4-Oxodecanoic acid reductase mutant and its application in the preparation of optically pure (R)-γ lactone

Abstract

The present invention belongs to the field of bioengineering technology, and specifically relates to a 4-oxodecanoic acid reductase mutant with significantly improved catalytic performance, a coding gene thereof, a recombinant expression vector and a recombinant expression transformant containing the gene, and the use of the 4-oxodecanoic acid reductase mutant or the recombinant expression transformant to catalyze a potential chiral carbonyl compound and prepare an optically pure (R)-γ lactone. Compared with the parent reductase mutant, the 4-oxodecanoic acid reductase mutant obtained by the present invention has a specific enzyme activity 16 times higher than that of the parent when targeting 4-oxodecanoic acid, a higher catalytic activity, and a better industrial application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and specifically relates to a 4-oxodecanoic acid reductase mutant with significantly improved catalytic performance, a coding gene thereof, a recombinant expression vector and a recombinant expression transformant containing the gene, and an application of the 4-oxodecanoic acid reductase mutant or the recombinant expression transformant in catalyzing a potential chiral carbonyl compound to prepare an optically pure (R)-γ lactone. Background Art

[0002] γ-Alkyl lactone is a five-membered lactone compound, a natural product with fragrance, which can be used as an important source of many fruit aromas. As a naturally occurring flavoring substance, this type of compound is widely used in fruity aromatic foods and beverages and daily chemical products. In recent years, as people have higher and higher requirements for the safety of food, etc., the demand for natural flavors is also increasing year by year.

[0003] Most natural flavors are extracted from animals and plants, but the content of alkyl lactone flavors in animals and plants is extremely small, and it is difficult to obtain them through natural extraction. They need to rely on synthetic technology, and pure chemical synthesis methods often have low yields and pollution and safety issues. At the same time, chemically synthesized flavors are mostly racemic bodies with a single and floating fragrance. The production process is prone to the mixing of toxic substances and the change of fragrance due to production processes and other reasons.

[0004] Xiao Yang et al. (Xiao Yang, Tian Hongyu, Zhang Shishi, Sun Baoguo. Asymmetric synthesis of chiral flavor compound γ-lactone. Surfactant Chemical Industry, 2010, 40: 194-198) used malonic acid and fatty aldehyde as starting materials and prepared a series of optically active γ-lactones through five steps of Knoevenagel condensation, esterification, Sharpless asymmetric dihydroxylation, elimination and reduction. The ee value of the obtained R-configuration γ-alkyl lactone was only about 90%, and more metal catalysts were involved. At the same time, due to the large number of synthesis steps and the high product loss, the final yield was not high. In the process of splitting after obtaining the racemate, Xie et al. (Xie JC, Cheng J, Han HL, Sun BG. Yanik GW. Resolution of racemic γ-lactone flavors on Chiralpak AD by packed column supercritical fluid chromatography. Food Chemistry, 2011, 124: 1107-1112) used a chiral chromatographic column in supercritical fluid chromatography to physically split the racemic lactone, but the splitting method had high requirements on chromatographic conditions; Enzelberger et al. (Enzelberger MM, Bornscheuer UT, Gatfield I, Schmid RD. Lipase-catalyzed resolution of γ-and δ-lactones. Journal of Biotechnology, 1997, 56: 129-133) The lipase extracted from the dyeing viscous bacteria can be used to split the alkyl lactones with long side chains, and the corresponding optically active δ-undecyl / dodecyl lactones are obtained according to the position and length of the side chains, but the γ-alkyl lactones cannot be split, and the optical purity of the products is low.

[0005] In summary, the enzyme-catalyzed preparation of chiral lactones can overcome the pollution problems caused by the use of inorganic catalysts and organic solvents in chemical catalysis. However, there is currently a lack of highly active and optically selective biocatalysts. Summary of the invention

[0006] Based on the current situation that the existing enzyme catalysis technology for preparing chiral lactones lacks a biocatalyst with high activity and high optical selectivity, the present invention provides a 4-oxodecanoic acid reductase mutant with significantly improved catalytic performance, its encoding gene, and a recombinant expression vector and a recombinant expression transformant containing the gene, as well as the use of the 4-oxodecanoic acid reductase mutant or the recombinant expression transformant in catalyzing a potentially chiral carbonyl compound to prepare an optically pure (R)-γ lactone.

[0007] The present invention provides a 4-oxodecanoic acid reductase mutant, which has the advantages of mild reaction conditions, extremely high product optical purity, and the like in catalyzing a reaction for synthesizing chiral lactones, and has strong industrial application prospects.

[0008] The purpose of the present invention can be achieved by the following technical solutions:

[0009] One of the technical solutions of the present invention is to provide a carbonyl reductase mutant with significantly improved catalytic performance and enantioselectivity, namely a 4-oxodecanoate reductase mutant.

[0010] The present invention uses carbonyl reductase OdCR2 from Saccharomyces cerevisiae as the parent, and screens mutants with improved catalytic performance through saturation mutation and semi-rational transformation. The amino acid sequence of carbonyl reductase OdCR2 is shown in SEQ ID No. 2. Carbonyl reductase OdCR2 is derived from a brewer's yeast Saccharomyces cerevisiae, denoted as S288C, and the brewer's yeast (Saccharomyces cerevisiae) S288C is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration, with a deposit number of CGMCC No. 22135, a deposit date of April 6, 2021, and a deposit location of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and is disclosed in patent CN113278599A.

[0011] In the present invention, the carbonyl reductase OdCR2 gene is used as the parent, and a site-directed saturation mutagenesis strategy is adopted to carry out directed evolution thereof, and a high-throughput primary screening by an ELISA reader and a secondary screening by an ultraviolet spectrophotometer are combined to identify 4-oxodecanoate reductase with significantly improved catalytic performance.

[0012] The 4-oxodecanoate reductase mutant (hereinafter referred to as OdCR2 mutant) provided by the present invention is a derivative protein with a new amino acid sequence formed by replacing one of the amino acid residues at position 134 of leucine, position 182 of aspartic acid, position 132 of glycine, and position 133 of serine in the amino acid sequence shown in SEQ ID No. 2 with other amino acid residues, and the derivative protein has higher catalytic performance and thermal stability than the protein composed of the amino acid sequence shown in SEQ ID No. 2.

[0013] The OdCR2 mutant has any one of the following sequences:

[0014] (1) replacing the leucine at position 134 of the amino acid sequence shown in SEQ ID No. 2 with valine;

[0015] (2) replacing the aspartic acid at position 182 of the amino acid sequence shown in SEQ ID No. 2 with serine;

[0016] (3) replacing the leucine at position 134 of the amino acid sequence shown in SEQ ID No. 2 with arginine;

[0017] (4) The aspartic acid at position 182 of the amino acid sequence shown in SEQ ID No. 2 is replaced by valine.

[0018] The second technical solution of the present invention is to provide a nucleic acid encoding the 4-oxodecanoate reductase mutant (OdCR2 mutant).

[0019] The nucleic acid encodes any one of the OdCR2 mutants described in one of the technical solutions.

[0020] The nucleotide sequence encoding the OdCR2 mutant is the nucleic acid sequence encoding the OdCR2 mutant as described in one of the technical solutions.

[0021] The method for obtaining the nucleic acid encoding the OdCR2 mutant in the present invention is a conventional method in the art, for example, the nucleic acid can be obtained by cloning the gene sequence of the OdCR2 mutant as described in one of the technical solutions through genetic engineering technology or by artificial full sequence synthesis.

[0022] The third technical solution of the present invention: provides a recombinant expression vector, which contains the nucleic acid as described in the second technical solution.

[0023] The recombinant expression vector can be obtained by conventional methods in the art. The optional method includes: connecting the nucleic acid encoding the OdCR2 mutant in the present invention to various commercially available empty vectors to construct it. The commercially available empty vector can be various plasmid vectors conventional in the art, as long as the recombinant expression vector can be normally replicated in the corresponding expression host and express the corresponding reductase. The preferred plasmid of this scheme is pET-28a(+).

[0024] Preferably, the recombinant expression vector of the present invention can be prepared by the following method: the DNA fragment of the OdCR2 mutant obtained by PCR amplification is double-digested with restriction endonucleases EcoR I and Xho I, and the empty plasmid pET-28a(+) is also double-digested with restriction endonucleases EcoR I and Xho I, the DNA fragment of the OdCR2 mutant and the empty plasmid after the above-mentioned enzyme digestion are recovered, and they are connected using T4 DNA ligase to construct a recombinant expression vector containing the nucleic acid sequence of the OdCR2 mutant.

[0025] Technical solution four of the present invention: provides a recombinant expression transformant, which comprises the recombinant expression vector described in technical solution three.

[0026] The recombinant expression transformant of the present invention can be obtained by transforming the recombinant expression vector into a host microorganism by conventional methods in the art. The host microorganism can be any conventional host microorganism in the art, as long as the recombinant expression vector can stably replicate itself and effectively express after being induced by an inducer. The host microorganism is preferably Escherichia coli BL21 (DE3).

[0027] The fifth technical solution of the present invention is to provide a recombinant 4-oxodecanoic acid reductase mutant (OdCR2 mutant) catalyst, wherein the recombinant 4-oxodecanoic acid reductase mutant (OdCR2 mutant) catalyst is any one of the following forms:

[0028] (1) culturing the recombinant expression transformant as described in the fourth technical solution, and isolating the transformant cells containing the 4-oxodecanoate reductase mutant (OdCR2 mutant);

[0029] (2) culturing the recombinant expression transformant as described in the fourth technical solution, and isolating the crude enzyme solution containing the 4-oxodecanoate reductase mutant (OdCR2 mutant);

[0030] (3) Drying the crude enzyme liquid of the 4-oxodecanoate reductase mutant (OdCR2 mutant) to obtain crude enzyme powder.

[0031] Wherein, in order to obtain the OdCR2 mutant catalyst, the culture method and conditions of the recombinant expression transformant are conventional methods and conditions in the art. The culture medium is any culture medium in the art that can make the transformant grow and produce 4-oxodecanoate reductase.

[0032] The method for determining the activity of the OdCR2 mutant described in the present invention is as follows: the reaction takes place in a 1 ml cuvette, 970 ul PBS (pH 7.0) + 10 ul pure enzyme + 10 ul NADPH (0.2 mM) + 10 ul substrate (2 mM), the reaction time is 1 min, and the decrease in absorbance is observed. The activity of the mutant is determined in a constant temperature spectrophotometer.

[0033] Enzyme activity (U) = EW × V × 103 / (6220 × l)

[0034] Where, EW is the change in absorbance at 340 nm within 1 min; V is the volume of the reaction solution, in mL; 6220 is the molar extinction coefficient of NADPH, in L / (mol·cm); l is the optical path distance, in cm. One enzyme activity unit (U) is defined as the amount of enzyme required to catalyze the oxidation of 1 μmol NADPH per minute under the above conditions.

[0035] Technical solution six of the present invention: providing a 4-oxodecanoate reductase mutant or a recombinant 4-oxodecanoate reductase mutant (OdCR2 mutant) catalyst for use as a catalyst in the asymmetric reduction of 4-oxodecanoate compounds.

[0036] The specific synthesis of optically pure γ-alkyl lactone is as follows:

[0037]

[0038] In some embodiments of the present invention, the 4-oxodecanoate reductase mutant or recombinant 4-oxodecanoate reductase mutant catalyst catalyzes the asymmetric reduction reaction of carbonyl compounds in the presence of glucose dehydrogenase, glucose, coenzyme NADPH or NADP + is being carried out.

[0039] NADPH or NADP required for the reaction + The dosage is 0.2-0.5 mmol / L. Glucose can be used as a cosubstrate during the reaction, and the coenzyme cycle of NADPH in the reaction system is realized by catalysis of BmGDH dehydrogenase. The dosage of glucose dehydrogenase can be 50-200 U / mmol of the potential chiral carbonyl compound, the dosage of glucose can be 1.5 times the molar amount of the potential chiral carbonyl compound, and the dosage of the coenzyme NADPH or NADP+ is 0.1-0.5 mmol / L. The concentration of the potential chiral carbonyl compound can be 2-20 mmol / L, and the dosage of the OdCR2 mutant or recombinant OdCR2 mutant catalyst can be selected to be 5-20 U / mmol of the potential chiral carbonyl compound.

[0040] The phosphate buffer required in the asymmetric reduction reaction is a conventional phosphate buffer in the art, preferably a PBS buffer, with a concentration of 100 mmol / L.

[0041] Preferably, the reaction is carried out under shaking or stirring conditions; the temperature is 20-40°C, more preferably 30°C; the reaction time is based on the time when the substrate is completely reacted or the reaction terminates on its own, and more preferably the reaction time is less than 24h.

[0042] The invention provides a 4-oxodecanoic acid reductase mutant with better catalytic performance, which can efficiently catalyze the asymmetric reduction of the carbonyl group of 4-oxodecanoic acid to prepare optically pure chiral lactone.

[0043] Compared with the prior art, the invention has the following innovations and beneficial effects:

[0044] (1) The present invention couples the OdCR2 mutant with glucose dehydrogenase to achieve in situ regeneration of the coenzyme, greatly reducing the amount of coenzyme used; and can achieve a conversion rate of 98% within 3 hours, and the optical purity of the product can reach 99% (R).

[0045] (2) Compared with the parent reductase mutant, the 4-oxodecanoate reductase mutant obtained in the present invention has a specific enzyme activity 16 times higher than that of the parent when targeting 4-oxodecanoate, has a higher catalytic activity, and has a better industrial application prospect. DETAILED DESCRIPTION

[0046] Each reaction or detection condition described in the content of the present invention can be combined or changed according to the common sense in the art, and can be verified by experiments. The technical scheme and technical effects of the present invention will be clearly and completely described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited to these embodiments, and any changes or equivalent substitutions that do not deviate from the concept of the present invention are included in the protection scope of the present invention.

[0047] Sources of materials in the following examples:

[0048] The parent recombinant plasmid pET28a-OdCR2 contains the nucleic acid sequence shown in SEQ ID No. 1 in the sequence listing, which is constructed by the inventors themselves and is also disclosed in patent CN113278599A "Saccharomyces cerevisiae carbonyl reductase and its application in the preparation of optically active alkyl lactones".

[0049] The plasmid vector pET28a was purchased from Novagen.

[0050] E. coli BL21 (DE3) competent cells, 2× Taq PCR MasterMix, and agarose gel DNA recovery kit were purchased from Beijing Tiangen Biochemical Technology Co., Ltd.

[0051] Restriction endonucleases EcoRI, XhoI, and SacI are all commercially available products from New England Biolabs (NEB).

[0052] Unless otherwise stated, the specific experiments in the following examples were performed according to conventional methods and conditions in the art, or in accordance with the commercial instructions of the kits.

[0053] Example 1 Semi-rational construction of OdCR2 mutants

[0054] Homology modeling was performed on carbonyl reductase OdCR2, and then molecular docking was performed with the modeled protein three-dimensional model and 4-oxodecanoic acid. According to the catalytic mechanism and binding energy of carbonyl reductase OdCR2, a suitable docking model was selected. The activity of the enzyme was further improved by performing site-directed saturation mutagenesis on the amino acid near the substrate pocket. In the three-dimensional structure of the OdCR2wt amino acid sequence shown in SEQ ID No. 2, the amino acid residues around the substrate-oxodecanoic acid binding site include: phenylalanine at position 130, serine at position 131, glycine at position 132, serine at position 133, leucine at position 134, etc. Site-directed saturation mutagenesis was performed on the amino acid residues at these sites, and the primers used were as shown in Table 1.

[0055] Table 1 Primers for mutation sites

[0056]

[0057]

[0058] Using pET28a-OdCR2wt as template, PrimeStar HS premix was used for PCR amplification. The PCR system was: 2× PrimeStar HS premix 10μL, upstream and downstream primers 1μL each, pET28a-OdCR2 plasmid 40ng, DMSO 1μL, and sterile distilled water to make up to 20μL. PCR reaction procedure: (1) 95℃ pre-denaturation for 5min; (2) 94℃ denaturation for 30s; (3) 55℃ annealing for 30s; (4) 72℃ extension for 6.3min; steps (2) to (4) were repeated for 30 cycles; and finally, 72℃ extension for 10min. Add 1 μL of Dpn I enzyme to 20 μL of PCR product and digest the template at 37°C for 3 hours. Transform the digestion product into E. coli BL21 (DE3) competent cells and evenly spread on LB agar plates containing 50 μg / mL kanamycin. Place in a 37°C incubator and culture for about 12 hours. Pick the obtained monoclonal colony into a 96-well deep-well plate for culture, break the cell wall, use NADPH as a coenzyme, perform high-throughput activity screening on the expressed protein in a 96-well plate, purify and characterize the mutants with higher activity, and sequence the corresponding genes.

[0059] Through the high-throughput screening using an ELISA instrument as described in Example 1, it was found that the mutant in which the leucine at position 134 was replaced by valine (L134V) had improved activity and selectivity for 4-oxodecanoic acid.

[0060] Method for determining the enzyme activity of the OdCR mutant: preheat 1 mL of a reaction system (100 mmol / L sodium phosphate buffer, pH 6.0) containing 2 mmol / L 4-oxodecanoic acid and 0.1 mmol / L NADPH to 40°C, then add an appropriate amount of the OdCR mutant enzyme solution, incubate the reaction at 40°C, detect the absorbance change at 340 nm on a spectrophotometer, record the absorbance change within 1 min, and calculate the enzyme activity.

[0061] Through screening, mutants with significantly improved activity on 4-oxodecanoic acid were obtained, and the sequences of these mutants and the activities of these mutants on 2-pentanoylbenzonitrile are listed in Table 2. Table 2 provides a list of carbonyl reductase OdCR mutants with specific sequences having relevant activities disclosed in the present invention. In Table 2, the mutant proteins have improved specific activity and enantioselectivity over the protein consisting of the amino acid sequence shown in SEQ ID No. 2.

[0062] Table 2 Crude enzyme activities of mutants

[0063]

[0064] The 4-oxodecanoate reductase mutant amino acid has one of the following sequences:

[0065] Y1 replaces the leucine at position 134 of the amino acid sequence shown in SEQ ID No. 2 with valine;

[0066] Y2 replaces the leucine at position 134 of the amino acid sequence shown in SEQ ID No. 2 with arginine;

[0067] Y3 replaces the aspartic acid at position 182 of the amino acid sequence shown in SEQ ID No. 2 with valine;

[0068] Y4 replaces the aspartic acid at position 182 of the amino acid sequence shown in SEQ ID No. 2 with serine.

[0069] Example 2 Recombinant E. coli BL21 (DE3) / pET28a-OdCR Y1 Expression and enzymatic preparation.

[0070] It is preferred that the OdCR2 obtained in Example 1 Y1 Recombinant E. coli BL21

[0071] (DE3) / pET28a-OdCR Y1Inoculate into LB medium containing 50μg / mL kanamycin, shake and culture at 37℃ for 12h, then inoculate into a 500mL Erlenmeyer flask containing 100mL LB medium (containing 50μg / ml kanamycin) at a 1% (v / v) inoculation amount, place in a 37℃, 180rpm shaker for shaking culture, when the OD600 of the culture reaches 0.6, add IPTG with a final concentration of 0.2mmol / L as an inducer, and induce at 16℃ for 24h. Centrifuge the culture at 8000×g for 10min, collect the cells, and wash twice with physiological saline to obtain resting cells. Suspend the cells obtained from 100mL of the culture in 15mL of sodium phosphate buffer (100mM, pH 7.0), and perform ultrasonic disruption in an ice-water bath as follows: 350W power, 4s working, 6s rest, 10min disruption time, centrifuge at 12000×g for 40min at 4℃, and collect the supernatant crude enzyme solution. In addition, the harvested crude enzyme liquid can be freeze-dried to obtain freeze-dried enzyme powder.

[0072] Example 3 Recombinant OdCR Y1 Catalytic synthesis of lactones

[0073] In a 250 mL three-necked round-bottom flask, add substrate 4-oxodecanoic acid (5 mmol), D-glucose (10 mmol), NADP+ (200 μmol), lyophilized cells of E. coli / BmGDH (400 mg), E. coli BL21(DE3) / pET28a-OdCR Y1 Wet cells (5 g) and 100 mL sodium phosphate buffer (100 mM, pH 6.0). The reaction was carried out at 30 ° C and pH 6.0, with mechanical stirring at 400 rpm for 16 hours. The reaction was titrated automatically with 1.0 mol / L NaOH aqueous solution to keep the pH of the reaction system constant at 6.0. After the reaction, the pH of the reaction system was adjusted to 2.0 with 20% (w / v) H2SO4 to acidify the substrate / product molecules, and then the mixture was heated at 90 ° C for 2 hours to inactivate the enzyme protein and denature it to precipitate, and promote the lactonization of the product. The product was extracted with ethyl acetate (4×50 mL), the organic phases were combined, washed with saturated brine, dried overnight (>8 h) over anhydrous Na2SO4, and the sodium sulfate solid was filtered out and concentrated by rotary evaporation under reduced pressure. The crude product was further purified by silica gel column chromatography using a mixture of petroleum ether and ethyl acetate (10:1, v / v) as the eluting solvent to obtain the product (R)-γ lactone.

[0074] It can be seen that the recombinant mutant enzyme preparation obtained by the method of the present invention can efficiently catalyze the reduction of 4-oxodecanoic acid to generate the corresponding hydroxy ester, and then form (R)-γ lactone through intramolecular cyclization. This optically pure compound can be used as a fragrance for industrial applications and has a high application value.

[0075] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A 4-oxodecanoate reductase mutant, It is characterized in that The amino acid sequence of the 4-oxodecanoate reductase mutant is selected from one of the following: (1) replacing the leucine at position 134 of the amino acid sequence shown in SEQ ID No. 2 with valine; (2) replacing aspartic acid at position 182 of the amino acid sequence shown in SEQ ID No. 2 with serine; (3) replacing the leucine at position 134 of the amino acid sequence shown in SEQ ID No. 2 with arginine; (4) The aspartic acid at position 182 of the amino acid sequence shown in SEQ ID No. 2 is replaced by valine.

2. An isolated nucleic acid, It is characterized in that The nucleic acid encodes the 4-oxodecanoate reductase mutant according to claim 1.

3. A recombinant expression vector, It is characterized in that The recombinant expression vector comprises the nucleic acid according to claim 2.

4. A recombinant expression transformant, It is characterized in that The recombinant expression transformant comprises the recombinant expression vector according to claim 3.

5. A recombinant 4-oxodecanoate reductase mutant catalyst, It is characterized in that The recombinant 4-oxodecanoate reductase mutant catalyst is any one of the following forms: (1) culturing the recombinant expression transformant according to claim 4, and isolating transformant cells containing the 4-oxodecanoate reductase mutant; (2) culturing the recombinant expression transformant according to claim 4, and isolating a crude enzyme solution containing the 4-oxodecanoate reductase mutant; (3) Drying the crude enzyme liquid of the 4-oxodecanoate reductase mutant to obtain crude enzyme powder.

6. Use of the 4-oxodecanoic acid reductase mutant according to claim 1 or the recombinant 4-oxodecanoic acid reductase mutant catalyst according to claim 5 as a catalyst in the asymmetric reduction of 4-oxodecanoic acid compounds.

7. The use according to claim 6, It is characterized in that The 4-oxodecanoic acid reductase mutant or the recombinant 4-oxodecanoic acid reductase mutant catalyst catalyzes the asymmetric reduction reaction of 4-oxodecanoic acid to generate (R)-γ lactone, which is carried out in glucose dehydrogenase, glucose, and coenzyme NADPH or NADP+.

8. The use according to claim 7, It is characterized in that The dosage of the glucose dehydrogenase is 50-200 U / mmol 4-oxodecanoic acid, the dosage of the glucose is 1.5 times the molar amount of 4-oxodecanoic acid, and the dosage of the coenzyme NADPH or NADP+ is 0.1-0.5 mmol / L.

9. The use according to claim 7, It is characterized in that The concentration of 4-oxodecanoic acid is 2-20 mmol / L, and the dosage of the 4-oxodecanoic acid reductase mutant or the recombinant 4-oxodecanoic acid reductase mutant catalyst is selected to be 5-20 U / mmol 4-oxodecanoic acid.

10. The use according to claim 7, It is characterized in that The reaction is carried out under shaking or stirring conditions; the temperature is 20-40°C, and the reaction time is based on the time when the substrate is completely reacted or the reaction terminates on its own.

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