Inositol oxygenase mutant and application thereof in preparation of D-glucuronic acid

Abstract

The invention discloses an inositol oxygenase mutant and application of the inositol oxygenase mutant in preparation of D-glucuronic acid, and belongs to the technical field of genetic engineering. The inositol oxygenase mutant is obtained by mutating P into G from the 116th amino acid of an amino acid sequence as shown in SEQ ID NO.1. The 116th amino acid of wild type inositol oxygenase is mutated, and the enzyme activity of the obtained inositol oxygenase mutant P116G is remarkably improved, so that the yield of D-glucuronic acid is further improved when the D-glucuronic acid is prepared.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to an inositol oxygenase mutant and its application in the preparation of D-glucuronic acid. Background Technology

[0002] D-Glucuronic acid is a naturally occurring uronic acid that is widely used in the pharmaceutical, cosmetic, and food industries.

[0003] In the pharmaceutical field, D-glucuronic acid is a substrate of UDP-glucuronyl transferase in the liver. It can bind to various endogenous or exogenous toxic substances to form more water-soluble glucuronic acid conjugates, promoting their excretion through urine or bile, thereby exerting a detoxification effect. In the cosmetics field, D-glucuronic acid can be used as a moisturizer, antioxidant, or as a precursor to hyaluronic acid, helping to improve skin hydration and elasticity. In the food field, D-glucuronic acid can be used as a functional food additive to improve liver metabolism and promote detoxification.

[0004] Traditional processes for preparing D-glucuronic acid typically involve first oxidizing starch with nitric acid. After oxidation, the nitric acid is not separated; instead, the residual acid is used to hydrolyze the oxidized starch into glucuronic acid under high temperature and pressure. However, this method consumes a large amount of nitric acid, produces high NO2 emissions, and causes severe equipment corrosion. Alternatively, inositol oxygenase can be used to catalyze the synthesis of D-glucuronic acid using muscle inositol as a substrate. However, this method is limited by the activity of inositol oxygenase, resulting in low yields of D-glucuronic acid. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide an inositol oxygenase mutant and its application in the preparation of D-glucuronic acid, so as to overcome the problem of low D-glucuronic acid yield caused by the limitation of inositol oxygenase activity in the prior art.

[0006] In a first aspect, the present invention provides an inositol oxygenase mutant, wherein the inositol oxygenase mutant is obtained by mutating the 116th amino acid of the amino acid sequence shown in SEQ ID NO.1 from P to G.

[0007] Compared with the prior art, the present invention mutates the 116th amino acid of wild-type inositol oxygenase, resulting in an inositol oxygenase mutant P116G with significantly increased enzyme activity, thereby further increasing the yield of D-glucuronic acid during the preparation of D-glucuronic acid.

[0008] Secondly, the present invention provides a nucleic acid molecule that encodes the above-mentioned inositol oxygenase mutant.

[0009] Optionally, the nucleotide sequence of the nucleic acid molecule encoding the inositol oxygenase mutant P116G is shown in SEQ ID NO.3.

[0010] Thirdly, the present invention provides an expression vector containing the above-mentioned nucleic acid molecules.

[0011] Fourthly, the present invention provides a recombinant strain containing the aforementioned nucleic acid molecule or expression vector.

[0012] Fifthly, the present invention provides a method for preparing an inositol oxygenase mutant, comprising the following steps: The above recombinant strains were cultured in a culture medium at 35-40℃ and 120-220 rpm for 12-16 h to obtain seed culture. The seed culture was inoculated into another culture medium at an inoculum volume of 1% to 5% for fermentation culture at 35 to 40°C and 120 to 220 rpm until OD reached. 600 The value was 0.6~0.8, then the temperature was lowered to 16~25℃ for induction culture, and the culture was induced to reach OD. 600 When the value reaches 2.0~5.0, a fermentation broth containing an inositol oxygenase mutant is obtained; Centrifuge the fermentation broth and collect the precipitate to obtain wet bacterial cells containing the inositol oxygenase mutant; or, The wet bacterial cells were resuspended, then the cells were broken, and the mixture was centrifuged again. The resulting supernatant was the crude enzyme solution of the inositol oxygenase mutant.

[0013] In a sixth aspect, the present invention provides the application of the above-mentioned inositol oxygenase mutant, nucleic acid molecule, expression vector or recombinant strain in the preparation of D-glucuronic acid.

[0014] Compared with the prior art, the inositol oxygenase mutant P116G of the present invention has significantly improved enzyme activity, thereby further increasing the yield of D-glucuronic acid during the preparation of D-glucuronic acid.

[0015] In a seventh aspect, the present invention provides a method for preparing glucuronic acid, using the above-mentioned inositol oxygenase mutant to catalyze the reaction of muscle inositol to generate D-glucuronic acid.

[0016] Compared with the prior art, the P116G mutant of inositol oxygenase of the present invention has a further improved enzyme activity, thereby further improving the conversion rate and the yield of D-glucuronic acid when catalyzing the reaction of muscle inositol to D-glucuronic acid.

[0017] Furthermore, the reaction system includes the following components: 50-120 mM buffer, 10-30 g / L muscle inositol, and 50-200 g / L wet bacterial cells containing the inositol oxygenase mutant.

[0018] Furthermore, the buffer may include HEPES buffer, Tris-HCl buffer, phosphate buffer, or sodium carbonate buffer.

[0019] Furthermore, the reaction temperature is 30~40℃, and the pH value is 7.0~8.0. Detailed Implementation

[0020] To make the technical problems to be solved, the technical solutions, and the beneficial effects of 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 for explaining the present invention and are not intended to limit the present invention.

[0021] It should be understood that, unless otherwise specified, all raw materials used in the following examples are commercially available.

[0022] Example 1 Construction of recombinant plasmid pET28a-miox Wild-type inositol oxygenase from Arabidopsis thaliana was selected, and its amino acid sequence is shown in SEQ ID NO.1. The gene sequence encoding wild-type inositol oxygenase was codon-optimized to obtain the optimized miox gene sequence, and its nucleotide sequence is shown in SEQ ID NO.2.

[0023] Using the optimized miox gene sequence as a template, upstream primer F1 and downstream primer R1 were designed for PCR amplification to obtain the miox gene fragment with homologous arms. The PCR amplification reaction system is shown in Table 1, and the PCR amplification reaction conditions are shown in Table 2.

[0024] F1: 5'-atgggtcgcggatccgaattcATGACCATTCTGATTGATCGCC-3' (SEQ ID NO. 5); R1: 5'-tggtggtgctcgagtgcggccgcCCATTTCAGTTTGCTCGGAAA-3' (SEQ ID NO. 6).

[0025] Table 1 Table 2 It should be understood that the above-mentioned pre-denaturation, final extension, and maintenance steps do not participate in the cycle and are performed only once in the entire PCR process.

[0026] After the PCR amplification was completed, the reaction product was recovered by agarose gel extraction to obtain a miox gene fragment with high purity.

[0027] The expression vector pET28a was double-digested with restriction endonucleases EcoRI and NotI at 37°C for 20 min. The double-digested products were recovered and purified to obtain the linearized vector pET28a. The double-digestion system is shown in Table 3.

[0028] Table 3 The miox gene fragment obtained by PCR amplification was ligated to the linearized vector pET28a after double enzyme digestion. The ligation was carried out at 37℃ for 20 min to obtain the recombinant plasmid pET28a-miox. The ligation system is shown in Table 4.

[0029] Table 4 After the above ligation was completed, the ligation product was transformed into E. coli DH5α competent cells using chemical transformation. Single colonies were picked for plasmid extraction, and the extracted plasmids were sequenced for DNA.

[0030] Example 2 Constructing mutant plasmids Using the recombinant plasmid pET28a-miox constructed in Example 1 as a template, reverse PCR amplification was performed using P116G-F and P116G-R primers to obtain the mutant plasmid pET28a-miox. P116G The reverse PCR amplification reaction system is shown in Table 5, and the amplification reaction conditions are shown in Table 6.

[0031] P116G-F: GGATGAAggcCAGATTGAACATCTGCTGCAGAC (SEQ ID NO.7); P116G-R: CAATCTGgccTTCATCCAGATCCGGATCGCTT (SEQ ID NO. 8).

[0032] Table 5 Table 6 It should be understood that the above-mentioned pre-denaturation, final extension, and maintenance steps do not participate in the cycle and are performed only once in the entire PCR process.

[0033] Template elimination: After the above reverse PCR reaction was completed, 2 μL of restriction endonuclease DpnⅠ was added to the reaction solution (25 μL), and the mixture was gently blown and aspirated. The mixture was reacted at 37 °C for 1 h to obtain the enzyme digestion solution. The enzyme digestion solution was verified by agarose gel electrophoresis.

[0034] Reverse PCR product self-circularization: Using the enzyme digestion solution verified above, prepare the reaction solution according to Table 7, mix gently, and incubate at 16℃ for 1 h to obtain the mutant plasmid pET28a-miox containing the inositol oxygenase mutant gene. P116G .

[0035] Table 7 Mutant plasmid verification: The mutant plasmid obtained after circularization was transformed into E. coli DH5α competent cells by chemical transformation. Single colonies on the plate were picked for plasmid extraction, and the extracted plasmid was sequenced for DNA.

[0036] Mutant plasmid pET28a-miox P116G The gene containing the inositol oxygenase mutant P116G has the nucleotide sequence shown in SEQ ID NO.3 and the encoded amino acid sequence shown in SEQ ID NO.4.

[0037] Example 3 Preparation of wet bacterial cells and crude enzyme solution Take 1 μL of the recombinant plasmid pET28a-miox that was correctly sequenced in Example 1 and the mutant plasmid pET28a-miox that was correctly sequenced in Example 2. P116G The bacteria were added separately to *E. coli* BL21(DE3) competent cells. The two mixtures were then incubated on ice for 25 min, followed by heat shock at 42°C for 45 s, and then on ice for 5 min. Afterward, each mixture was transferred to 500 μL of LB broth and incubated at 37°C with shaking for 1 h. 100 μL of each culture was plated, and positive transformants were selected, yielding recombinant strains containing the recombinant plasmid pET28a-miox and mutant strains containing the pET28a-miox plasmid, respectively. P116G mutant strains.

[0038] The recombinant strains containing the recombinant plasmid pET28a-miox and the mutant strains containing the pET28a-miox were screened. P116G The mutant strains were seed cultured in liquid LB medium at 37°C and 120 rpm for 12 h to obtain recombinant strains containing the recombinant plasmid pET28a-miox and mutant strains containing the pET28a-miox mutant plasmid.P116G Seed culture of mutant strains.

[0039] Seed cultures of the two strains were inoculated separately into new liquid culture medium at a 2% (v / v) ratio for fermentation. LB liquid medium could be selected as the new liquid culture medium. The cultures were incubated at 37°C and 120 rpm until OD (Organic Degrees Per Minute). 600 The value was 0.7, then the temperature was lowered to 20℃, and induction culture was performed separately. The induction culture conditions were as follows: IPTG was added to a final concentration of 0.5 mM for induction culture, and the culture was induced to OD. 600 When the value reached 3.0, fermentation broth containing wild-type inositol oxygenase and fermentation broth containing the inositol oxygenase mutant P116G were obtained, respectively.

[0040] The two fermentation broths were centrifuged at 4℃ and 4000r / min for 15min, and the wet cells were collected to obtain wet cells of recombinant strain containing wild-type inositol oxygenase and wet cells of mutant strain containing inositol oxygenase mutant P116G, respectively.

[0041] Wet cells of the recombinant strain containing wild-type inositol oxygenase and the mutant strain containing the inositol oxygenase mutant P116G were taken separately. The wet cells were resuspended in phosphate buffer (pH 7.5), and then the cells were disrupted using an ultrasonic cell disruptor at 450W for 20 minutes with a 2-second interval between disruptions and a 3-second pause. After disruption, the cells were centrifuged at 8000 rpm for 30 minutes at 4°C to remove cell debris. The supernatants were collected to obtain crude enzyme solutions of wild-type inositol oxygenase and P116G, respectively. The concentrations of both wild-type and P116G crude enzymes in the crude enzyme solutions were 3 mg / mL.

[0042] Example 4 Measurement of relative enzyme activity Enzyme activity (U): The amount of enzyme required to catalyze the synthesis of 1 μmol of D-glucuronic acid per unit time (min) under standard reaction conditions.

[0043] Enzyme activity assay method: In the standard reaction system, react at 37℃ for 30 min, and then boil for 10 min to terminate the reaction.

[0044] Standard reaction system 1: A solution containing 50 mM Tris-HCl buffer (pH 7.5), 2 mM L-cysteine, and 1 mM ferrous sulfate (to provide Fe). 2+Add crude wild-type inositol oxygenase solution to the 20mM muscle inositol reaction system to make the concentration of crude wild-type inositol oxygenase in the reaction system 200μg / mL.

[0045] Standard reaction system 2: A solution containing 50 mM Tris-HCl buffer (pH 7.5), 2 mM L-cysteine, and 1 mM ferrous sulfate (to provide Fe). 2+ Add crude enzyme solution of inositol oxygenase mutant P116G to the 20mM muscle inositol reaction system to make the concentration of crude inositol oxygenase mutant P116G in the reaction system 200μg / mL.

[0046] After the reaction was completed, the two reaction products were centrifuged at 12000 rpm for 10 min, and the supernatant was collected and filtered through a 0.22 μm pore membrane. The filtrate was then diluted 10 times and analyzed by HPLC.

[0047] HPLC detection method: Detector: RID; Column: Aminex HPX-87H (300×7.8mm); Column temperature: 30℃; Mobile phase: sulfuric acid aqueous solution (5mM); Flow rate: 0.5 mL / min.

[0048] The relative enzyme activity of the inositol oxygenase mutant P116G was calculated with the wild-type inositol oxygenase activity as 100%, and the results are shown in Table 8.

[0049] Table 8 The results above show that, compared with wild-type inositol oxygenase, the enzyme activity of the inositol oxygenase mutant P116G obtained by the present invention is significantly improved.

[0050] Example 5 Preparation of D-glucuronic acid Muscle inositol and the recombinant strain containing wild-type inositol oxygenase prepared in Example 3 above were added to 100 mL of 100 mM phosphate buffer (pH 7.5) to make the concentration of muscle inositol in the reaction system 25 g / L and the concentration of recombinant strain wet cells 120 g / L.

[0051] Muscle inositol and the wetted bacterial cells of the mutant strain containing the inositol oxygenase mutant P116G prepared in Example 3 above were added to 100 mL of 100 mM phosphate buffer (pH 7.5) to make the concentration of muscle inositol in the reaction system 25 g / L and the concentration of the wetted bacterial cells of the mutant strain 120 g / L.

[0052] The two reaction systems were reacted at 37℃ for 12 h. After the reaction was completed, the two reaction products were centrifuged at 12000 rpm for 10 min. The supernatant was collected and filtered through a 0.22 μm pore membrane. The filtrate was diluted 10 times and the concentration of D-glucuronic acid in the reaction system was detected by HPLC and the conversion rate was calculated. The results are shown in Table 9.

[0053] Conversion rate =C P / [C S0 ×(M P / M S )]×100%.

[0054] Among them, C P = Concentration of D-glucuronic acid (g / L), C S0 =Initial concentration of muscle inositol (g / L), M P = Molar mass of D-glucuronic acid, M S =Molar mass of muscle inositol.

[0055] HPLC detection method: Detector: RID; Column: Aminex HPX-87H (300×7.8mm); Column temperature: 30℃; Mobile phase: sulfuric acid aqueous solution (5mM); Flow rate: 0.5 mL / min.

[0056] Table 9 The results above show that, compared with wild-type inositol oxygenase, the inositol oxygenase mutant P116G contained in the wet cell of the mutant strain of this invention has higher enzyme activity, thereby further improving the conversion rate and the yield of D-glucuronic acid.

[0057] The above description is merely a preferred embodiment of the present invention and is 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 make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An inositol oxygenase mutant, characterized in that, The inositol oxygenase mutant is obtained by mutating amino acid P to G in the 116th amino acid position of the amino acid sequence shown in SEQ ID NO.

1.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the inositol oxygenase mutant of claim 1.

3. An expression carrier, characterized in that, The expression vector contains the nucleic acid molecule as described in claim 2.

4. A recombinant bacterial strain, characterized in that, The recombinant strain contains the nucleic acid molecule of claim 2 or the expression vector of claim 3.

5. A method for preparing an inositol oxygenase mutant, used to prepare the inositol oxygenase mutant of claim 1, characterized in that, Includes the following steps: The recombinant strain described in claim 4 was cultured in a culture medium at 35-40°C and 120-220 rpm for 12-16 h to obtain a seed culture. The seed culture was inoculated into another culture medium at an inoculum volume of 1% to 5% for fermentation culture at 35 to 40°C and 120 to 220 rpm until OD reached. 600 The value was 0.6~0.8, then the temperature was lowered to 16~25℃ for induction culture, and the culture was induced to reach OD. 600 When the value reaches 2.0~5.0, a fermentation broth containing an inositol oxygenase mutant is obtained; Centrifuge the fermentation broth and collect the precipitate to obtain wet bacterial cells containing the inositol oxygenase mutant; or, The wet bacterial cells were resuspended, then the cells were broken, and the mixture was centrifuged again. The resulting supernatant was the crude enzyme solution of the inositol oxygenase mutant.

6. The use of the inositol oxygenase mutant of claim 1, the nucleic acid molecule of claim 2, the expression vector of claim 3, or the recombinant strain of claim 4 in the preparation of D-glucuronic acid.

7. A method for preparing glucuronic acid, characterized in that, Using the inositol oxygenase mutant of claim 1, the reaction of muscle inositol is catalyzed to produce D-glucuronic acid.

8. The preparation method according to claim 7, characterized in that, The reaction system includes the following components: Buffer solution 50-120mM, muscle inositol 10-30g / L, wet bacterial cells containing inositol oxygenase mutant 50-200g / L.

9. The preparation method according to claim 8, characterized in that, The buffer solution includes HEPES buffer, Tris-HCl buffer, phosphate buffer, or sodium carbonate buffer.

10. The preparation method according to any one of claims 7 to 9, characterized in that, The reaction temperature is 30~40℃, and the pH value is 7.0~8.0.

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