Glucose isomerase and method for improving activity of glucose isomerase

Abstract

The invention discloses a glucose isomerase which is glucose isomerase Pd-xylA, and the amino acid sequence of the glucose isomerase is shown as SEQ ID NO. 1. The invention belongs to the technical field of enzyme engineering, the affinity of the provided glucose isomerase to substrate glucose is higher than that of the existing glucose isomerase, and the conversion rate of fructose generated by glucose isomerization can reach about 50% after the glucose isomerase reacts for 30 h at the temperature of 50 DEG C and is higher than the conversion rate of about 40% of the existing glucose isomerase. Besides, the glucose isomerase provided by the invention has a resistance effect on most organic solvents, and the catalytic activity of the glucose isomerase Pd-xylA can be remarkably improved by adding Tween 80 and / or Tween 20 with a certain concentration.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme engineering, and in particular relates to glucose isomerase and a method for improving the activity of glucose isomerase. Background Art

[0002] High fructose corn syrup (HFCS), also known as high fructose syrup, is a mixture of glucose and fructose. It has the advantages of good solubility, high sweetness, not easy to crystallize at low temperatures, and low price. As a sweetener, it is widely used in food, medicine and other fields, and has replaced sucrose in a large number of application scenarios.

[0003] At present, the methods for producing fructose syrup include acid hydrolysis and bio-enzyme conversion. The acid hydrolysis method uses strong acid to hydrolyze fructose. Its reaction conditions need to be under high temperature conditions, which forms more by-products and is not environmentally friendly. Compared with the acid hydrolysis method, the bio-enzyme conversion method has the advantages of low cost, simple operation, few by-products, and environmental friendliness, and has gradually become the main method for industrial production of fructose syrup. The steps of producing fructose syrup by bio-enzyme conversion include: using corn starch as raw material, liquefying it with α-amylase, then saccharifying it with amyloglucosidase and pullulanase, and finally isomerizing it with glucose isomerase to produce fructose syrup. It can be seen that glucose isomerase (xylose isomerase, xylA) plays an important role in the production of fructose syrup.

[0004] There are literature reports on Thermoanaerobacter ethanolicus (GenBank: AKM94132.1), Thermotoga (GenBank: WP_015919215.1), Actinoplanes sp.ATCC 31351 (GenBank: AAA92578.1), Streptomyces rubiginosus (GenBank: AAA26838.1), Streptomycesviolaceusniger (GenBank: AAA26839.1), Bacillus subtilis (GenBank: AOR98272.1), Levilactobacillusbrevis (GenBank: ARW51820.1), Lactiplantibacillus pentosus(GenBank:WP_279490182.1) and Staphylococcus xylosus (GenBank: GEQ10674.1), and studied the enzyme activity encoded by it. According to the fructose content, high fructose corn syrup is divided into HFCS-42 (containing 42% fructose), HFCS-55 (containing 55% fructose) and HFCS-90 (containing 90% fructose), among which HFCS-55 is the most widely used in the fields of food and medicine. However, the direct production of HFCS-55 by bioenzymatic conversion currently has problems such as low conversion rate. The existing glucose isomerase can only reach a conversion rate of about 40% at most, resulting in HFCS-55 being prepared by mixing HFCS-42 and HFCS-90, which increases the production and application costs. It is urgent to develop a glucose isomerase suitable for the direct production of HFCS-55 with a high conversion rate.

[0005] Heterologous expression refers to the use of DNA or RNA from another organism as an exogenous gene or expressor in one organism, so that the gene or its product can produce specific functions or traits. Heterologous expression of prokaryotic proteins is to introduce a plasmid with an inserted foreign gene into an engineered bacterium for stable genetic and transcriptional expression of the target protein. The main steps include: first, obtaining the target gene from the whole genome of the microorganism by PCR, connecting the target gene fragment with the pGEM-T vector overnight with T4 ligase to complete TA cloning, heat-shock transforming the TA ligation product into the DH5α host bacteria, and performing blue-white shift screening after plate activation coating. The positive clones with correct sequencing and the pET-28a expression vector are subjected to double restriction digestion and agarose gel electrophoresis purification, and the fragments with the same sticky ends and the pET-28a expression vector are connected with T4 DNA ligase and then heat-shock transformed into the Escherichia coli BL21 (DE3) host bacteria. A certain concentration of inducer IPTG is added to allow the Escherichia coli BL21 (DE3) host bacteria to express the target protein in large quantities, and finally a recombinant target protein with a histidine tag (His-tag) is obtained. The applicant's previous application CN117384893A discloses a method for expressing and purifying N-acetylglucosamine deacetylase from Pantoea dispersa DJL-B (deposited in Guangdong Microbial Culture Collection Center in April 2022, with a deposit number of GDMCC No: 62372) by heterologous expression and affinity chromatography. At present, there are few studies on Pantoea dispersa, and there are no reports on the glucose isomerase gene (Pd-xylA) from Pantoea dispersa, and it is difficult to know its activity.

[0006] Therefore, it is of great significance to provide a glucose isomerase derived from Pantoea dispersa and a method for improving the activity of glucose isomerase. Summary of the invention

[0007] In order to solve the problems existing in the prior art, the present invention screened out the glucose isomerase gene (Pd-xylA) from the previously preserved strain Pantoea dispersa DJL-B (preserved in Guangdong Provincial Microbiological Culture Collection Center in April 2022, with the preservation number GDMCC No: 62372), and used heterologous expression of prokaryotic proteins and affinity chromatography technology to achieve soluble expression and purification of glucose isomerase in Escherichia coli BL21 (DE3), providing a new method for industrially obtaining glucose isomerase derived from Pantoea dispersa. By characterizing the glucose isomerase from Pantoea dispersa, it was unexpectedly found that the kinetic parameter Km of glucose isomerase Pd-xylA is 27.685, and its affinity for substrate glucose is higher than that of existing glucose isomerases. The conversion rate of glucose isomerization to fructose can reach about 50% after reacting for 30 hours at 50°C and containing cobalt ions, which is higher than the conversion rate of about 40% of existing glucose isomerases. In addition, the addition of a certain concentration of Tween 80 and / or Tween 20 can significantly improve the catalytic activity of glucose isomerase Pd-xylA. Based on the above findings, the present invention is completed and provides a glucose isomerase and a method for improving the activity of glucose isomerase.

[0008] The purpose of the present invention will be further illustrated by the following detailed description.

[0009] The invention provides a glucose isomerase, which is glucose isomerase Pd-xylA, and its amino acid sequence is as follows: MHAYFDQLERVGYEGANTTNALAFRHYNPQEVILGKTMAEHLRFAACYWHTFCWNGADMFGVGAFDRPWQKNGDALQLAKLKADVAFEFFYKLNVPYYCFHDVDVSPEGDSLRSYQENLAVMTDKLLEKQQETGVRLLWGTANCFTHPRFAAGAATSPDPEIFAWAASQVCSAMQATQTLGGENYVLWGGREGYETLLNTD LRQEREQIGRFMQMVVEHKHKIGFQGMLLIEPKPQEPTKHQYDFDVATVYGFLRQFGLEKEIKVNVEANHATLAGHSFHHEIATAIALGIFGSVDANRGDSQCGWDTDQFPNSVEENALVMY EILKAGGFTTGGLNFDAKVRRQSTDKYDLFYGHIGAMDTMALALKVAARMVSDGELDKRVAQRYSGWNGEFGQQILKGEFSLASLAAHAQQQQLNPQHRSGRQEQLENLVNHYLYNF (such as SEQ IDNO.1).

[0010] Preferably, the optimum reaction temperature is 48-52°C.

[0011] Preferably, its optimum pH is 8.5-9.3.

[0012] Accordingly, the heterologous expression method of glucose isomerase comprises the following steps: 1) extracting genomic DNA of Pantoea dispersa, using the forward full-length primer xylA-FF as shown in SEQ ID NO.2 and the reverse full-length primer xylA-FR as shown in SEQ ID NO.3 to PCR amplify the target Pd-xylA gene, and recovering the Pd-xylA gene fragment containing the A tail by agarose gel electrophoresis and cutting the gel, and then connecting it with the pGEM-T vector and transforming it into Escherichia coli DH5α to complete TA cloning; 2) obtaining positive clones by blue-white screening, extracting plasmids after sequencing verification, digesting them with the pGBTNH2 empty vector by NotI and EcoRI restriction endonucleases, and then connecting the Pd-xylA gene fragment with the restriction site with the linearized pGBTNH2 by T4 DNA ligase to obtain a connection product; 3) transforming the connection product into competent cells by heat shock method, and conducting an induced expression experiment, and culturing the positive clones in LB medium containing ampicillin resistance to OD 600 After the p-value is 0.6-0.8, IPTG is added and cultured in a 27-29°C incubator for 12-18h for induction; 4) the induced recombinant cells are centrifuged, the supernatant is removed, and the cells are resuspended in PBS buffer containing 0.08%-0.12% volume concentration of Triton X-100 and 0.15-0.25mM phenylmethylsulfonyl fluoride, ultrasonically disrupted, centrifuged, and the supernatant is collected and purified by metal ion chelate affinity chromatography using a Ni-NTA agarose column to collect the purified glucose isomerase.

[0013] Preferably, the dispersed Pantoea is Pantoea dispersa DJL-B, which is deposited in Guangdong Microbiological Culture Collection Center with a deposit number of GDMCC No: 62372.

[0014] Preferably, the competent cells are Escherichia coli BL21 (DE3).

[0015] In addition, the present invention also provides a method for improving the activity of glucose isomerase, comprising the following steps: adding Tween to an enzymatic reaction system containing the glucose isomerase, a substrate and metal ions; the Tween is Tween 80 and / or Tween 20, and its volume percentage content in the reaction system is 0.8%-1.2%.

[0016] Preferably, the Tween is Tween 80.

[0017] Preferably, the metal ions include cobalt ions. More preferably, the concentration of the cobalt ions is 2.5-5.5 mM.

[0018] More preferably, the metal ions include cobalt ions and magnesium ions. Glucose isomerase is a metal ion-dependent enzyme, and cobalt ions and magnesium ions play a role in promoting enzyme activity. Cobalt ions will not affect the determination of the hydrochloric acid resorcinol method.

[0019] Preferably, the temperature of the reaction system is 48-52° C., and the pH is 8.5-9.3.

[0020] Compared with the prior art, the beneficial effects of the present invention include:

[0021] (1) The present invention screened out the glucose isomerase gene (Pd-xylA) from the previously preserved strain Pantoea dispersa DJL-B, and used the heterologous expression of prokaryotic proteins and affinity chromatography technology to achieve soluble expression and purification of glucose isomerase in Escherichia coli BL21 (DE3), providing a new method for industrially obtaining glucose isomerase from Pantoea dispersa.

[0022] (2) The present invention characterizes the glucose isomerase from Pantoea dispersa by enzymatic characterization and finds that the kinetic parameter Km of the glucose isomerase Pd-xylA is 27.685. The glucose isomerase has a higher affinity for the substrate glucose than the existing glucose isomerase. The glucose isomerase can be isomerized to fructose at a conversion rate of about 50% (determined by the hydrochloric acid resorcinol method) at 50°C for 30 hours, which is higher than the conversion rate of about 40% of the existing glucose isomerase. In addition, the optimum reaction temperature of the glucose isomerase provided by the present invention is significantly lower than the optimum reaction temperature of the existing glucose isomerase (80-100°C), which is beneficial to reduce the occurrence of side reactions and is expected to play an important role in the production of high fructose corn syrup.

[0023] (3) The glucose isomerase provided by the present invention is resistant to most organic solvents. Adding a certain concentration of Tween 80 and / or Tween 20 can significantly improve the catalytic activity of glucose isomerase Pd-xylA, thereby providing a method for improving the activity of glucose isomerase. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1Heterologous expression and purification SDS-PAGE map of glucose isomerase Pd-xylA; wherein, 1-A is the SDS-PAGE map of heterologous expression of glucose isomerase Pd-xylA, and M refers to marker; 1-B is the SDS-PAGE map of purification of glucose isomerase Pd-xylA, and M refers to marker, CL refers to bacterial lysate, FT refers to column flow-through, W1 refers to washing solution without imidazole, and E1-E6 refer to elution solutions containing imidazole for 1-6 times, respectively.

[0025] Figure 2 Fructose conversion rate of glucose isomerase Pd-xylA under different conditions; 2-A is the fructose conversion rate under different enzyme addition amounts, and 2-B is the fructose conversion rate under different substrate concentrations.

[0026] Figure 3 The temperature and pH investigation results of glucose isomerase Pd-xylA; among them, 3-A is the optimal reaction temperature result diagram, 3-B is the optimal reaction pH result diagram, 3-C is the temperature stability result diagram, and 3-D is the pH stability result diagram.

[0027] Figure 4 The results of the effects of different chemical reagents on the activity of glucose isomerase Pd-xylA.

[0028] Figure 5 The results of the effects of different metal ions on the activity of glucose isomerase Pd-xylA.

[0029] Figure 6 The effect of different cobalt ion concentrations on the activity of glucose isomerase Pd-xylA.

[0030] Figure 7 The results of the effect of different cobalt salts with the same cobalt ion concentration on the activity of glucose isomerase Pd-xylA.

[0031] Figure 8 The result of the effect of the combination of cobalt ions and magnesium ions on the activity of glucose isomerase Pd-xylA.

[0032] Fig. 9 The results of multiple sequence comparison between glucose isomerase Pd-xylA and 13 existing glucose isomerases from different sources.

[0033] Fig.10 A partial schematic diagram of the docking of glucose isomerase Pd-xylA with the substrate glucose molecule; wherein 10-A is a schematic diagram of the interaction between the enzyme and the substrate, 10-B is a schematic diagram of the interaction between the enzyme and the magnesium ion, and 10-C is a schematic diagram of the interaction between the enzyme and the cobalt ion. DETAILED DESCRIPTION

[0034] The present invention is further described in detail below with reference to the accompanying drawings and embodiments.

[0035] In the present invention, the materials, reagents and culture media involved are all conventional commercial products, or can be obtained by conventional technical means in the art. For example, IPTG inducer was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. If not explicitly stated, % refers to volume percentage. The experimental data in the present invention are expressed as mean ± standard deviation (n = 3), and GraphPadPrism 9.5.0 statistical software was used for data analysis and processing; each group was evaluated by one-way analysis of variance, followed by Dunnett's multiple comparison test, and statistical significance was determined by *p<0.05, **p<0.01, ***p<0.001 or ****p<0.0001.

[0036] Example 1 Heterologous Expression and Purification of Pd-xylA Enzyme from Pantoea dispersa

[0037] The Pantoea dispersa DJL-B screened in the previous study was activated overnight and the whole genome was extracted using a kit. The target Pd-xylA gene was amplified by PCR using the Pd-xylA specific forward full-length primer xylA-FF (CGGAATTCATGCACGCTTATTTCG ACCAGC, as shown in SEQ ID NO.2) and the reverse full-length primer xylA-FR (ATAAGAATGCGGCCGCTTAGA AATTATACAGATAGTGATTCAC, as shown in SEQ ID NO.3). The Pd-xylA gene with an A tail was purified by agarose gel electrophoresis, the Pd-xylA fragment was recovered by gel excision, and the fragment was connected to the pGEM-T vector and transformed into E coli DH5α to complete TA cloning. The white positive clone colonies were screened, and after sequencing verification, the extracted plasmids were digested with NotI and EcoRI restriction endonucleases for 3.5 h with the pGBTNH2 empty vector, and the Pd-xylA gene fragment with restriction sites was connected with the linearized pGBTNH2 by T4 DNA ligase at 16°C overnight to obtain the connection product; the connection product was transformed into E coli BL21 (DE3) by heat shock method, and the induced expression experiment was carried out. The positive clones were cultured in LB medium containing ampicillin resistance until OD 600After the concentration of 0.6, IPTG with a final concentration of 0.1mM was added and induced overnight at 28°C. The induced recombinant cells were centrifuged at 6000rpm for 10min to remove the supernatant culture medium components, and PBS buffer containing 0.1% volume concentration of Triton X-100 and 0.2mM phenylmethylsulfonyl fluoride was added to the bacterial precipitate, and the cells were crushed in an ultrasonic cell crusher for 20min (crushing conditions: 250W, working 2s, interval 6s, temperature 28°C). The crushed cells were centrifuged at 10000rpm, and the precipitate was the inclusion body part. The supernatant was the soluble recombinant protein Pd-xylA. The expression of the recombinant protein was detected by polyacrylamide gel electrophoresis (SDS-PAGE). The supernatant after crushing (containing the soluble protein His-tag protein) was purified by metal ion chelation affinity chromatography using a Ni-NTA agarose column to collect the purified glucose isomerase.

[0038] Heterologous expression and purification of glucose isomerase Pd-xylA SDS-PAGE pattern Figure 1 As shown. Figure 1 -A, Pd-xylA enzyme (GenBank: WP_222951480.1) is expressed in a soluble form, and SDS-PAGE shows that the molecular weight is around 49 kDa, which is consistent with the theoretical molecular weight of Pd-xylA enzyme 49 kDa. The target protein can be successfully eluted by imidazole by purifying the His-tagged Pd-xylA recombinant protein using metal ion chelate affinity chromatography, as shown in Figure 1. Figure 1 -B.

[0039] Example 2 Activity determination and enzymatic property characterization of glucose isomerase Pd-xylA

[0040] 10mg / ml glucose was dissolved in 49μL of glycine sodium hydroxide buffer solution, 50μL of enzyme solution (the supernatant after the disruption in Example 1, crude enzyme), 0.5MCo 2+ 1μL, react at 50℃ for 60min, and terminate the reaction in an ice water bath. Determination of enzyme activity: Add hydrochloric acid resorcinol colorimetric solution to the reaction solution, mix well, color at 100℃ for 5min, terminate the reaction in an ice water bath, and measure the absorbance at 480nm. The fructose content was calculated based on the fructose standard curve. Definition of enzyme activity: Under the standard conditions for enzyme activity determination, the amount of glucose isomerase required to produce 1μmol D-fructose per minute is defined as one enzyme activity unit, expressed as U. The effect of different enzyme solution addition amounts on the fructose conversion rate in the reaction system is shown in the following table. Figure 2 -A, the fructose conversion rate was the highest when 2 mL of enzyme solution was added to the reverse system; the effect of different substrate concentrations on the fructose conversion rate is as follows Figure 2 -B shows that Pd-xylA has the highest conversion rate to 0.02 M glucose.

[0041] The enzyme activity of Pd-xylA at different temperatures was measured. In a standard reaction system (50 μL 0.04 mM glucose dissolved in sodium dihydrogen phosphate citrate solution at pH 7, 48 μL enzyme solution, 1 μL 500 mM cobalt sulfate, 1 μL 500 mM magnesium sulfate), Pd-xylA was reacted at 40-100°C for 1 hour, cooled in an ice water bath, and the amount of fructose produced was measured. The highest enzyme activity was taken as 100%, and the relative enzyme activity was calculated to determine the optimal reaction temperature. The results are shown in Figure 2. Figure 3 -A, it can be seen that 50 degrees is the optimal reaction temperature of the enzyme. When the reaction temperature is 100 degrees, the enzyme is inactivated. Pd-xylA is a mesophilic enzyme.

[0042] At the optimum reaction temperature, the enzyme activity of Pd-xylA enzyme in buffers with different pH values ​​was measured. Under the standard reaction system, Pd-xylA enzyme was reacted in buffers with pH values ​​of 5.0-12.0 for 1 hour, and the reaction was terminated in an ice-water bath. The amount of fructose produced was measured, and the relative enzyme activity was calculated with the highest enzyme activity as 100% to determine the optimum reaction pH. The results are shown in the figure. Figure 3 -B, it can be seen that pH 9 is the optimal pH for Pd-xylA enzyme. The enzyme activity of Pd-xylA enzyme is very low under acidic conditions, indicating that Pd-xylA is not acid-resistant and is inactivated as the pH increases to 11 under alkaline conditions.

[0043] After the Pd-xylA enzyme was incubated at 30, 40, 50, and 60°C for 30 and 60 min, the residual enzyme activity of the standard reaction system was measured at the optimal reaction temperature and optimal pH. The initial enzyme activity was taken as 100%, and the relative enzyme activity was calculated to determine the effect of temperature on enzyme stability. The results are shown in Figure 2. Figure 3 As shown in Figure 3-C, it can be seen that after incubation at 30°C and 40°C (without adding cobalt ions) for 1 hour, the relative enzyme activity of Pd-xylA enzyme decreased to about 80%, and after incubation at 50°C for 1 hour, the enzyme activity decreased to about 30%.

[0044] After the Pd-xylA enzyme diluted with pH 6.0-10.0 buffer was incubated at 37°C for 1 hour, the residual enzyme activity of the standard reaction system was measured at the optimal reaction temperature and optimal pH. The initial enzyme activity was taken as 100% and the relative enzyme activity was calculated. The results are as follows: Figure 3 -D, it can be seen that the enzyme activity of Pd-xylA can be maintained at about 80% after incubation in pH 8 buffer for 1 hour, and the enzyme activity drops to about 30% after incubation in pH 10 buffer for 1 hour, indicating that Pd-xylA is relatively stable at around pH 8.

[0045] Example 3 Effects of different chemical reagents on the activity of glucose isomerase Pd-xylA

[0046] Under standard conditions for enzyme activity determination, 5 mM urea, 5 mMSDS, 50 mM imidazole, 1% volume percentage of β-mercaptoethanol, dimethyl sulfoxide, ethanol, methanol, acetonitrile, glycerol, Tween 20, Tween 60, Tween 80, and Triton X-100 were added to the reaction system (50 μL 0.04 mM glucose dissolved in sodium dihydrogen phosphate citrate solution at pH 7, 48 μL enzyme solution, 1 μL 500 mM cobalt sulfate, and 1 μL 500 mM magnesium sulfate) respectively at a final concentration, and the enzyme activity was determined after reacting at 50°C for 1 hour. The enzyme activity of the group without adding chemical reagents was taken as 100%. The results are as follows: Figure 4 shown.

[0047] The present invention uses 13 kinds of chemical reagents to study the influence of different chemical reagents on Pd-xylA enzyme, including common detergents, organic solvents, surfactants, etc.; urea, SDS, and β-mercaptoethanol have certain inhibition on enzyme activity, and β-mercaptoethanol completely loses enzyme activity. β-mercaptoethanol (β-ME) and urea (Urea) are respectively a reducing agent and a denaturing agent; urea may dissolve part of Pd-xylA enzyme protein, and its three-dimensional structure is destroyed; and imidazole also has certain inhibition on enzyme activity, so it is necessary to dialyze the enzyme solution after purification by nickel column to remove imidazole; most common organic solvents such as ethanol, methanol, dimethyl sulfoxide, and acetonitrile have slight inhibition on enzyme activity, and glycerol has the greatest inhibition on enzyme activity; most surfactants have little effect on enzyme activity, but some reagents can promote enzyme activity, among which Tween 80 has the greatest effect on promoting enzyme activity, increasing the relative enzyme activity to about 140%, and Tween 20 can increase the relative enzyme activity to about 120%, indicating that Pd-xylA has certain tolerance to surfactants.

[0048] Example 4 Effect of different metal ions on the activity of glucose isomerase Pd-xylA

[0049] Under the standard conditions for enzyme activity determination, metal ions (K + 、Na + Mg 2+ , Mn 2+ , Cu 2+ , Ca 2+ , Ba 2+ , Fe 2+ 、Co 2+ 、Zn 2+ , EDTA), react at the optimal reaction temperature and optimal reaction pH for 1 hour, and measure the enzyme activity. The enzyme activity without adding metal ions is taken as 100%. The results are as follows: Figure 5 shown.

[0050] from Figure 5It can be seen that 5mM cobalt ions have a great activating effect on enzyme activity. As the cobalt ion concentration increases to 10mM, the enzyme activity decreases slightly, indicating that after exceeding a certain range, the increase in cobalt ion concentration becomes a burden on the enzyme; secondly, the promoting effect of magnesium ions on enzyme activity is slightly lower than that of cobalt ions. 5mM manganese ions can also promote enzyme activity. When the manganese ion concentration increases to 10mM, the enzyme activity decreases slightly; different iron ions can also have a slight promoting effect on enzyme activity, and different concentrations of calcium ions, zinc ions and barium ions have a significant inhibitory effect on enzyme activity. In addition, EDTA has a significant inhibitory effect on enzyme activity, and with the increase of EDTA concentration, the enzyme activity is almost completely inhibited, indicating that Pd-xylA enzyme is a metal-dependent enzyme.

[0051] Furthermore, the present invention investigated the effect of cobalt ion concentration on enzyme activity: under standard conditions for enzyme activity determination, cobalt ions at final concentrations of 0.5 mM, 1 mM, 2.5 mM, 5 mM, and 10 mM were added to the reaction system, reacted at the optimal reaction temperature and optimal pH for 1 hour, and the enzyme activity was determined, with the enzyme activity of the group without adding cobalt ions as 100%. The results are as follows: Figure 6 As shown. In the range of 0.5-2.5mM, the enzyme activity increased with the increase of cobalt ion concentration; when the cobalt ion concentration increased to 5mM, the relative enzyme activity increased slightly compared with that at 2.5mM. Figure 6 The ns index difference was not statistically significant; however, when the cobalt ion concentration reached 10 mM, the enzyme activity was greatly reduced, which may be due to the fact that the cobalt ions burdened the active sites of the enzyme, thereby inhibiting the enzyme activity. 5 mM cobalt ions had the best effect on promoting enzyme activity.

[0052] Furthermore, the present invention investigates the effect of different cobalt salts with the same cobalt ion concentration on enzyme activity: under standard conditions for enzyme activity determination, cobalt sulfate heptahydrate, cobalt chloride hexahydrate, and cobalt acetate with a final concentration of 5 mM are added to the reaction system, and the reaction is carried out at the optimal reaction temperature and the optimal reaction pH for 1 hour, and the enzyme activity is determined, and the enzyme activity of the group without adding cobalt salt is taken as 100%. The results are as follows: Figure 7 As shown, different cobalt salts with the same concentration of cobalt ions have little effect on enzyme activity and can all promote enzyme activity, while cobalt acetate has a relatively better effect on promoting enzyme activity.

[0053] Furthermore, the present invention investigated the effects of different metal ion combinations on enzyme activity: under standard conditions for enzyme activity determination, a final concentration of 5 mM and 2 mM were added to the reaction system. 2+ , 5mMCo 2+ With 1mMMg 2+ , 5mMCo 2+ With 5mMMg 2+ , 5mMCo 2+ With 10mMMg 2+The reaction was carried out at the optimum reaction temperature and the optimum reaction pH for 1 h, and the enzyme activity was determined, with the enzyme activity of the group without adding any metal ions as 100%. Figure 8 As shown. It can be seen that the combination of magnesium ions and cobalt ions at different concentrations has different effects on enzyme activity. The combination of magnesium ions and cobalt ions is better than adding cobalt ions alone. Among them, 5mM magnesium ions and 5mM cobalt ions have the best effect on promoting enzyme activity, indicating that glucose isomerase may have two metal ion binding sites M1 and M2. It is speculated that the cobalt ion bound to M1 is a structural ion. The synergistic use of magnesium ions and cobalt ions can also promote enzyme activity. It is speculated that the magnesium ion bound to M2 is a catalytic ion.

[0054] Example 5 Comparative analysis of sequences of glucose isomerases from different microorganisms

[0055] Thirteen different sources of glucose isomerase have been confirmed to have glucose isomerase activity, namely, from Thermoanaerobacter ethanolicus (GenBank: AKM94132.1), Thermoanaerobacteriumthermosulfuri genes (GenBank: 1A0C), Thermotogota (GenBank: WP_015919215.1), Actinoplanes missouriensis (GenBank: WP_014441152.1), Actinoplanes sp.ATCC31351 (GenBank: AAA92578.1), Streptomyces rubiginosus (GenBank: AAA26838.1), Streptomyces violaceusniger (GenBank: AAA26839.1), Bacillus subtilis (GenBank: AOR98272.1), Levilactobacillus brevis (GenBank: ARW51820.1), Lactiplantibacillus pentosus (GenBank: WP_279490182.1), Thermoanaerobacterium thermosulfuri genes (GenBank: AAA23285.1), Staphylococcus xylosus (GenBank: GEQ10674.1), Escherichiacoli (GenBank: WP_336480169.1). The glucose isomerase Pd-xylA provided by the present invention was compared with the existing glucose isomerases from 13 different sources. The results are as follows Fig. 9 It can be seen that the amino acid sequence of the glucose isomerase Pd-xylA provided by the present invention is significantly different from that of the existing glucose isomerase.

[0056] Example 6 Investigation of the interaction between glucose isomerase Pd-xylA and ligands

[0057] Discover studio software was used to dock the Pd-xylA enzyme with the substrate glucose molecule. The local structure is shown in Fig.10 Combined with the results of multiple sequence alignment analysis Fig. 9 , W49, E232, and E268 form hydrogen bonds with the substrate glucose, and K234 forms a salt bridge with the substrate, e.g. Fig.10 -A; The metal ion Mg was found by docking2+ It forms coordination bonds with amino acids E232, E268, D296, and D339, such as Fig.10 -B; and the metal ion Co 2+ It forms coordination bonds with H271 and D307, such as Fig.10 -C, which is consistent with the speculation in Example 4.

[0058] The above contents are further detailed descriptions of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as falling within the protection scope of the present invention.

Claims

1. A glucose isomerase, characterized in that: It is glucose isomerase Pd-xylA, and its amino acid sequence is shown in SEQ ID NO.

1.

2. The glucose isomerase according to claim 1, characterized in that: The optimum reaction temperature is 48-52°C.

3. Glucose isomerase according to claim 1, characterized in that: Its optimum pH is 8.5-9.

3.

4. The method for heterologous expression of glucose isomerase according to claim 1, characterized in that: The method comprises the following steps: 1) extracting genomic DNA of Pantoea dispersa, using a forward full-length primer xylA-FF as shown in SEQ ID NO.2 and a reverse full-length primer xylA-FR as shown in SEQ ID NO.3 to perform PCR amplification of the target Pd-xylA gene, and recovering the Pd-xylA gene fragment containing the A tail by gel excision through agarose gel electrophoresis, and then connecting the fragment with a pGEM-T vector and transforming it into Escherichia coli DH5α to complete TA cloning; 2) Positive clones were obtained by blue-white screening, and plasmids were extracted after sequencing verification. The pGBTNH2 empty vector was digested with NotI and EcoRI restriction endonucleases, and then the Pd-xylA gene fragment with restriction sites was connected to the linearized pGBTNH2 by T4 DNA ligase to obtain a ligation product; 3) The ligation product was transformed into competent cells by heat shock method to perform an induced expression experiment, and the positive clones were cultured in LB medium containing ampicillin resistance until OD 600 After the p-value is 0.6-0.8, IPTG is added and cultured in a 27-29°C incubator for 12-18h for induction; 4) the induced recombinant cells are centrifuged, the supernatant is removed, and the cells are resuspended in PBS buffer containing 0.08%-0.12% volume concentration of Triton X-100 and 0.15-0.25mM phenylmethylsulfonyl fluoride, ultrasonically disrupted, centrifuged, and the supernatant is collected and purified by metal ion chelate affinity chromatography using a Ni-NTA agarose column to collect the purified glucose isomerase.

5. The method for heterologous expression of glucose isomerase according to claim 4, characterized in that: The dispersed Pantoea is Pantoea dispersa DJL-B, which is preserved in Guangdong Microbial Culture Collection Center with a preservation number of GDMCC No: 62372.

6. The method for heterologous expression of glucose isomerase according to claim 4, characterized in that: The competent cells used were Escherichia coli BL21 (DE3).

7. A method for improving glucose isomerase activity, characterized in that: The method comprises the following steps: adding Tween into an enzymatic reaction system containing the glucose isomerase according to any one of claims 1 to 6, a substrate and metal ions; the Tween is Tween 80 and / or Tween 20, and its volume percentage in the reaction system is 0.8%-1.2%.

8. The method for improving glucose isomerase activity according to claim 7, characterized in that: The Tween is Tween 80.

9. The method for improving glucose isomerase activity according to claim 7, characterized in that: The metal ions include cobalt ions.

10. The method for improving glucose isomerase activity according to claim 7, characterized in that: The temperature of the reaction system is 48-52° C., and the pH is 8.5-9.3.

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