An inositol oxidase mutant and its application

Abstract

The present invention discloses an inositol oxidase mutant and its application, belonging to the technical field of genetic engineering. The inositol oxidase mutant is obtained by mutating the 113th amino acid of the amino acid sequence shown in SEQ ID NO.1, and the mutation is: the 113th amino acid is mutated from T to A. The inositol oxidase mutant of the present invention has significantly improved enzyme activity, good thermal stability and long half-life, and can improve the yield and conversion rate of glucuronic acid.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and particularly relates to an inositol oxidase mutant and its application. Background Art

[0002] Glucuronic acid is a uronic acid formed by oxidizing the C-6 hydroxyl group of glucose to a carboxyl group, with the chemical formula C6H 10 O7. Glucuronic acid is widely present in animals and plants, especially in certain fruits and vegetables. Glucuronic acid is generated through the glucose metabolic pathway in the body. D-glucose is first converted to α-D-glucose-1-phosphate, and then through a series of enzymatic reactions to generate UDP-glucuronic acid (UDPGA), which finally participates in the detoxification reaction. Glucuronic acid is not only a traditional liver detoxifying agent and immune function regulator, but also an important intermediate for synthesizing many pharmaceuticals, and is commonly used as an additive in functional beverages, weight loss drugs, cosmetics, etc.

[0003] Currently, the production of glucuronic acid using the biocatalytic method generally uses inositol as a substrate, and glucuronic acid is generated through the catalysis of inositol oxidase. However, due to the need for a long reaction time during industrial production, and the low catalytic activity of inositol oxidase towards inositol, as well as the poor thermal stability and short half-life of this enzyme, it limits the practical application of producing glucuronic acid using the biocatalytic method. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide an inositol oxidase mutant and its application, which are used to overcome the problems of low catalytic activity of inositol oxidase towards inositol, poor thermal stability, and short half-life in the prior art.

[0005] In a first aspect, the present invention provides an inositol oxidase mutant, which is obtained by mutating the 113th amino acid of the amino acid sequence shown in SEQ ID NO.1, and the mutation is: the 113th amino acid is mutated from T to A.

[0006] Compared with the prior art, the enzyme activity of the inositol oxidase mutant provided by the present invention is significantly improved, and it has good thermal stability and a long half-life.

[0007] Furthermore, the amino acid sequence of the inositol oxidase mutant is as shown in SEQ ID NO.2.

[0008] Preferably, the gene sequence of the inositol oxidase mutant is as shown in SEQ ID NO.4.

[0009] In a second aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned inositol oxidase mutant.

[0010] In a third aspect, the present invention provides a recombinant vector or recombinant strain containing the above nucleic acid molecule.

[0011] Fourthly, the present invention provides a method for preparing an inositol oxidase mutant for preparing the above-mentioned inositol oxidase mutant, comprising the following steps:

[0012] Culturing the above-mentioned recombinant strain in LB medium for seed culture, and obtaining a seed solution after culturing for 12 - 16 h;

[0013] Inoculating the seed solution into another LB medium for fermentation culture, and after culturing until the OD 600 value is 0.6 - 0.8, cooling down and adding IPTG for induction culture to induce the recombinant strain to express the inositol oxidase mutant, and obtaining a fermentation broth containing the inositol oxidase mutant.

[0014] Further, the temperature of the seed culture is 35 - 40 °C; and / or, the temperature of the fermentation culture is 35 - 40 °C.

[0015] Further, cooling down means cooling down to 16 - 20 °C.

[0016] Fifthly, the present invention provides the application of the above-mentioned inositol oxidase mutant in the production of glucuronic acid.

[0017] Compared with the prior art, using the inositol oxidase mutant of the present invention to produce glucuronic acid can improve the yield and conversion rate of glucuronic acid.

[0018] Further, the inositol oxidase mutant catalyzes the synthesis of glucuronic acid using inositol as a substrate.

[0019] Further, the reaction temperature for the catalytic synthesis is 35 - 40 °C, and the pH value is 7.5 - 8.5. Description of the Drawings

[0020] Figure 1 Thermal stability and half-life of inositol oxidase at 30 °C in Example 5.

[0021] Figure 2 Thermal stability and half-life of inositol oxidase at 35 °C in Example 5.

[0022] Figure 3 Thermal stability and half-life of inositol oxidase at 40 °C in Example 5. Detailed Embodiments

[0023] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0024] It should be understood that the raw materials used in the following examples are all commercially available raw materials unless otherwise specified. Example 1

[0025] Construct a mutant plasmid containing the inositol oxidase mutant gene, and the specific method is as follows:

[0026] S1. Using the recombinant plasmid B: pBAD / HisB-MIOX (disclosed in Patent CN 109423469 A) as a template, perform inverse PCR on the recombinant plasmid B pBAD / HisB-MIOX with a primer pair. The reaction system and reaction conditions of the inverse PCR are shown in Table 1 and Table 2. The recombinant plasmid B contains the gene of inositol oxidase B, and the gene sequence of the inositol oxidase B is shown in SEQ ID NO.3.

[0027] Forward amplification primer:

[0028] 5'-AAACAGGCCGTGGCGCATAACCTGGCTGCCCG-3' (SEQ ID NO.5).

[0029] Reverse amplification primer:

[0030] 5'-TGCGCCACGGCCTGTTTGGCGTGCTGCTCCGC-3' (SEQ ID NO.6).

[0031] Table 1 Inverse PCR reaction system (20 μL)

[0032]

[0033] Table 2 Inverse PCR reaction conditions

[0034]

[0035] S2. Eliminate the template: After the above inverse PCR reaction is completed, obtain the reaction solution. Pipette 2 μL of the restriction endonuclease DpnⅠ into the reaction solution, gently pipette the reaction solution with a pipette to fully mix the enzyme and the reaction solution, then place it at 37 °C for 1 h to obtain the digestion solution. Verify the digestion solution using agarose gel electrophoresis.

[0036] S3. Self-cyclization of the PCR product. Prepare the reaction solution according to Table 3, gently mix it evenly, and place it at 16 °C for 1 hour to obtain a mutant plasmid containing the inositol oxidase mutant gene.

[0037] Table 3 Product cyclization system (15 μL)

[0038]

[0039] S4. Verification of the mutant plasmid: The reaction product of step S3 above was transformed into E. coli DH5α competent cells by the chemical transformation method. The specific method was as follows: Take the reaction product of step S3 above and add it to E. coli DH5α competent cells. Place the mixed reaction system on ice for 30 min, then heat shock it in a 42°C water bath for 60 s, and then incubate it on ice for 5 min. Transfer it to 500 μL of LB liquid medium and shake it at 37°C for 1 h to recover. Take 100 μL of the bacterial solution and spread it on a plate.

[0040] Pick monoclonal colonies on the plate for plasmid extraction and DNA sequencing. The successfully mutated plasmid was named M. Example 2

[0041] Construction and induced expression of the mutant strain:

[0042] Take 1 μL of the correctly sequenced mutant plasmid M in Example 1 and add it to E. coli BL21 competent cells. Place the mixed reaction system on ice for 30 min, then heat shock it in a 42°C water bath for 60 s, and then incubate it on ice for 5 min. Transfer it to 500 μL of LB liquid medium and shake it at 37°C for 1 h to recover. Take 100 μL of the bacterial solution and spread it on a plate.

[0043] Screen out the positive transformants containing the mutant plasmid M (i.e., the mutant strain), and perform seed culture in liquid LB medium. Culture it at 37°C and 120 rpm for 14 h to obtain the seed solution. Inoculate the seed solution into another LB medium for fermentation culture at an inoculation amount of 2% by volume. Culture it at 37°C until the OD 600 value reaches 0.7, then cool it down to 18°C, add IPTG with a final concentration of 1.0 mM for induced culture. After 17 h of induced culture, a fermentation broth containing the inositol oxidase mutant was obtained.

[0044] Compared with the inositol oxidase B (SEQ ID NO.1) encoded in the recombinant plasmid B, the inositol oxidase mutant in the present invention has a mutation at the 113th amino acid of the amino acid sequence shown in SEQ ID NO.1, from T (threonine) to A (alanine). Therefore, this inositol oxidase mutant was named T113A. Example 3

[0045] Isolation and purification of the inositol oxidase mutant:

[0046] Take the fermentation broth in Example 2 and centrifuge it at 4°C and 8000 rpm for 15 min. Discard the supernatant, collect the mutant strains in the fermentation broth, resuspend the mutant strains in a phosphate buffer with a pH of 8.0 and a concentration of 0.2 mM, then use a high-pressure homogenizer to break the bacterial cells. Centrifuge at 4°C and 12000 r / min for 1 h, discard the cell debris precipitate, and the collected supernatant is the crude enzyme solution of inositol oxidase mutant T113A. After performing Ni affinity chromatography on this crude enzyme solution, the eluted protein is replaced into a phosphate buffer with a pH of 8.0 and a concentration of 0.2 mM using an ultrafiltration tube to remove imidazole and other metal ions in the eluent, and obtain the enzyme solution of inositol oxidase mutant T113A. Example 4

[0047] Relative enzyme activity determination:

[0048] Enzyme activity definition (U): Under standard reaction conditions, the amount of enzyme required to catalyze the synthesis of 1 μmol of glucuronic acid per unit time (min).

[0049] Enzyme activity determination method: In the standard reaction system, react at 37°C for 30 min, and terminate the reaction by boiling for 10 min.

[0050] Standard reaction system: 50 mM Tris-HCl buffer (pH value 8.0), 2 mM L-cysteine, 1 mM ferrous sulfate (providing Fe 2+ ), 20 mM inositol, 100 μg / mL inositol oxidase enzyme solution.

[0051] After centrifuging the reaction product at 12000 rmp for 10 min, collect the supernatant and filter it through a 0.22 μm microporous filter membrane. Dilute the filtrate 10 times and perform HPLC detection.

[0052] HPLC detection method: Chromatograph: Agilent1260; Detector: RID; Chromatographic column Aminex HPX-87H (300×7.8 mm); Column temperature 30°C; Mobile phase: Sulfuric acid aqueous solution (5 mM); Flow rate 0.5 mL / min.

[0053] Taking the enzyme activity of inositol oxidase B encoded in recombinant plasmid B as 100%, the detection results are shown in Table 4.

[0054] Table 4

[0055]

[0056] As can be seen from Table 1: The enzyme activity of inositol oxidase mutant T113A in the present invention is significantly improved. Example 5

[0057] Thermal stability and half-life determination:

[0058] The myo-inositol oxidase enzyme solution was incubated statically at 30 °C, 35 °C, and 40 °C respectively. Taking the enzyme activity at 0 h as 100%, after static incubation for 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h respectively, samples were taken to detect the enzyme activity, and the thermal stability and half-life of myo-inositol oxidase at these three temperatures were obtained. The detection results are as Figure 1 , Figure 2 , Figure 3 shown.

[0059] It can be seen from Figure 1 that: at 30 °C, the thermal stability of the myo-inositol oxidase mutant T113A is better than that of myo-inositol oxidase B. Moreover, at 30 °C, compared with the half-life of 5 h of myo-inositol oxidase B, the half-life of the myo-inositol oxidase mutant T113A is increased to 10 h.

[0060] It can be seen from Figure 2 that: at 35 °C, the thermal stability of the myo-inositol oxidase mutant T113A is better than that of myo-inositol oxidase B. Moreover, at 35 °C, compared with the half-life of 4 h of myo-inositol oxidase B, the half-life of the myo-inositol oxidase mutant T113A is increased to 8.4 h.

[0061] It can be seen from Figure 3 that: at 40 °C, the thermal stability of the myo-inositol oxidase mutant T113A is better than that of myo-inositol oxidase B. Moreover, at 40 °C, compared with the half-life of 4 h of myo-inositol oxidase B, the half-life of the myo-inositol oxidase mutant T113A is increased to 7 h. Example 6

[0062] Preparation of glucuronic acid:

[0063] The recombinant plasmid B was introduced into Escherichia coli BL21 according to the method of Example 2 to obtain the recombinant strain B, and induced culture was carried out according to the method of Example 2 to obtain the fermentation broth B.

[0064] The fermentation broth in Example 2 and the above fermentation broth B were taken respectively, centrifuged at 4 °C and 8000 rpm for 15 min, the supernatant was discarded, and the wet cell precipitates were collected respectively.

[0065] Using 50 g / L of myo-inositol as the substrate in 100 mL of 0.2 mM phosphate buffer (pH value 8), 25 g of wet cells were added, and the reaction was carried out at 37 °C for 12 h. After the reaction, HPLC was used to detect the concentration and conversion rate of glucuronic acid in the reaction system. The detection results are shown in Table 5.

[0066] Table 5

[0067]

[0068] As can be seen from Table 2, compared with inositol oxidase B, the inositol oxidase mutant T113A in the present invention increases the yield of glucuronic acid and increases the conversion rate of glucuronic acid from 91.3% to 96.0%.

[0069] The foregoing are only the preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or perform equivalent replacements on some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An inositol oxidase mutant, characterized in that, The inositol oxidase mutant is obtained by mutating the 113th amino acid of the amino acid sequence shown in SEQ ID NO.1, and the mutation is that the 113th amino acid is mutated from T to A; The amino acid sequence of the inositol oxidase mutant is shown in SEQ ID NO.2; the gene sequence of the inositol oxidase mutant is shown in SEQ ID NO.

4.

2. A nucleic acid molecule encoding the inositol oxidase mutant according to claim 1.

3. A recombinant vector or recombinant strain containing the nucleic acid molecule according to claim 2.

4. A method for preparing an inositol oxidase mutant, which is used to prepare the inositol oxidase mutant described in claim 1, and is characterized in that, Comprising the following steps: Carrying out seed culture on the recombinant strain described in claim 3 in LB medium, and obtaining a seed solution after culturing for 12-16 h; Inoculating the seed solution into another LB medium for fermentation culture, after culturing until the OD600 value is 0.6-0.8, cooling down and adding IPTG for induction culture to induce the recombinant strain to express the inositol oxidase mutant, and obtaining a fermentation broth containing the inositol oxidase mutant.

5. The preparation method according to claim 4, characterized in that, The temperature of the seed culture is 35-40 °C; and / or, the temperature of the fermentation culture is 35-40 °C.

6. The preparation method according to claim 4, characterized in that The cooling down is to cool down to 16-20 °C.

7. Use of the inositol oxidase mutant according to claim 1 in the production of glucuronic acid.

8. The application according to claim 7, wherein The inositol oxidase mutant catalyzes the synthesis of glucuronic acid using inositol as a substrate.

9. The application according to claim 8, wherein The reaction temperature for the catalytic synthesis is 35-40 °C, and the pH value is 7.5-8.5.

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