme-catalyzed reaction is 50-75°C, the stirring speed is 70-300 rpm, and the time is 3-8 hours.

8. The use according to any one of claims 5 to 7, characterized in that: The glucose is D-glucose, and the fructose is D-fructose.