Inositol oxidase mutant and application thereof

Abstract

The invention discloses an inositol oxidase mutant and application thereof, and belongs to the technical field of gene engineering. The inositol oxidase mutant is obtained by mutating 113th amino acid of an amino acid sequence shown as SEQ ID NO.1, and the mutation is that the 113th amino acid is mutated from T to A. The enzyme activity of the inositol oxidase mutant is remarkably improved, the thermal stability is good, the half-life period is long, and the yield and conversion rate of glucuronic acid can be improved.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to an inositol oxidase mutant and application thereof. Background Art

[0002] Glucuronic acid is a uronic acid formed by oxidation of the C-6 hydroxyl group of glucose to a carboxyl group. Its chemical formula is C6H 10 O7. Glucuronic acid is widely present in animals and plants, especially in some fruits and vegetables. Glucuronic acid is produced in the body through the glucose metabolism pathway. D-glucose is first converted into 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 detoxifier and immune function regulator, but also an important intermediate for the synthesis of many medicines. It is often used as an additive for functional beverages, diet pills, cosmetics, etc.

[0003] At present, the production of glucuronic acid by bioenzymatic method generally uses inositol as substrate and generates glucuronic acid by catalysis of inositol oxidase. However, since it takes a long time to react in industrial production, and inositol oxidase has low catalytic activity on inositol and poor thermal stability and short half-life, the practical application of bioenzymatic method for producing glucuronic acid is limited. Summary of the invention

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

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

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

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

[0008] Preferably, the gene sequence of the inositol oxidase mutant is 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 a recombinant strain containing the above nucleic acid molecule.

[0011] In a fourth aspect, the present invention provides a method for preparing an inositol oxidase mutant, which is used to prepare the above-mentioned inositol oxidase mutant, comprising the following steps: The recombinant strain was cultured in LB medium to obtain seed solution after culturing for 12-16 hours; The seed solution was inoculated into another LB medium for fermentation and cultured until OD 600 After the pH value reaches 0.6-0.8, the temperature is lowered and IPTG is added for induction culture to induce the recombinant strain to express the inositol oxidase mutant, thereby obtaining a fermentation broth containing the inositol oxidase mutant.

[0012] Furthermore, the temperature of the seed culture is 35-40°C; and / or the temperature of the fermentation culture is 35-40°C.

[0013] Furthermore, the temperature is lowered to 16-20°C.

[0014] In a fifth aspect, the present invention provides use of the above-mentioned inositol oxidase mutant in producing glucuronic acid.

[0015] Compared with the prior art, the production of glucuronic acid using the inositol oxidase mutant of the present invention can increase the yield and conversion rate of glucuronic acid.

[0016] Furthermore, the inositol oxidase mutant catalyzes the synthesis of glucuronic acid using inositol as a substrate.

[0017] Furthermore, the reaction temperature of the catalytic synthesis is 35-40° C., and the pH value is 7.5-8.5. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The thermal stability and half-life of the inositol oxidase in Example 5 at 30°C.

[0019] Figure 2 The thermal stability and half-life of the inositol oxidase in Example 5 at 35°C.

[0020] Figure 3 The thermal stability and half-life of the inositol oxidase in Example 5 at 40°C. DETAILED DESCRIPTION

[0021] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with 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.

[0022] It should be understood that the raw materials used in the following examples are all commercially available raw materials unless otherwise specified. Example 1

[0023] The mutant plasmid containing the inositol oxidase mutant gene was constructed as follows: S1. Using recombinant plasmid B: pBAD / HisB-MIOX (disclosed in patent CN 109423469 A) as a template, the recombinant plasmid B pBAD / HisB-MIOX was subjected to inverse PCR using primers. The reaction system and reaction conditions of 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.

[0024] Forward amplification primer: 5'-AAACAGGCCGTGGCGCATAACCTGGCTGCCCG-3' (SEQ ID NO. 5).

[0025] Reverse amplification primer: 5'-TGCGCCACGGCCTGTTTGGCGTGCTGCTCCGC-3' (SEQ ID NO. 6).

[0026] Table 1 Reverse PCR reaction system (20 μL)

[0027] Table 2 Reaction conditions of inverse PCR

[0028] S2. Eliminate the template: After the above reverse PCR reaction is completed, obtain the reaction solution, draw 2 μL of restriction endonuclease DpnⅠ and add it to the reaction solution, use a pipette to gently blow the reaction solution to fully mix the enzyme and the reaction solution, place it at 37°C for 1 h to obtain a digestion solution. Use agarose gel electrophoresis to verify the digestion solution.

[0029] S3. Self-cyclization of PCR products: Prepare the reaction solution according to Table 3, mix gently, and react at 16° C. for 1 hour to obtain a mutant plasmid containing the mutant gene of inositol oxidase.

[0030] Table 3 Product cyclization system (15 μL)

[0031] S4. Mutant plasmid verification: The reaction product of step S3 above was transformed into E. coli DH5α competent cells by chemical transformation. The specific method is as follows: the reaction product of step S3 above was added to E. coli DH5α competent cells, the mixed reaction system was placed on ice for 30 min, heat-shocked in a 42°C water bath for 60 s, and then incubated on ice for 5 min, transferred to 500 μL of LB liquid culture medium, shaken and revived at 37°C for 1 h, and 100 μL of bacterial solution was plated.

[0032] Single clone colonies on the plate were picked for plasmid extraction and DNA sequencing, and the plasmid with successful mutation was named M. Example 2

[0033] Construction of mutant strains and induced expression: Take 1 μL of the mutant plasmid M sequenced correctly in Example 1 and add it to the competent cells of E. coli BL21. Place the mixed reaction system on ice for 30 min, 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 culture medium and shake it at 37°C for 1 h, and take 100 μL of the bacterial solution to plate.

[0034] The positive transformants (i.e. mutant strains) containing mutant plasmid M were screened out and seed cultured in liquid LB medium at 37°C and 120 rpm for 14 h to obtain seed solution. The seed solution was inoculated with another LB medium at a volume ratio of 2% for fermentation culture and cultured at 37°C until OD 600 The value was 0.7, then the temperature was lowered to 18°C, IPTG with a final concentration of 1.0 mM was added for induction culture, and the fermentation broth containing the inositol oxidase mutant was obtained after 17 hours of induction culture.

[0035] Compared with the inositol oxidase B encoded in the recombinant plasmid B (SEQ ID NO.1), the inositol oxidase mutant in the present invention has a mutation at the 113th amino acid in the amino acid sequence shown in SEQ ID NO.1, from T (threonine) to A (alanine), so the inositol oxidase mutant is named T113A. Example 3

[0036] Isolation and purification of inositol oxidase mutants: The fermentation broth in Example 2 was centrifuged at 4°C and 8000rpm for 15min, the supernatant was discarded, the mutant strain in the fermentation broth was collected, the mutant strain was resuspended in a phosphate buffer with a pH of 8.0 and a concentration of 0.2mM, the bacterial cells were broken using a high-pressure homogenizer, and the mixture was centrifuged at 4°C and 12000r / min for 1h, the cell debris precipitate was discarded, and the collected supernatant was the crude enzyme solution of the inositol oxidase mutant T113A. After the crude enzyme solution was subjected to Ni affinity chromatography, the eluted protein was replaced into a phosphate buffer with a pH of 8.0 and a concentration of 0.2mM using an ultrafiltration tube to remove imidazole and other metal ions in the eluate, and the inositol oxidase mutant T113A enzyme solution was obtained. Example 4

[0037] Relative enzyme activity determination: Definition of enzyme activity (U): The amount of enzyme required to catalyze the synthesis of 1 μmol of glucuronic acid per unit time (min) under standard reaction conditions.

[0038] Enzyme activity determination method: In the standard reaction system, react at 37°C for 30 min and boil for 10 min to terminate the reaction.

[0039] Standard reaction system: 50 mM Tris-HCl buffer (pH 8.0), 2 mM L-cysteine, 1 mM ferrous sulfate (providing Fe 2+ ), 20 mM inositol, 100 μg / mL inositol oxidase enzyme solution.

[0040] The reaction product was centrifuged at 12000 rpm for 10 min, the supernatant was collected and filtered through a 0.22 μm microporous filter membrane, and the filtrate was diluted 10 times and then subjected to HPLC detection.

[0041] HPLC detection method: chromatograph: Agilent1260; detector: RID; chromatographic column Aminex HPX-87H (300×7.8mm); column temperature 30℃; mobile phase: aqueous sulfuric acid solution (5mM); flow rate 0.5 mL / min.

[0042] The detection results are shown in Table 4, with the enzyme activity of inositol oxidase B encoded in recombinant plasmid B being 100%.

[0043] Table 4

[0044] It can be seen from Table 1 that the enzyme activity of the inositol oxidase mutant T113A in the present invention is significantly improved. Example 5

[0045] Thermal stability and half-life determination: The inositol oxidase enzyme solution was incubated at 30℃, 35℃ and 40℃, respectively. The enzyme activity at 0h was taken as 100%. After incubation for 2h, 4h, 6h, 8h, 10h and 12h, samples were taken to detect the enzyme activity, and the thermal stability and half-life of inositol oxidase at these three temperatures were obtained. The test results are as follows: Figure 1 , Figure 2 , Figure 3 shown.

[0046] Depend on Figure 1 It can be seen that at 30°C, the thermal stability of the inositol oxidase mutant T113A is better than that of inositol oxidase B. Moreover, at 30°C, compared with the half-life of 5h of inositol oxidase B, the half-life of the inositol oxidase mutant T113A is increased to 10h.

[0047] Depend on Figure 2 It can be seen that at 35°C, the thermal stability of the inositol oxidase mutant T113A is better than that of inositol oxidase B. Moreover, at 35°C, compared with the half-life of inositol oxidase B of 4 hours, the half-life of the inositol oxidase mutant T113A is increased to 8.4 hours.

[0048] Depend on Figure 3 It can be seen that at 40°C, the thermal stability of the inositol oxidase mutant T113A is better than that of inositol oxidase B. Moreover, at 40°C, compared with the half-life of inositol oxidase B of 4h, the half-life of the inositol oxidase mutant T113A is increased to 7h. Example 6

[0049] Preparation of glucuronic acid: The recombinant plasmid B was introduced into Escherichia coli BL21 according to the method of Example 2 to obtain recombinant strain B, and induced culture was performed according to the method of Example 2 to obtain fermentation broth B.

[0050] The fermentation broth in Example 2 and the fermentation broth B were respectively centrifuged at 4°C and 8000 rpm for 15 min, the supernatant was discarded, and the wet bacterial precipitates were respectively collected.

[0051] In 100 mL, 0.2 mM phosphate buffer (pH 8), 50 g / L inositol was used as the substrate, 25 g of wet bacteria were added, and the reaction was carried out at 37°C for 12 hours. After the reaction was completed, the concentration and conversion rate of glucuronic acid in the reaction system were detected by HPLC. The test results are shown in Table 5.

[0052] Table 5

[0053] It can be seen from Table 2 that 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%.

[0054] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An inositol oxidase mutant, characterized in that The inositol oxidase mutant is obtained by a mutation in the 113th amino acid of the amino acid sequence shown in SEQ ID NO.1, wherein the mutation is: the 113th amino acid is mutated from T to A.

2. The inositol oxidase mutant according to claim 1, characterized in that The amino acid sequence of the inositol oxidase mutant is shown in SEQ ID NO.

2.

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

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

5. A method for preparing an inositol oxidase mutant, for preparing the inositol oxidase mutant according to claim 1 or 2, characterized in that: The following steps are involved: Carrying out seed culture of the recombinant strain described in claim 4 in LB medium, and obtaining seed solution after culturing for 12 to 16 hours; The seed solution was inoculated into another LB medium for fermentation and cultured until OD 600 After the pH value reaches 0.6 to 0.8, the temperature is lowered and IPTG is added for induction culture to induce the recombinant strain to express the inositol oxidase mutant, thereby obtaining a fermentation liquid containing the inositol oxidase mutant.

6. The preparation method according to claim 5, 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.

7. The preparation method according to claim 5, characterized in that: The cooling is to cool to 16-20°C.

8. Use of the inositol oxidase mutant according to claim 1 or 2 in producing glucuronic acid.

9. The use according to claim 8, characterized in that: The inositol oxidase mutant catalyzes the synthesis of glucuronic acid using inositol as a substrate.

10. The use according to claim 9, characterized in that: The reaction temperature of the catalytic synthesis is 35-40° C., and the pH value is 7.5-8.5.

Images