D-mangiferoline dehydrogenase mtOEPa mutant and application thereof

Abstract

The application discloses a D-mangiferoline dehydrogenase MtOEPa mutant and application thereof, and belongs to the technical field of genetic engineering. The amino acid sequence of the D-mangiferoline dehydrogenase MtOEPa mutant is shown as SEQ ID NO. 4. The D-mangiferoline dehydrogenase MtOEPa mutant has stronger affinity to a substrate myo-inositol, higher catalytic efficiency and higher enzyme activity, and can significantly improve the conversion rate of a reaction and the yield of D-chiro-inositol when catalyzing the reaction of myo-inositol to generate D-chiro-inositol.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a D-manganese dehydrogenase MtOEPa mutant and its applications. Background Technology

[0002] D-Chiro-inositol (DCI) is an important stereoisomer of inositol that has received much attention in the field of metabolic health in recent years. D-Chiro-inositol has a significant effect on improving polycystic ovary syndrome (PCOS) and insulin resistance.

[0003] The general route for producing D-chiral inositol via enzymatic catalysis involves using inositol dehydrogenase and ketoisomerase to catalyze the reaction of muscle inositol to D-chiral inositol. This route tends to accumulate intermediates, resulting in generally low yields and conversion efficiencies of the final D-chiral inositol. In contrast, studies have demonstrated that D-monosodium dehydrogenase (MtOEPa) and D-pineol dehydrogenase (MtOEPb) from alfalfa, expressed heterologously in Corynebacterium glutamicum, have enabled the reversible conversion of muscle inositol to D-chiral inositol using substrate heterogeneity, establishing a new pathway for D-chiral inositol synthesis. However, this pathway is limited by the activity of D-monosodium dehydrogenase (MtOEPa), leaving significant room for improvement in catalytic efficiency. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a D-monomentol dehydrogenase MtOEPa mutant and its application, in order to overcome the problem of low enzyme activity of D-monomentol dehydrogenase MtOEPa in the prior art.

[0005] In a first aspect, the present invention provides a D-monomentol dehydrogenase MtOEPa mutant, the amino acid sequence of which is shown in SEQ ID NO.4.

[0006] Secondly, the present invention provides a nucleic acid molecule that encodes the aforementioned D-mangmenol dehydrogenase MtOEPa mutant.

[0007] Optionally, the nucleotide sequence of the above-mentioned nucleic acid molecule encoding the D-monomentol dehydrogenase MtOEPa mutant is shown in SEQ ID NO.3.

[0008] Thirdly, the present invention provides an expression vector containing the above-mentioned nucleic acid molecules.

[0009] Fourthly, the present invention provides a recombinant strain containing the above-mentioned nucleic acid molecule or the above-mentioned expression vector.

[0010] Fifthly, the present invention provides a method for preparing a D-monomentol dehydrogenase MtOEPa mutant, which includes the following steps:

[0011] The above recombinant strain was subjected to seed culture to obtain seed solution;

[0012] The seed culture was inoculated into the fermentation medium for fermentation culture until the OD reached... 600 The value was 0.6~0.8, the temperature was lowered, and an inducer was added for induction culture for 12~16h to obtain fermentation broth containing the D-monomentol dehydrogenase MtOEPa mutant;

[0013] The fermentation broth was centrifuged to collect the cells. After the cells were resuspended, they were broken up and centrifuged again. The collected supernatant was the crude enzyme solution of the D-manganese dehydrogenase MtOEPa mutant.

[0014] In a sixth aspect, the present invention provides the application of the above-mentioned D-manganese dehydrogenase MtOEPa mutant, the above-mentioned nucleic acid molecule, the above-mentioned expression vector, or the above-mentioned recombinant strain in the preparation of D-chiral inositol.

[0015] In a seventh aspect, the present invention provides a method for preparing D-chiral inositol, which uses the above-mentioned D-manganese dehydrogenase MtOEPa mutant and D-pinel dehydrogenase MtOEPb to catalyze the reaction of the substrate muscle inositol to generate D-chiral inositol in the presence of a coenzyme factor.

[0016] Further, muscle inositol, coenzyme factor, crude enzyme solution of D-monomentol dehydrogenase MtOEPa mutant, and enzyme solution of D-pineol dehydrogenase MtOEPb were added to a 20-100mM buffer solution, so that the concentration of muscle inositol in the reaction system was 20-120 mg / mL, the concentration of crude enzyme of D-monomentol dehydrogenase MtOEPa mutant was 2-12 mg / mL, and the concentration of D-pineol dehydrogenase MtOEPb was 2-12 mg / mL.

[0017] Furthermore, the buffer solution includes phosphate buffer, Tris-HCl buffer, or HEPES buffer.

[0018] Furthermore, coenzyme factors include NAD. ＋ NADP ＋ In the reaction system, NAD ＋ The concentration is 0.5~2.0mM, NADP ＋ The concentration is 0.5~2.0mM.

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

[0020] Compared with the prior art, the present invention has the following specific beneficial effects:

[0021] (1) Compared with the existing wild-type D-monomentol dehydrogenase MtOEPa, the present invention mutates the 245th amino acid of the wild-type D-monomentol dehydrogenase MtOEPa from R to K to obtain the D-monomentol dehydrogenase MtOEPa mutant R245K. The D-monomentol dehydrogenase MtOEPa mutant R245K has a stronger affinity for the substrate muscle inositol, higher catalytic efficiency, and higher enzyme activity. When catalyzing the reaction of muscle inositol to generate D-chiral inositol, it can significantly improve the conversion rate and the yield of D-chiral inositol.

[0022] (2) The preparation method of the present invention can easily and quickly prepare crude enzyme solution of D-manganese dehydrogenase MtOEPa mutant with catalytic activity.

[0023] (3) The present invention uses D-manganese dehydrogenase MtOEPa mutant and D-pineol dehydrogenase MtOEPb with specific sequences to catalyze the reaction of the substrate muscle inositol to generate D-chiral inositol in the presence of coenzyme factors, which effectively improves the conversion rate of the reaction and the yield of D-chiral inositol. Detailed Implementation

[0024] 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.

[0025] It should be understood that, unless otherwise specified, all raw materials used in the following examples are commercially available.

[0026] Example 1

[0027] Construction of recombinant plasmid pET28a-MtOEPA

[0028] Based on the codon preference of Escherichia coli, the amino acid sequence of wild-type D-ononitol dehydrogenase MtOEPa (Medicago truncatula D-ononitol dehydrogenase) derived from alfalfa (as shown in SEQ ID NO.1) was reverse-translated into a DNA sequence to obtain the gene sequence of wild-type D-ononitol dehydrogenase MtOEPa (as shown in SEQ ID NO.2).

[0029] Using the gene sequence of the wild-type D-manganese dehydrogenase MtOEPa as a template, PCR amplification was performed with F1 as the upstream primer and R1 as the downstream primer. The reaction system is shown in Table 1.

[0030] F1: 5'-cagcaaatgggtcgcggatccATGAGCAAAACCGTGTCG-3' (SEQ ID NO. 5).

[0031] R1: 5'-ctcgagtgcggccgcaagcttCTACACCAGGCCGCGGCT-3' (SEQ ID NO. 6).

[0032] Table 1

[0033] Components Volume (μL) template 2μL Upstream primer F1 (10 pmol / μL) 1μL Downstream primer R1 (10 pmol / μL) 1μL DNA polymerase 1μL 2×PCR buffer 25μL <![CDATA[Sterile ddH2O]]> 20μL

[0034] The reaction program for the above PCR amplification reaction is as follows: pre-denaturation: 98℃, 30s; denaturation-annealing-extension: 98℃, 10s, 60℃, 10s, 72℃, 20s, cycle number 35; complete extension: 55℃, 5.5min; incubation: 16℃.

[0035] After the PCR amplification reaction was completed, the target gene MtOEPA with homologous arms was recovered by agarose gel extraction to obtain a high-purity sample.

[0036] The expression vector pET28a was double-digested with restriction endonucleases BamHⅠ and XhoⅠ at 37℃ for 20 min to obtain the linearized expression vector pET28a. The digestion system is shown in Table 2.

[0037] Table 2

[0038] Components Volume (μL) expression vector pET28a 25μL BamHⅠ 2μL XhoⅠ 2μL 10× buffer solution 5μL <![CDATA[ddH2O]]> 16μL

[0039] The linearized expression vector pET28a after enzyme digestion was ligated with the target gene MtOEPA obtained by the above PCR amplification reaction using recombinase Exnase II. The ligation temperature was 37℃ and the ligation time was 30 min. The ligation system is shown in Table 3.

[0040] Table 3

[0041] Components Volume (μL) Linearized expression vector pET28a 4.5μL Target gene MtOEPA 1.5μL Recombinase Exnase II 2μL 5×CEⅡ Buffer 4μL <![CDATA[ddH2O]]> 8μL

[0042] After the above ligation reaction is completed, the ligation product is transformed into E. coli DH5α competent cells using chemical transformation. Single colonies are picked and plasmids are extracted and sent for sequencing. The recombinant plasmid pET28a-MtOEPA is the one that is correctly sequenced.

[0043] Example 2

[0044] Construction of mutant plasmid pET28a-MtOEPA R245K

[0045] Using the recombinant plasmid pET28a-MtOEPA as a template, with F2 as the upstream primer and R2 as the downstream primer, a reverse PCR amplification reaction was performed. The reaction system is shown in Table 4.

[0046] F2: 5'-GAGCGGCaaaCATCTGTGCGTGGAAGCGATTC-3' (SEQ ID NO. 7).

[0047] R2: 5'-ACAGATGtttGCCGCTCGCTTTTTTCTGTTCA-3' (SEQ ID NO. 8).

[0048] Table 4

[0049] Components Volume (μL) template 2μL Upstream primer F2 (10 pmol / μL) 2μL Downstream primer R2 (10 pmol / μL) 2μL KOD-Plus DNA polymerase 2μL 2mM deoxynucleoside triphosphates (dNTPs) 5μL 10×PCR buffer 5μL <![CDATA[Sterile ddH2O]]> 32μL

[0050] The reverse PCR amplification reaction procedure is as follows: pre-denaturation: 94℃, 2min; denaturation-annealing-extension: 98℃, 10s, 55℃, 30s, 68℃, 30s, cycle number 10; incubation: 4℃.

[0051] Template digestion: After the above reverse PCR amplification reaction is completed, 2 μL of restriction endonuclease DpnⅠ is added to the reaction solution (50 μL) and gently mixed by pipetting; the reaction is carried out at 37℃ for 1 h to obtain the enzyme digestion solution, and the enzyme digestion solution is verified by agarose gel electrophoresis.

[0052] Reverse PCR product self-cyclization reaction: Take 2 μL of the verified enzyme digestion solution, 1 μL of high-efficiency ligation reagent, 2 μL of T4 polynucleotide kinase, and 6 μL of ddH2O, mix gently, and react at 16℃ for 1 hour to obtain the mutant plasmid pET28a-MtOEPA. R245K .

[0053] Mutant plasmid verification: The mutant plasmid pET28a-MtOEPA obtained after the above circularization was... R245K The cells were transformed into E. coli DH5α competent cells using chemical transformation. Single colonies on the plates were picked for plasmid extraction, and the extracted plasmids were sequenced for DNA.

[0054] Mutant plasmid pET28a-MtOEPA R245K The enzyme contains the encoding gene for the D-monomentol dehydrogenase MtOEPa mutant R245K, the nucleotide sequence of which is shown in SEQ ID NO.3. The amino acid sequence of the D-monomentol dehydrogenase MtOEPa mutant R245K is shown in SEQ ID NO.4.

[0055] Example 3

[0056] Preparation of crude enzyme solution

[0057] Take 1 μL of the mutant plasmid pET28a-MtOEPA R245K Add the mixture to E. coli BL21(DE3) competent cells, place the mixture on ice for 25 min, then heat shock it in a 42°C water bath for 60 s, followed by incubation on ice for 5 min. Transfer the mixture to 500 μL of LB liquid medium and incubate at 37°C with shaking for 1 h. Spread 100 μL of the bacterial culture onto an LB agar plate containing 50 μg / mL kanamycin and incubate upside down for 12 h to obtain the mutant strain BL21-pET28a-MtOEPA. R245K .

[0058] Mutant strain BL21-pET28a-MtOEPA R245K Seed culture was carried out by inoculating the culture medium into liquid LB medium (containing 50 μg / mL kanamycin) and culturing it at 37℃ and 200 rpm for 12 h to obtain seed culture.

[0059] The above seed culture was inoculated into fresh LB liquid medium (containing 50 μg / mL kanamycin) at a 2% (v / v) inoculum and cultured at 37°C until OD500. 600 The value was 0.7, then the temperature was lowered to 25℃, and IPTG with a final concentration of 0.01mM was added for induction culture for 14h to obtain fermentation broth containing the D-monomentol dehydrogenase MtOEPa mutant R245K.

[0060] The fermentation broth was centrifuged at 4℃ and 4000 r / min for 15 min to collect the cells. The collected cells were resuspended in solution 1, and this process was repeated twice to remove residual culture medium and impurities. Solution 1 consisted of 20 mM Tris-HCl, 200 mM NaCl, and 10 mM imidazole, and its pH was 8.0.

[0061] After resuspension, the bacterial cells in the resuspension were disrupted using an ultrasonic cell disruptor. The ultrasonic power was 450W, with a 2-second break and a 3-second pause, for a total of 30 minutes of ultrasonic disruption. After ultrasonic disruption, the cells were centrifuged at 4℃ and 12000r / min for 60 minutes to remove cell debris. The supernatant was collected to obtain the crude enzyme solution of the D-monomenthol dehydrogenase MtOEPa mutant R245K.

[0062] Take 1 μL of the recombinant plasmid pET28a-MtOEPA constructed in Example 1 and prepare the crude enzyme solution of wild-type D-monomentol dehydrogenase MtOEPa using the same method as described above.

[0063] Example 4

[0064] Dynamic parameter detection

[0065] The crude enzyme solutions of the D-monosodium dehydrogenase MtOEPa mutant R245K and the crude enzyme solution of wild-type D-monosodium dehydrogenase MtOEPa prepared in Example 3 above were subjected to Ni-NTA affinity chromatography to obtain purified protein solutions of the D-monosodium dehydrogenase MtOEPa mutant R245K and the wild-type D-monosodium dehydrogenase MtOEPa, respectively. The specific steps are as follows:

[0066] Add 1 mL of Ni-NTA resin to the open column and equilibrate the resin with 2 column volumes of Solution 1.

[0067] Take 40 mL of crude D-manganese dehydrogenase MtOEPa enzyme solution and mix it with 1 mL of equilibrated resin. Place the mixture at 4℃ for 60 min to bind. Pass the bound mixture through an open column, and the resin containing the bound protein is retained in the column.

[0068] Rinse the resin with two column volumes of solution 1 to remove non-specifically bound proteins.

[0069] The target protein was eluted with 20 mL of solution 2, which consisted of 20 mM Tris-HCl, 100 mM NaCl, and 200 mM imidazole, and had a pH of 8.0.

[0070] The eluted protein solution was replaced with 20 mM phosphate buffer (pH 7.5) using a 10 kDa ultrafiltration tube to remove imidazole and other ions, resulting in a purified protein solution.

[0071] Prepare a reaction system containing the D-manganese dehydrogenase MtOEPa mutant R245K: Add muscle inositol and NAD to the reaction system. ＋ The purified protein solution of the D-monomenthol dehydrogenase MtOEPa mutant R245K was added to a reaction volume of 10 mL with 20 mM phosphate buffer (pH 7.5). NAD+ was added to the reaction system. ＋ The concentration of the purified D-monomentol dehydrogenase MtOEPa mutant R245K protein solution was 5 mM, and the concentration of the protein solution was 100 μL / mL.

[0072] Eight reaction systems were prepared according to the above method, with muscle inositol concentrations of 0.1 mM, 0.2 mM, 0.5 mM, 1 mM, 2 mM, 5 mM, 8 mM, and 12 mM, respectively. After mixing each system thoroughly, the reactions were carried out at 37°C. Samples were taken from each system at 0 min, 3 min, 6 min, and 9 min, respectively. The reactions were then terminated by heating in a boiling water bath, centrifuged at 12000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm filter membrane. The filtrates were then analyzed by high-performance liquid chromatography (HPLC).

[0073] Configure a reaction system containing wild-type D-manganese dehydrogenase MtOEPa: Add muscle inositol and NAD to the reaction system. ＋ The purified wild-type D-monosodium dehydrogenase MtOEPa protein solution was adjusted to a reaction volume of 10 mL using 20 mM phosphate buffer (pH 7.5). NAD+ was added to the reaction system. ＋ The concentration of the purified wild-type D-manganese dehydrogenase MtOEPa protein solution was 100 μL / mL, with a concentration of 5 mM.

[0074] Eight reaction systems were prepared according to the above method, with muscle inositol concentrations of 0.1 mM, 0.2 mM, 0.5 mM, 1 mM, 2 mM, 5 mM, 8 mM, and 12 mM, respectively. After mixing each system thoroughly, the reactions were carried out at 37°C. Samples were taken from each system at 0 min, 3 min, 6 min, and 9 min, respectively. The reactions were then terminated by heating in a boiling water bath, centrifuged at 12000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm filter membrane. The filtrates were then analyzed by high-performance liquid chromatography (HPLC).

[0075] High performance liquid chromatography (HPLC) analysis methods:

[0076] The chromatographic column was a 4.6 × 250 mm, 5 μm amino column; the mobile phase was acetonitrile: 50 mM ammonium acetate aqueous solution = 75: 25 (volume ratio); the column temperature was set at 30℃ and the flow rate was 1.0 mL / min.

[0077] The kinetic parameters of D-monomentol dehydrogenase MtOEPa mutant R245K and wild-type D-monomentol dehydrogenase MtOEPa on the substrate muscle inositol were calculated, and the results are shown in Table 5.

[0078] Table 5

[0079]

[0080] From the above results, we can conclude that:

[0081] Compared to wild-type D-monomentol dehydrogenase MtOEPa, the K of the D-monomentol dehydrogenase MtOEPa mutant R245K is significantly different. m The lower value indicates that the D-monomenthol dehydrogenase MtOEPa mutant R245K has a higher affinity for the substrate muscle inositol. cat A higher value indicates that the D-monomenthol dehydrogenase MtOEPa mutant R245K has a faster catalytic rate, K cat / Km The higher value indicates that the overall catalytic performance of the MtOEPa mutant of D-monomentol dehydrogenase R245K is better.

[0082] Example 5

[0083] Preparation of D-chiral inositol

[0084] Add muscle inositol and NAD to a 20mM phosphate buffer solution with a pH of 7.5. + NADP + Wild-type D-manganese dehydrogenase MtOEPa crude enzyme solution and D-pinel dehydrogenase MtOEPb enzyme solution were used to make the concentration of muscle inositol in the reaction system 100 mg / mL, NAD+ ＋ The concentration was 2.0 mM NADP. ＋ The concentration of the enzyme was 2.0 mM, the concentration of wild-type D-manganese dehydrogenase MtOEPa crude enzyme was 10 mg / mL, and the concentration of D-pineol dehydrogenase MtOEPb was 10 mg / mL.

[0085] Add muscle inositol and NAD to a 20mM phosphate buffer solution with a pH of 7.5. + NADP + The crude enzyme solution of D-monosodium dehydrogenase MtOEPa mutant R245K and the enzyme solution of D-pinesol dehydrogenase MtOEPb were used to make the concentration of muscle inositol in the reaction system 100 mg / mL, and NAD+. ＋ The concentration was 2.0 mM NADP. ＋ The concentration of D-manganese dehydrogenase MtOEPa mutant R245K was 2.0 mM, the concentration of crude D-manganese dehydrogenase MtOEPb was 10 mg / mL, and the concentration of D-pineol dehydrogenase MtOEPb was 10 mg / mL.

[0086] The two reaction systems were reacted at 35℃ for 60 min. After the reaction was completed, the content of D-chiral inositol in the reaction solution was measured and the conversion rate was calculated. The results are shown in Table 6.

[0087] Conversion rate: D-chiral inositol content after reaction ÷ initial muscle inositol content in the reaction system × 100%.

[0088] Detection method: High performance liquid chromatography (HPLC) was used. The chromatographic column was a 4.6 × 250 mm, 5 μm amino column. The mobile phase was acetonitrile: 50 mM ammonium acetate aqueous solution = 75: 25 (volume ratio). The column temperature was set at 30℃ and the flow rate was 1.0 mL / min.

[0089] Table 6

[0090] D-Momentin dehydrogenase MtOEPa D-chiroinositol content (mg / mL) Conversion rate (%) Wild-type D-manganese dehydrogenase MtOEPa 9.55 mg / mL 9.55% D-Momentin dehydrogenase MtOEPa mutant R245K 13.23 mg / mL 13.23%

[0091] From the above results, we can conclude that:

[0092] Compared to wild-type D-monomentol dehydrogenase MtOEPa, the D-monomentol dehydrogenase MtOEPa mutant has higher enzyme activity and better catalytic performance, thus effectively improving the yield and conversion rate of D-chiral inositol.

[0093] 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. A D-monomentol dehydrogenase MtOEPa mutant, characterized in that, The amino acid sequence of the D-monomentol dehydrogenase MtOEPa mutant is shown in SEQ ID NO.

4.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the MtOEPa mutant of D-manganese dehydrogenase as described in 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 a D-monomentol dehydrogenase MtOEPa mutant, characterized in that, The preparation of the D-monomentol dehydrogenase MtOEPa mutant according to claim 1 includes the following steps: The recombinant strain described in claim 4 was subjected to seed culture to obtain a seed solution; The seed culture was inoculated into a fermentation medium for fermentation culture until the OD value reached... 600 The value was 0.6~0.8, the temperature was lowered, and an inducer was added for induction culture for 12~16h to obtain fermentation broth containing the D-monomentol dehydrogenase MtOEPa mutant; The fermentation broth was centrifuged to collect the bacterial cells. After the bacterial cells were resuspended, they were broken up and centrifuged again. The collected supernatant was the crude enzyme solution of the D-monomentol dehydrogenase MtOEPa mutant.

6. The use of the D-manganese dehydrogenase MtOEPa 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-chiral inositol.

7. A method for preparing D-chiral inositol, characterized in that, Using the D-manganese dehydrogenase MtOEPa mutant and D-pinel dehydrogenase MtOEPb described in claim 1, in the presence of coenzyme factors, the substrate muscle inositol is catalyzed to react and generate D-chiral inositol.

8. The preparation method according to claim 7, characterized in that, Add muscle inositol, coenzyme factor, crude enzyme solution of D-monomentol dehydrogenase MtOEPa mutant, and enzyme solution of D-pineol dehydrogenase MtOEPb to a 20-100 mM buffer solution, so that the concentration of muscle inositol in the reaction system is 20-120 mg / mL, the concentration of crude enzyme of D-monomentol dehydrogenase MtOEPa mutant is 2-12 mg / mL, and the concentration of D-pineol dehydrogenase MtOEPb is 2-12 mg / mL.

9. The preparation method according to claim 8, characterized in that, The buffer solution includes phosphate buffer, Tris-HCl buffer, or HEPES buffer; and / or, The coenzyme factor includes NAD. ＋ NADP ＋ In the reaction system, the NAD ＋ The concentration of NADP is 0.5~2.0 mM. ＋ The concentration is 0.5~2.0mM.

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

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