CN115433747A - Enzymatic synthesis method of puerarin 6'' -O-acetate - Google Patents

Abstract

The invention provides two enzymatic synthesis methods of puerarin 6 '-O-acetate, in particular relates to a method for synthesizing puerarin 6' -O-acetate by catalyzing puerarin and diacerein or acetyl coenzyme A by taking maltose-O-acyltransferase (MAT) as a biocatalyst, and belongs to the field of biological catalysis. Through condition optimization and MAT directed evolution, the transformation efficiency of mutants MAT-E125V, MAT-E125M and MAT-E125R on puerarin can be improved from 68.10 percent to 94.03 percent. The conversion efficiency of mutants MAT-E125N, MAT-E125T and MAT-E125P to puerarin can be improved from 29.62 percent to 68.53 percent by taking acetyl coenzyme A as an acyl donor.

Description

Enzymatic synthesis method of puerarin 6' -O-acetate

Technical Field

The invention belongs to the field of biocatalysis, and particularly relates to a method for enzymatically synthesizing puerarin 6 '-O-acetate by taking diacerein as an acyl donor and a method for enzymatically synthesizing puerarin 6' -O-acetate by taking acetyl coenzyme A as an acyl donor.

Background

Puerarin (puerarin) is an isoflavone compound separated from radix Puerariae. At present, puerarin injection is developed and marketed, and is commonly used for treating diseases such as coronary heart disease, angina pectoris, sudden deafness and the like in clinic. In addition, puerarin also has the functions of protecting the nervous system, repairing liver injury and the like, and is also used for combined medication of diseases such as diabetes and complications thereof, alzheimer disease, alcoholic liver injury and the like. With the expansion of clinical application range, adverse reactions caused by puerarin are gradually reported, including drug fever, allergy, hemolysis and the like. The adverse reactions are mostly related to poor solubility and low bioavailability of puerarin.

The puerarin has special structure, and the 4' -position and 7-position phenolic hydroxyl groups of the puerarin easily form molecular hydrogen bonds, so that the acting force among puerarin molecules is enhanced, the solubility of the puerarin in an oil system and a water-soluble system is reduced, the puerarin hardly reaches effective concentration, and the clinical curative effect is reduced. In order to improve the solubility and bioavailability of puerarin, puerarin is often structurally modified to obtain puerarin derivatives with improved properties.

The acylation modification can help to improve the lipid solubility of the puerarin and strengthen the biological activity and the drug effect of the puerarin. Because puerarin has a polyhydroxy structure, the biological catalysis based on enzymatic synthesis becomes a powerful tool for single-site acylation modification of puerarin due to high selectivity. Based on the current research situation, the development of an enzymatic synthesis method of puerarin mono-acylated derivatives is urgently needed.

Disclosure of Invention

The invention aims to provide a method for enzymatically synthesizing puerarin 6' -O-acetate by taking diacerein as an acyl donor.

The invention provides a method for enzymatically synthesizing puerarin 6 '-O-acetate by taking diacerein as an acyl donor, which takes Maltose-O-acyltransferase (MAT, access No: ACT 42309.1) as a catalyst to catalyze puerarin and diacerein to generate puerarin 6' -O-acetate, and has the following specific reaction formula:

specifically, in the above reaction process, 1 molecule of the acyl donor diacerein can provide 2 molecules of the acetyl group.

The maltose-O-acyltransferase MAT is derived from escherichia coli BL21 (DE 3), and the amino acid sequence and the nucleotide sequence of the maltose-O-acyltransferase MAT are shown as SEQ ID NO.1 and SEQ ID NO.2.

In the enzyme catalysis system, the molar ratio of puerarin to acyl donor diacerein is 1; the reaction temperature is 20-80 ℃; the reaction pH is 3-9.

Preferably, the reaction temperature is 20 to 60 ℃; the reaction pH is 4-8. More preferably, the reaction temperature is 30 to 60 ℃; the reaction pH is 4.5-7. Most preferably, the reaction temperature is 30-50 ℃; the reaction pH is 4.5-6.

Under the most preferable conditions, the invention also provides that the conversion efficiency of enzymatic synthesis of puerarin 6' -O-acetate by taking maltose-O-acyltransferase MAT as a biocatalyst and diacerein as an acyl donor is 68.10%.

Furthermore, in order to improve the synthesis efficiency of the method, the invention also provides maltose-O-acyltransferase mutants MAT-E125V, MAT-E125M and MAT-E125R which are obtained by directed evolution and have obviously improved activities. The synthesis efficiency of the puerarin 6' -O-acetate is improved to 94.03%,92.81% and 92.18% respectively by taking diacerein as an acyl donor.

Specifically, the amino acid sequences of maltose-O-acyltransferase mutants MAT-E125V, MAT-E125M and MAT-E125R are selected from SEQ ID NO.3, SEQ ID NO.5 and SEQ ID NO.7, and the nucleotide sequences are selected from SEQ ID NO.4, SEQ ID NO.6 and SEQ ID NO.8.

Another objective of the present invention is to provide a method for enzymatically synthesizing puerarin 6' -O-acetate using acetyl-CoA as an acyl donor.

The invention provides a method for enzymatic synthesis of puerarin 6' -O-acetate by taking acetyl coenzyme A as an acyl donor. Takes maltose-O-acyltransferase MAT as a catalyst to catalyze puerarin and acetyl coenzyme A to generate puerarin 6' -O-acetate, and the specific reaction formula is as follows:

specifically, the maltose-O-acyltransferase is derived from Escherichia coli BL21 (DE 3), and the amino acid sequence and the nucleotide sequence of the maltose-O-acyltransferase are shown as SEQ ID NO.1 and SEQ ID NO.2.

In the enzyme catalysis system, the molar ratio of puerarin to acyl donor acetyl coenzyme A is 1; the reaction temperature is 20-80 ℃; the reaction pH is 3-7.

Preferably, the reaction temperature is 20 to 70 ℃; the reaction pH is 3-6. More preferably, the reaction temperature is 30 to 60 ℃; the reaction pH is 3-5. Most preferably, the reaction temperature is 30-50 ℃; the reaction pH is 3.5-5.

Under the most preferred conditions, the invention also provides that the conversion efficiency of enzymatic synthesis of puerarin 6' -O-acetate by using maltose-O-acyltransferase MAT as biocatalyst and acetyl-CoA as acyl donor is 29.62%.

Furthermore, in order to improve the synthesis efficiency of the method, the invention also provides maltose-O-acyltransferase mutants MAT-E125N, MAT-E125T and MAT-E125P which are obtained by directed evolution and have obviously improved activity. The synthesis efficiency of puerarin 6' -O-acetate is improved to 68.53%,57.37% and 56.41% respectively by taking acetyl coenzyme A as an acyl donor.

Specifically, the amino acid sequences of maltose-O-acyltransferase mutants MAT-E125N, MAT-E125T and MAT-E125P are selected from SEQ ID NO.9, SEQ ID NO.11 and SEQ ID NO.13, and the nucleotide sequences are selected from SEQ ID NO.10, SEQ ID NO.12 and SEQ ID NO.14.

The invention also provides an expression vector containing the nucleotide sequence, which is selected from pET-28a (+).

The invention also provides a host cell of the expression vector. Preferred host cells thereof are selected from the group consisting of E.coli BL21 (DE 3).

The beneficial technical effects are as follows:

the invention provides two methods for enzymatically synthesizing puerarin 6' -O-acetate, wherein maltose-O-acyltransferase MAT and a mutant thereof are respectively used as biocatalysts, and diacerein or acetyl coenzyme A are used as acyl donors. The two methods can prepare puerarin 6' -O-acetate in one step, avoid complex protection and deprotection processes in chemical synthesis, and have the advantages of high site selectivity, easiness in preparation, mild conditions, simplicity and convenience in operation and the like. Particularly, in the method for preparing puerarin 6' -O-acetate by taking diacerein as an acyl donor, 1 molecule of diacerein can provide 2 molecules of acetyl, so that the method is expected to realize the aims of high-efficiency synthesis and cost reduction, and can be widely applied to the fields of medicines, foods, cosmetics and the like.

Drawings

FIG. 1: analyzing a Coomassie brilliant blue staining result by MAT recombinant protein SDS-PAGE, wherein M is a protein molecular weight standard; 1 is BL21 (DE 3) [ pET28a-MAT ] induction result; 2 is the induction result of BL21 (DE 3) [ pET28a (+) ] and 3 is the purified MAT recombinant protein.

FIG. 2: and (3) a detection result of synthesizing puerarin 6' -O-acetate by catalyzing puerarin and acetyl coenzyme A by MAT recombinase: (A) Liquid phase detection results, wherein a is a control group (MAT pure enzyme is not added); b is the reaction group catalyzed by MAT. (B) Ultraviolet spectrum comparison chart of puerarin and acetylated derivative 1a thereof.

FIG. 3: and (3) a detection result of synthesizing puerarin 6' -O-acetate by catalyzing puerarin and diacerein by MAT recombinase: (A) And (3) detecting the result in a liquid phase, wherein a is a control group (MAT-free pure enzyme) and b is a reaction group catalyzed by MAT. (B) The ultraviolet spectrum contrast chart of puerarin and its acetylated derivative 1a, and the ultraviolet spectrum chart of diacerein and its deacetylated compound.

FIG. 4: liquid mass analysis of synthesis of puerarin 6' -O-acetate catalyzed by MAT recombinase.

FIG. 5 is a schematic view of: MAT recombinase catalyzed puerarin 6' -O-acetate synthesis ¹ H-NMR spectrum.

FIG. 6: MAT recombinase catalyzed puerarin 6' -O-acetate synthesis ¹³ C-NMR spectrum.

FIG. 7: influence of pH on the synthesis of puerarin 6 "-O-acetate with acetyl-CoA as acyl donor.

FIG. 8: temperature effects on the synthesis of puerarin 6 "-O-acetate with acetyl-CoA as acyl donor.

FIG. 9: influence of pH on the synthesis of puerarin 6' -O-acetate with diacerein as acyl donor.

FIG. 10: temperature effect on the synthesis of puerarin 6 "-O-acetate with diacerein as acyl donor.

FIG. 11: and (3) comparing the efficiency of synthesizing puerarin 6' -O-acetate catalyzed by MAT alanine mutant by taking acetyl coenzyme A as acyl donor.

FIG. 12: and (3) comparing the catalytic synthesis efficiency of puerarin 6' -O-acetate by using diacerein as an acyl donor and using the MAT alanine mutant.

FIG. 13 is a schematic view of: and (3) comparing the catalytic synthesis efficiency of puerarin 6' -O-acetate by using acetyl coenzyme A as an acyl donor and using MAT 125 site saturated mutant.

FIG. 14: and (3) comparing the catalytic synthesis efficiency of puerarin 6' -O-acetate by using diacerein as an acyl donor and using MAT 125 site saturated mutant.

Detailed Description

The invention is further illustrated by the following examples which are intended to be illustrative only and not to limit the scope of the claims in any way.

The experimental procedures in the present invention are conventional unless otherwise specified.

BL21 (DE 3) competent, fast Mutagenis System kits of the invention were purchased from Kyoto Kogyo gold Biotech, inc., beijing.

Puerarin and diacerein used in the present invention were obtained from Kyoto pusi Biotech Co., ltd, and acetyl coenzyme A was obtained from Sigma Co., ltd.

The catalytic reaction of the invention is monitored and analyzed by a high performance liquid chromatograph Agilent 1200, the model number of a chromatographic column is SILGREEN C18 column,250 multiplied by 4.6mm,5 mu m, beijing, china, and the detection wavelength is 254nm. HPLC analysis conditions for the synthesis of puerarin 6' -O-acetate with acetyl-CoA as acyl donor: phase A is water; phase B is 100% acetonitrile. The HPLC analysis condition for synthesizing puerarin 6' -O-acetate by taking diacerein as an acyl donor is as follows: phase A is 0.1% phosphoric acid aqueous solution; phase B is 100% acetonitrile.

Example 1 inducible expression of MAT recombinant protein

mu.L of the previously constructed plasmid pET28a-MAT was added to the freshly thawed 100. Mu.L of BL21 (DE 3) competence, ice-washed for 30min, heat-shocked in a water bath at 42 ℃ for 45s, and immediately ice-washed for 2min. The conversion product was applied to a solution containing 50. Mu.g/mL ^-1 Kanamycin on LB plate, 37 degrees C inverted culture overnight. The single clone was picked up in 10mL of 50. Mu.g.mL ^-1 Culturing at 37 ℃ and 220rpm for 6h in an LB culture medium containing kanamycin, transferring into 100mL of LB culture medium containing kanamycin at the same concentration according to the proportion of 1 ₆₀₀ Approximately 0.6, isopropyl-. Beta. -D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.2mM and incubated at 18 ℃ for 18 hours at 160 rpm.

Example 2 SDS-PAGE detection of MAT recombinant protein

1.5mL of the induced bacterial solution was collected by centrifugation. Adding 100 μ L double distilled water to resuspend the thallus precipitate, and crushing thallus suspension by ultrasonication for 3s and 3s, wherein the total crushing time is 2min. Then, centrifugation is carried out at 12000rpm for 2min, 40 mu L of supernatant is taken, 10 mu L of 5 Xloading buffer is added, and after uniform mixing, boiling water bath is carried out for 5min, so that protein denaturation is complete. A6. Mu.L protein sample was subjected to SDS-PAGE analysis, and protein was developed by Coomassie Brilliant blue. The results show (FIG. 1) that there is a strong protein band around 25kDa in the sample group, consistent with the size of the molecular weight of the MAT recombinant protein. Since the recombinant MAT has His tag fused to its N-terminus, its protein size is equal to the sum of the molecular weights of the peptides expressed by the MAT protein and the pET-28a (+) vector sequence. No band was observed at the corresponding position in the control group, indicating successful expression of MAT protein in E.coli.

Example 3 purification and quantification of recombinant MAT protein

Centrifuging at 6566rpm and 4 deg.C for 6min by using large high speed centrifuge, and collecting thallus. The pellet was washed with PBS buffer (pH =8.0, 20 mM) at a volume ratio of 1L of zymogen solution to PBS buffer: the washed cells were resuspended at a volume of 30 mL. And (3) carrying out ultrasonic crushing under an ice bath condition, wherein parameters are set to 5s of ultrasonic treatment, the intermittent treatment is carried out for 5s, and the total time is 40min. Crushing a large amount of thallus by using a high-pressure cell crusher, and circularly crushing for 3 times under the pressure of 800 bar; centrifuging at 10000rpm and 4 deg.C for 30min. The supernatant of the disrupted broth was collected, and 2.5. Mu.L of recombinant DNase I (RNase-free) (TaKaRa Co.) was added to each liter of the supernatant of the disrupted broth, and the mixture was digested at 4 ℃ overnight to remove nucleic acids. Then, the mixture was centrifuged again at 10000rpm at 4 ℃ for 30min, and the resulting supernatant was purified by affinity chromatography on a Ni gel purification resin column. Firstly, adding the processed protein sample solution into pre-balanced Ni glue purification resin to combine the protein and the glue. The non-binding and non-specific binding proteins were then washed with elution buffer (pH 8.0, 20mM PBS buffer,10-30mM imidazole). The recombinant MAT protein with a histidine tag bound to the Ni gel was then eluted with an elution buffer (pH 8.0, 20mM PBS buffer,60-300mM imidazole), and the eluate was collected as purified MAT. SDS-PAGE analysis of 10. Mu.L of the effluent showed a single clear band around 25kDa (FIG. 1), indicating a higher purity of the purified protein sample.

The eluate containing the target protein was concentrated using a 10kD ultrafiltration tube to remove imidazole, phosphate and water. The ethanol stock solution was washed clean with deionized water before using the ultrafiltration tube, and then equilibrated with PBS buffer (pH =8.0, 20 mM), 10mL protein eluent was added for each ultrafiltration, and centrifuged at 6361rpm for 15min. The purified and concentrated protein is preserved at-20 deg.C by adding 20% glycerol. The Protein concentration is determined by quantitative experiments of purified and concentrated MAT Protein by a Bradford method, and the specific process refers to a Super-Bradford Protein Assay Kit which is purchased from Beijing kang, a century Biotechnology Co., ltd. Standard concentration bovine serum albumin BSA (2000. Mu.g. ML) was diluted with a diluent identical to the protein sample to be tested ^-1 ) Gradually diluting with deionized water to form gradient concentration (1500, 1000, 500, 250, 125 μ g. Per mL) ^-1 ) Taking 0. Mu.g/mL ^-1 The concentration is blank control; respectively adding 5 mu L of diluted BSA standard substance and a protein sample to be detected (stock solution or diluent) into a 96-well plate, adding 250 mu L of Bradford protein Assay reagent, repeating each group for 3 times in parallel, fully mixing, standing at room temperature for 10min, and detecting the light absorption value at the wavelength of 595nm by using an enzyme-linked immunosorbent Assay; in terms of protein content (μ g. ML) ^-1 ) As abscissa, absorbance value (A) ₅₉₅ ) And drawing a standard curve for a vertical coordinate, and calculating the concentration of the sample to be measured. According to the equation for the concentration of the standard BSA protein obtained: the concentration of MAT protein after purification is calculated to be 97.60 mg/mL ^-1 。

Example 4 catalytic Synthesis of Puerarin 6 "-o-acetate with acetyl-CoA as acyl Donor

Using purified MAT protein as a catalyst, a reaction system of 100. Mu.L of citric acid/sodium citrate buffer (pH 4.0, 10 mM) was established, including puerarin with a final concentration of 1mM, 1mM of acetyl coenzyme A and 127.61. Mu.g of purified MAT recombinant protein, reacted in a water bath at 40 ℃ for 2h, then quenched with 100. Mu.L of methanol, centrifuged at 12000rpm for 2min, the supernatant was filtered through a 0.22. Mu.L filter, and 30. Mu.L of the filtrate was taken for HPLC analysis. The blank control group was not added with MAT pure enzyme, and the other components and the treatment operation after the reaction were completely the same as those of the sample group. The blank and MAT catalyzed reaction groups were tested by HPLC elution conditions as shown in table 1. As shown in fig. 2A, a is a blank control group without MAT-added pure enzyme; b is the reaction result of puerarin and acetyl coenzyme A catalyzed by MAT. The results show that puerarin (1) is converted into a new compound (1 a) under the action of MAT pure enzyme compared with a control group without MAT pure enzyme, the new compound 1a and puerarin have similar ultraviolet spectrums (figure 2B), the 1a and puerarin have similar chemical structures, and puerarin acetylated derivatives are presumed.

TABLE 1 HPLC detection conditions for the catalytic synthesis of puerarin 6 "-O-acetate with acetyl-CoA as acyl donor

Example 5 catalytic Synthesis of Puerarin 6 "-O-acetate with diacerein as acyl Donor

Diacerein is a novel and efficient acetyl donor discovered by the research of the invention, and is not reported before. A reaction system of 100. Mu.L of citric acid/sodium citrate buffer (pH 5.5, 10 mM) was set up and puerarin (1 mM), diacerein (1 mM) and MAT pure enzyme (127.61. Mu.g) were incubated at 50 ℃ for 2h. In the control group, only diacerein and puerarin were added, and MAT pure enzyme was replaced with water. The control and experimental groups were run under the same conditions, after completion of the reaction, the reaction was stopped with 100. Mu.L of methanol, centrifuged at 12000rpm for 2min, the supernatant was filtered through a 0.22. Mu.L filter and 30. Mu.L of the filtrate was analyzed by HPLC. The samples were assayed by Agilent 1200 HPLC and the HPLC elution conditions are shown in Table 2.

As shown in FIG. 3A, in the control reaction without MAT pure enzyme, spontaneous trace hydrolysis of diacerein was observed to produce 4-acetylrhein (2 a), 5-acetylrhein (2 b), and rhein (2 c), which is consistent with the literature report. In the reaction group, the diacerein was found to be completely converted into rhein (2 c), indicating that diacerein has two acetyl groups removed in the reaction, i.e. 1 molecule of diacerein can provide 2 molecules of acetyl groups during the reaction. Compared with the blank control group, the compound peak (1 a) of the suspected puerarin acetylated derivative is also obviously observed in the reaction group, and the generation amount of the new compound is obviously higher than that of the reaction group using acetyl coenzyme A as an acyl donor. The ultraviolet absorption spectrum of the new compound 1a is similar to that of the substrate puerarin, which indicates that the structure of the new compound is similar to that of the puerarin.

TABLE 2 HPLC detection conditions for the catalytic synthesis of puerarin 6' -O-acetate with diacerein as acyl donor

Example 6 structural identification of Puerarin 6 "-O-acetate

1. Liquid mass analysis

ESI-MS shows the excimer ion peak m/z 459.13260[ m ] +H ] of the suspected puerarin acetylated derivative 1a] ⁺ Molecular formula of C ₂₃ H ₂₃ O ₁₀ (figure 4), the molecular weight and the structural formula of the puerarin monoacetylated derivative are consistent.

2. Nuclear magnetic analysis

In addition, a suspected puerarin acetylated derivative 1a was prepared, and the site of acetylation of the puerarin derivative was confirmed by nuclear magnetic resonance spectroscopy (fig. 5 and 6).

By CD ₃ OD-d ₄ As a solvent, from ¹³ The carbonyl carbon (C = O) and methyl (-CH) groups in the acetyl group are clearly observed in the C-NMR spectrum ₃ ) The characteristic signal peak of (1) was a chemical group which does not exist in the substrate puerarin, and thus it was judged that the resulting puerarin derivative 1a contained an acetyl group in the molecular structure (table 3). Furthermore, the chemical shift of the carbon atom on the mother nucleus of puerarin is not significantly changed compared with the standard chemical shift, but the C5 "and C6" in the glucose group are shifted correspondingly, wherein the chemical shift of C6 "is shifted to low field and the chemical shift of C5" is shifted to high field. According to the nuclear magnetic data shown in Table 3, it is shown that acetylation modification occurs on the C6 "-OH of puerarin. The above experimental data show that in MAT pure enzymeUnder the catalysis, acetyl coenzyme A or diacerein can be used as acyl donor to effectively synthesize puerarin monoacetylated derivative, i.e. puerarin 6' -O-acetate.

TABLE 3 preparation of Puerarin 6 "-O-acetate ¹ H and ¹³ c NMR data

Example 7 determination of Water partition coefficient of Puerarin and its derivative Puerarin 6' -O-acetate oil

The oil-water distribution condition of puerarin and puerarin 6 '-O-acetate derivatives thereof is determined by adopting a n-octanol-water distribution coefficient (log P) method so as to observe the lipid solubility of the puerarin and puerarin 6' -O-acetate derivatives thereof. A quantity of puerarin and puerarin 6' -O-acetate are mixed well in the same volume of hydrated n-octanol. Vortex and shake for 2min, then ultrasonic sound for 15min, after mixing well, 12000rpm, centrifuge for 5min. The solution is divided into an upper layer and a lower layer, 40 mu L of the solution is respectively taken from the upper layer and the lower layer, the corresponding concentrations C1 and C2 of the compound in the n-octanol solution and the water solution are calculated by HPLC, and the oil-water distribution coefficient is calculated according to a formula P = C1/C2. The results showed that the log P of puerarin was-0.413, while the log P (-0.354) of puerarin 6 "-O-acetate was improved, thus indicating that the lipid solubility of puerarin 6" -O-acetate obtained by the above enzymatic method was higher than that of puerarin.

EXAMPLE 8 determination of optimum reaction conditions for Synthesis of Puerarin 6' -O-acetate Using acetyl-CoA as acyl Donor 1, determination of optimum pH

In order to obtain the maximum conversion efficiency, the optimum reaction conditions for catalytically synthesizing puerarin 6' -O-acetate by using acetyl coenzyme A as an acyl donor are determined. Design 8 parallel reaction pH gradients: 3. 3.5, 4, 4.5, 5, 5.5, 6, 7, wherein each set was provided with 3 parallel samples. Wherein the pH is 3-5.5 and the pH is 10mM citrate buffer solution, and the pH is 6-7 and the pH is 20mM phosphate buffer solution. A reaction system of 100. Mu.L was established using a 1.5mL EP tube, and the reaction system and the termination method were referred to in example 4 and reacted for 2 hours. The peak area of the product 1a at a wavelength of 254nm was measured by HPLC-UV, and the HPLC conditions were as shown in Table 1. The relative values of each group at other pH conditions were calculated by calculating the product concentration from a standard curve of puerarin 6 "-O-acetate, defining the average of the conversion of 3 parallel reactions at the optimum pH as 100%. As shown in FIG. 7, the optimum pH for the catalytic synthesis of puerarin 6 "-O-acetate by MAT using acetyl-CoA as acyl donor is 4.0. The pH range of the reaction is narrow, the catalytic activity is strong only between pH3.5 and 4.5, and the activity is immediately and greatly reduced slightly beyond the range, so that the pH condition of the reaction needs to be strictly controlled to keep the high activity of MAT.

2. Determination of optimum temperature

7 parallel reaction temperature gradients were designed: 20 ℃, 30 ℃,40 ℃,50 ℃,60 ℃, 70 ℃ and 80 ℃, wherein each group is provided with 3 parallel samples. A reaction system of 100. Mu.L was prepared in a 1.5mL EP tube, and the reaction was carried out for 2 hours in accordance with example 4. The peak area of the product 1a at a wavelength of 254nm was measured by HPLC-UV, and the HPLC conditions are shown in Table 1. The concentration of the product was also calculated from the standard curve for puerarin 6 "-O-acetate, and the data processing method was identical to the determination of the optimum pH. As shown in FIG. 8, the optimal reaction temperature for MTA to catalyze puerarin acetylation is 40 ℃. The temperature adaptation range of MAT acetylation activity is wide, and when the temperature reaches 80 ℃, the reaction efficiency is still maintained at about 60 percent under the optimal temperature condition.

Example 9 determination of optimum reaction conditions for the Synthesis of Puerarin 6 "-O-acetate Using diacerein as acyl Donor

1. Determination of the optimum pH

The optimal reaction condition for synthesizing puerarin 6' -O-acetate under the catalysis of MAT is determined by taking diacerein as an acyl donor. The specific reaction system is as in example 5, 10 parallel reaction pH gradients are designed: 3. 3.5, 4, 4.5, 5, 5.5, 6, 7, 8, 9, each set being provided with 3 parallel samples. The product concentration was calculated by puerarin 6 "-O-acetate standard curve and the data processing method refers to example 8. As shown in fig. 9, MAT exerted the highest activity at pH 5.5-6.0. The reaction method also has strong pH sensitivity, and the pH value of the reaction system needs to be strictly controlled during reaction.

2. Determination of optimum temperature

The optimum temperature for the synthesis of puerarin 6' -O-acetate by MAT was tested in 10mM citric acid/sodium citrate buffer at pH 5.5 using diacerein as the acyl donor. 7 temperature gradients were set: 20. 30, 40, 50, 60, 70 and 80 ℃, and the specific reaction system is the same as that of the example 5. As shown in FIG. 10, the optimum reaction temperature for MAT was 50 ℃. When the temperature is higher than 50 ℃ the conversion of the product drops abruptly compared to lower temperatures (20-40 ℃).

Example 10 determination of enzymatic kinetic parameters

The enzymatic kinetic parameters of the catalytic synthesis of puerarin 6 "-O-acetate by MAT were determined at optimum pH and temperature with acetyl-coa and diacerein as acyl donors, respectively (table 4). The results show K of MAT on diacerein _m Is significantly lower than acetyl coenzyme A, which indicates that MAT has higher affinity for the acyl donor diacerein than acetyl coenzyme A.

TABLE 4 enzymatic kinetic parameters for MAT catalysis of puerarin 6 "-O-acetate synthesis

EXAMPLE 11 alanine mutation of MAT

1. Construction of MAT alanine mutants

In order to further improve the transformation efficiency of puerarin 6' -O-acetate, an MAT mutant with improved activity is screened by a directed evolution method. First, alanine scans were performed for 5 candidate key sites around the MAT active center. Site-directed mutagenesis was performed using the Fast mutagenesis System (Transgen) kit. Primer design is carried out according to the instructions in the kit, and each primer comprises a mutation site, a 5 'end overlapping region and a 3' end extension region. PCR was carried out using plasmid pET28a-MAT as a template and a mutant primer (SEQ ID NO.15-SEQ ID NO. 60) as a specific primer by a procedure and system in which 1. Mu.L of the template pET28a-MAT, 1. Mu.L of 10. Mu.M primers FET28aMAT and RET28aMAT, respectively, and 1. Mu.L of 2 XStart FastPfu PCR Supermix 25. Mu.L, dd H were contained in a 50. Mu.L system ₂ And (4) complementing O. PCR procedure: 94Pre-denaturation at deg.C for 5min; denaturation at 94 ℃ for 20s, annealing at 55-60 ℃ for 20s, and extension at 72 ℃ for 3min for 20 cycles; final extension at 72 ℃ for 10min and incubation at 4 ℃. The PCR product was digested with DMT demethylase. After colony PCR screening, sequencing verification is carried out and corresponding mutants are obtained.

2. Comparison of catalytic efficiency of MAT alanine mutants

The M101A, E125A, F81A, N83A and W137A mutants were compared for their efficiency in catalyzing the synthesis of puerarin 6 "-O-acetate. Under the best reaction condition, puerarin is taken as a receptor substrate, and equal amount of acetyl coenzyme A or diacerein is respectively added into the system, and the specific reaction system refers to examples 4 and 5. As a result, as shown in FIGS. 11 and 12, the catalytic activity of MAT-E125A was significantly increased compared to that of wild-type MAT, while the activities of the other 4 mutants were decreased to different degrees. The experimental data show that MAT-E125A has a promoting effect on the reaction efficiency in the two puerarin 6' -O-acetate enzymatic synthesis methods developed by the invention.

3. Catalytic efficiency comparison of MAT 125 site saturation mutant

The efficiency of catalyzing and synthesizing puerarin 6' -O-acetate by MAT 125 glutamic acid and 19 saturation mutants thereof is further determined. 16 mutants with improved activity were selected from the acyl donor acetyl-CoA (FIG. 13). Compared with the conversion efficiency (29.62%) of wild MAT to puerarin 6 '-O-acetate, the conversion rates of the mutant MAT-E125N, MAT-E125T and MAT-E125P to puerarin 6' -O-acetate are respectively increased to 68.53%,57.37% and 56.41%.

In addition, 11 mutants exhibiting higher activity were selected using diacerein as an acyl donor (fig. 14). The efficiency of synthesizing puerarin 6 '-O-acetate under the catalysis of wild MAT is 68.10%, and the conversion rates of the puerarin 6' -O-acetate by MAT-E125V, MAT-E125M and MAT-E125R are respectively increased to 94.03%,92.81% and 92.18% through directed evolution.

The results show that the catalytic efficiency of taking diacerein as an acyl donor is obviously higher than that of acetyl coenzyme A in the process of catalyzing and synthesizing puerarin 6' -O-acetate by MAT and mutants thereof. The enzymatic synthesis of puerarin 6 "-O-acetate using diacerein as acyl donor is a significant advantage since diacerein can provide double acetyl donor and is less expensive than acetyl-coa.

<110> institute of medicine of Chinese academy of medical sciences

<120> enzymatic synthesis method of puerarin 6'' -O-acetate

<160> 56

<170> SIPOSequenceListing 1.0

<210> 1

<211> 183

<212> PRT

<213> Escherichia coli (Escherichia coli)

<220>

<223> maltose-O-acyltransferase (MAT) amino acid sequence

<400> 1

Met Ser Thr Glu Lys Glu Lys Met Ile Ala Gly Glu Leu Tyr Arg Ser

1 5 10 15

Ala Asp Glu Thr Leu Ser Arg Asp Arg Leu Arg Ala Arg Gln Leu Ile

20 25 30

His Arg Tyr Asn His Ser Leu Ala Glu Glu His Thr Leu Arg Gln Gln

35 40 45

Ile Leu Ala Asp Leu Phe Gly Gln Val Thr Glu Ala Tyr Ile Glu Pro

50 55 60

Thr Phe Arg Cys Asp Tyr Gly Tyr Asn Ile Phe Leu Gly Asn Asn Phe

65 70 75 80

Phe Ala Asn Phe Asp Cys Val Met Leu Asp Val Cys Pro Ile Arg Ile

85 90 95

Gly Asp Asn Cys Met Leu Ala Pro Gly Val His Ile Tyr Thr Ala Thr

100 105 110

His Pro Ile Asp Pro Val Ala Arg Asn Ser Gly Ala Glu Leu Gly Lys

115 120 125

Pro Val Thr Ile Gly Asn Asn Val Trp Ile Gly Gly Arg Ala Val Ile

130 135 140

Asn Pro Gly Val Thr Ile Gly Asp Asn Val Val Val Ala Ser Gly Ala

145 150 155 160

Val Val Thr Lys Asp Val Pro Asp Asn Val Val Val Gly Gly Asn Pro

165 170 175

Ala Arg Ile Ile Lys Lys Leu

180

<210> 2

<211> 552

<212> DNA

<213> Escherichia coli (Escherichia coli)

<220>

<223> maltose-O-acyltransferase (MAT) nucleotide sequence

<400> 2

atgagcacag aaaaagaaaa gatgattgct ggtgagttgt atcgctcggc agatgagacg 60

ttatctcgcg atcgcctgcg cgctcgtcag cttattcacc gatacaatca ttccctggcg 120

gaagagcaca cattacgcca gcaaattctc gctgatctat tcggtcaggt gacagaggct 180

tatattgagc caacgtttcg ctgtgactat ggctataaca tttttctcgg taataatttt 240

ttcgccaact tcgattgcgt gatgcttgat gtctgcccta ttcgcatcgg tgataactgt 300

atgttggcac caggcgttca tatctacacg gcaacacatc ccatcgaccc tgtagcacgt 360

aatagcggtg ctgaactggg gaaacccgtc accatcggta ataacgtctg gattggcgga 420

cgcgcggtca ttaaccctgg tgtgaccatt ggtgataacg tcgtggtagc ctcaggtgca 480

gttgtcacaa aagatgtccc ggacaacgtt gtcgtgggcg gtaatccagc cagaataatt 540

aaaaaattgt aa 570

<210> 3

<211> 183

<212> PRT

<213> Artificial sequence

<220>

<223> MAT-E125V amino acid sequence

<400> 3

Met Ser Thr Glu Lys Glu Lys Met Ile Ala Gly Glu Leu Tyr Arg Ser

1 5 10 15

Ala Asp Glu Thr Leu Ser Arg Asp Arg Leu Arg Ala Arg Gln Leu Ile

20 25 30

His Arg Tyr Asn His Ser Leu Ala Glu Glu His Thr Leu Arg Gln Gln

35 40 45

Ile Leu Ala Asp Leu Phe Gly Gln Val Thr Glu Ala Tyr Ile Glu Pro

50 55 60

Thr Phe Arg Cys Asp Tyr Gly Tyr Asn Ile Phe Leu Gly Asn Asn Phe

65 70 75 80

Phe Ala Asn Phe Asp Cys Val Met Leu Asp Val Cys Pro Ile Arg Ile

85 90 95

Gly Asp Asn Cys Met Leu Ala Pro Gly Val His Ile Tyr Thr Ala Thr

100 105 110

His Pro Ile Asp Pro Val Ala Arg Asn Ser Gly Ala Val Leu Gly Lys

115 120 125

Pro Val Thr Ile Gly Asn Asn Val Trp Ile Gly Gly Arg Ala Val Ile

130 135 140

Asn Pro Gly Val Thr Ile Gly Asp Asn Val Val Val Ala Ser Gly Ala

145 150 155 160

Val Val Thr Lys Asp Val Pro Asp Asn Val Val Val Gly Gly Asn Pro

165 170 175

Ala Arg Ile Ile Lys Lys Leu

180

<210> 4

<211> 552

<212> DNA

<213> Artificial sequence

<220>

<223> MAT-E125V nucleotide sequence

<400> 4

atgagcacag aaaaagaaaa gatgattgct ggtgagttgt atcgctcggc agatgagacg 60

ttatctcgcg atcgcctgcg cgctcgtcag cttattcacc gatacaatca ttccctggcg 120

gaagagcaca cattacgcca gcaaattctc gctgatctat tcggtcaggt gacagaggct 180

tatattgagc caacgtttcg ctgtgactat ggctataaca tttttctcgg taataatttt 240

ttcgccaact tcgattgcgt gatgcttgat gtctgcccta ttcgcatcgg tgataactgt 300

atgttggcac caggcgttca tatctacacg gcaacacatc ccatcgaccc tgtagcacgt 360

aatagcggtg ctgtgctggg gaaacccgtc accatcggta ataacgtctg gattggcgga 420

cgcgcggtca ttaaccctgg tgtgaccatt ggtgataacg tcgtggtagc ctcaggtgca 480

gttgtcacaa aagatgtccc ggacaacgtt gtcgtgggcg gtaatccagc cagaataatt 540

aaaaaattgt aa 552

<210> 5

<211> 183

<212> PRT

<213> Artificial sequence

<220>

<223> MAT-E125M amino acid sequence

<400> 5

Met Ser Thr Glu Lys Glu Lys Met Ile Ala Gly Glu Leu Tyr Arg Ser

1 5 10 15

Ala Asp Glu Thr Leu Ser Arg Asp Arg Leu Arg Ala Arg Gln Leu Ile

20 25 30

His Arg Tyr Asn His Ser Leu Ala Glu Glu His Thr Leu Arg Gln Gln

35 40 45

Ile Leu Ala Asp Leu Phe Gly Gln Val Thr Glu Ala Tyr Ile Glu Pro

50 55 60

Thr Phe Arg Cys Asp Tyr Gly Tyr Asn Ile Phe Leu Gly Asn Asn Phe

65 70 75 80

Phe Ala Asn Phe Asp Cys Val Met Leu Asp Val Cys Pro Ile Arg Ile

85 90 95

Gly Asp Asn Cys Met Leu Ala Pro Gly Val His Ile Tyr Thr Ala Thr

100 105 110

His Pro Ile Asp Pro Val Ala Arg Asn Ser Gly Ala Met Leu Gly Lys

115 120 125

Pro Val Thr Ile Gly Asn Asn Val Trp Ile Gly Gly Arg Ala Val Ile

130 135 140

Asn Pro Gly Val Thr Ile Gly Asp Asn Val Val Val Ala Ser Gly Ala

145 150 155 160

Val Val Thr Lys Asp Val Pro Asp Asn Val Val Val Gly Gly Asn Pro

165 170 175

Ala Arg Ile Ile Lys Lys Leu

180

<210> 6

<211> 552

<212> DNA

<213> Artificial sequence

<220>

<223> MAT-E125M nucleotide sequence

<400> 6

atgagcacag aaaaagaaaa gatgattgct ggtgagttgt atcgctcggc agatgagacg 60

ttatctcgcg atcgcctgcg cgctcgtcag cttattcacc gatacaatca ttccctggcg 120

gaagagcaca cattacgcca gcaaattctc gctgatctat tcggtcaggt gacagaggct 180

tatattgagc caacgtttcg ctgtgactat ggctataaca tttttctcgg taataatttt 240

ttcgccaact tcgattgcgt gatgcttgat gtctgcccta ttcgcatcgg tgataactgt 300

atgttggcac caggcgttca tatctacacg gcaacacatc ccatcgaccc tgtagcacgt 360

aatagcggtg ctatgctggg gaaacccgtc accatcggta ataacgtctg gattggcgga 420

cgcgcggtca ttaaccctgg tgtgaccatt ggtgataacg tcgtggtagc ctcaggtgca 480

gttgtcacaa aagatgtccc ggacaacgtt gtcgtgggcg gtaatccagc cagaataatt 540

aaaaaattgt aa 552

<210> 7

<211> 183

<212> PRT

<213> Artificial sequence

<220>

<223> MAT-E125R amino acid sequence

<400> 7

Met Ser Thr Glu Lys Glu Lys Met Ile Ala Gly Glu Leu Tyr Arg Ser

1 5 10 15

Ala Asp Glu Thr Leu Ser Arg Asp Arg Leu Arg Ala Arg Gln Leu Ile

20 25 30

His Arg Tyr Asn His Ser Leu Ala Glu Glu His Thr Leu Arg Gln Gln

35 40 45

Ile Leu Ala Asp Leu Phe Gly Gln Val Thr Glu Ala Tyr Ile Glu Pro

50 55 60

Thr Phe Arg Cys Asp Tyr Gly Tyr Asn Ile Phe Leu Gly Asn Asn Phe

65 70 75 80

Phe Ala Asn Phe Asp Cys Val Met Leu Asp Val Cys Pro Ile Arg Ile

85 90 95

Gly Asp Asn Cys Met Leu Ala Pro Gly Val His Ile Tyr Thr Ala Thr

100 105 110

His Pro Ile Asp Pro Val Ala Arg Asn Ser Gly Ala Arg Leu Gly Lys

115 120 125

Pro Val Thr Ile Gly Asn Asn Val Trp Ile Gly Gly Arg Ala Val Ile

130 135 140

Asn Pro Gly Val Thr Ile Gly Asp Asn Val Val Val Ala Ser Gly Ala

145 150 155 160

Val Val Thr Lys Asp Val Pro Asp Asn Val Val Val Gly Gly Asn Pro

165 170 175

Ala Arg Ile Ile Lys Lys Leu

180

<210> 8

<211> 552

<212> DNA

<213> Artificial sequence

<220>

<223> MAT-E125R nucleotide sequence

<400> 8

atgagcacag aaaaagaaaa gatgattgct ggtgagttgt atcgctcggc agatgagacg 60

ttatctcgcg atcgcctgcg cgctcgtcag cttattcacc gatacaatca ttccctggcg 120

gaagagcaca cattacgcca gcaaattctc gctgatctat tcggtcaggt gacagaggct 180

tatattgagc caacgtttcg ctgtgactat ggctataaca tttttctcgg taataatttt 240

ttcgccaact tcgattgcgt gatgcttgat gtctgcccta ttcgcatcgg tgataactgt 300

atgttggcac caggcgttca tatctacacg gcaacacatc ccatcgaccc tgtagcacgt 360

aatagcggtg ctcgcctggg gaaacccgtc accatcggta ataacgtctg gattggcgga 420

cgcgcggtca ttaaccctgg tgtgaccatt ggtgataacg tcgtggtagc ctcaggtgca 480

gttgtcacaa aagatgtccc ggacaacgtt gtcgtgggcg gtaatccagc cagaataatt 540

aaaaaattgt aa 552

<210> 9

<211> 183

<212> PRT

<213> Artificial sequence

<220>

<223> MAT-E125N amino acid sequence

<400> 9

Met Ser Thr Glu Lys Glu Lys Met Ile Ala Gly Glu Leu Tyr Arg Ser

1 5 10 15

Ala Asp Glu Thr Leu Ser Arg Asp Arg Leu Arg Ala Arg Gln Leu Ile

20 25 30

His Arg Tyr Asn His Ser Leu Ala Glu Glu His Thr Leu Arg Gln Gln

35 40 45

Ile Leu Ala Asp Leu Phe Gly Gln Val Thr Glu Ala Tyr Ile Glu Pro

50 55 60

Thr Phe Arg Cys Asp Tyr Gly Tyr Asn Ile Phe Leu Gly Asn Asn Phe

65 70 75 80

Phe Ala Asn Phe Asp Cys Val Met Leu Asp Val Cys Pro Ile Arg Ile

85 90 95

Gly Asp Asn Cys Met Leu Ala Pro Gly Val His Ile Tyr Thr Ala Thr

100 105 110

His Pro Ile Asp Pro Val Ala Arg Asn Ser Gly Ala Asn Leu Gly Lys

115 120 125

Pro Val Thr Ile Gly Asn Asn Val Trp Ile Gly Gly Arg Ala Val Ile

130 135 140

Asn Pro Gly Val Thr Ile Gly Asp Asn Val Val Val Ala Ser Gly Ala

145 150 155 160

Val Val Thr Lys Asp Val Pro Asp Asn Val Val Val Gly Gly Asn Pro

165 170 175

Ala Arg Ile Ile Lys Lys Leu

180

<210> 10

<211> 552

<212> DNA

<213> Artificial sequence

<220>

<223> MAT-E125N nucleotide sequence

<400> 10

atgagcacag aaaaagaaaa gatgattgct ggtgagttgt atcgctcggc agatgagacg 60

ttatctcgcg atcgcctgcg cgctcgtcag cttattcacc gatacaatca ttccctggcg 120

gaagagcaca cattacgcca gcaaattctc gctgatctat tcggtcaggt gacagaggct 180

tatattgagc caacgtttcg ctgtgactat ggctataaca tttttctcgg taataatttt 240

ttcgccaact tcgattgcgt gatgcttgat gtctgcccta ttcgcatcgg tgataactgt 300

atgttggcac caggcgttca tatctacacg gcaacacatc ccatcgaccc tgtagcacgt 360

aatagcggtg ctaacctggg gaaacccgtc accatcggta ataacgtctg gattggcgga 420

cgcgcggtca ttaaccctgg tgtgaccatt ggtgataacg tcgtggtagc ctcaggtgca 480

gttgtcacaa aagatgtccc ggacaacgtt gtcgtgggcg gtaatccagc cagaataatt 540

aaaaaattgt aa 570

<210> 11

<211> 183

<212> PRT

<213> Artificial sequence

<220>

<223> MAT-E125T amino acid sequence

<400> 11

Met Ser Thr Glu Lys Glu Lys Met Ile Ala Gly Glu Leu Tyr Arg Ser

1 5 10 15

Ala Asp Glu Thr Leu Ser Arg Asp Arg Leu Arg Ala Arg Gln Leu Ile

20 25 30

His Arg Tyr Asn His Ser Leu Ala Glu Glu His Thr Leu Arg Gln Gln

35 40 45

Ile Leu Ala Asp Leu Phe Gly Gln Val Thr Glu Ala Tyr Ile Glu Pro

50 55 60

Thr Phe Arg Cys Asp Tyr Gly Tyr Asn Ile Phe Leu Gly Asn Asn Phe

65 70 75 80

Phe Ala Asn Phe Asp Cys Val Met Leu Asp Val Cys Pro Ile Arg Ile

85 90 95

Gly Asp Asn Cys Met Leu Ala Pro Gly Val His Ile Tyr Thr Ala Thr

100 105 110

His Pro Ile Asp Pro Val Ala Arg Asn Ser Gly Ala Thr Leu Gly Lys

115 120 125

Pro Val Thr Ile Gly Asn Asn Val Trp Ile Gly Gly Arg Ala Val Ile

130 135 140

Asn Pro Gly Val Thr Ile Gly Asp Asn Val Val Val Ala Ser Gly Ala

145 150 155 160

Val Val Thr Lys Asp Val Pro Asp Asn Val Val Val Gly Gly Asn Pro

165 170 175

Ala Arg Ile Ile Lys Lys Leu

180

<210> 12

<211> 552

<212> DNA

<213> Artificial sequence

<220>

<223> MAT-E125T nucleotide sequence

<400> 12

atgagcacag aaaaagaaaa gatgattgct ggtgagttgt atcgctcggc agatgagacg 60

ttatctcgcg atcgcctgcg cgctcgtcag cttattcacc gatacaatca ttccctggcg 120

gaagagcaca cattacgcca gcaaattctc gctgatctat tcggtcaggt gacagaggct 180

tatattgagc caacgtttcg ctgtgactat ggctataaca tttttctcgg taataatttt 240

ttcgccaact tcgattgcgt gatgcttgat gtctgcccta ttcgcatcgg tgataactgt 300

atgttggcac caggcgttca tatctacacg gcaacacatc ccatcgaccc tgtagcacgt 360

aatagcggtg ctaccctggg gaaacccgtc accatcggta ataacgtctg gattggcgga 420

cgcgcggtca ttaaccctgg tgtgaccatt ggtgataacg tcgtggtagc ctcaggtgca 480

gttgtcacaa aagatgtccc ggacaacgtt gtcgtgggcg gtaatccagc cagaataatt 540

aaaaaattgt aa 552

<210> 13

<211> 183

<212> PRT

<213> Artificial sequence

<220>

<223> MAT-E125P amino acid sequence

<400> 13

Met Ser Thr Glu Lys Glu Lys Met Ile Ala Gly Glu Leu Tyr Arg Ser

1 5 10 15

Ala Asp Glu Thr Leu Ser Arg Asp Arg Leu Arg Ala Arg Gln Leu Ile

20 25 30

His Arg Tyr Asn His Ser Leu Ala Glu Glu His Thr Leu Arg Gln Gln

35 40 45

Ile Leu Ala Asp Leu Phe Gly Gln Val Thr Glu Ala Tyr Ile Glu Pro

50 55 60

Thr Phe Arg Cys Asp Tyr Gly Tyr Asn Ile Phe Leu Gly Asn Asn Phe

65 70 75 80

Phe Ala Asn Phe Asp Cys Val Met Leu Asp Val Cys Pro Ile Arg Ile

85 90 95

Gly Asp Asn Cys Met Leu Ala Pro Gly Val His Ile Tyr Thr Ala Thr

100 105 110

His Pro Ile Asp Pro Val Ala Arg Asn Ser Gly Ala Pro Leu Gly Lys

115 120 125

Pro Val Thr Ile Gly Asn Asn Val Trp Ile Gly Gly Arg Ala Val Ile

130 135 140

Asn Pro Gly Val Thr Ile Gly Asp Asn Val Val Val Ala Ser Gly Ala

145 150 155 160

Val Val Thr Lys Asp Val Pro Asp Asn Val Val Val Gly Gly Asn Pro

165 170 175

Ala Arg Ile Ile Lys Lys Leu

180

<210> 14

<211> 552

<212> DNA

<213> Artificial sequence

<220>

<223> MAT-E125P nucleotide sequence

<400> 14

atgagcacag aaaaagaaaa gatgattgct ggtgagttgt atcgctcggc agatgagacg 60

ttatctcgcg atcgcctgcg cgctcgtcag cttattcacc gatacaatca ttccctggcg 120

gaagagcaca cattacgcca gcaaattctc gctgatctat tcggtcaggt gacagaggct 180

tatattgagc caacgtttcg ctgtgactat ggctataaca tttttctcgg taataatttt 240

ttcgccaact tcgattgcgt gatgcttgat gtctgcccta ttcgcatcgg tgataactgt 300

atgttggcac caggcgttca tatctacacg gcaacacatc ccatcgaccc tgtagcacgt 360

aatagcggtg ctccgctggg gaaacccgtc accatcggta ataacgtctg gattggcgga 420

cgcgcggtca ttaaccctgg tgtgaccatt ggtgataacg tcgtggtagc ctcaggtgca 480

gttgtcacaa aagatgtccc ggacaacgtt gtcgtgggcg gtaatccagc cagaataatt 540

aaaaaattgt aa 552

<210> 15

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer MAT-F81AF

<400> 15

ctcggtaata attttgcggc caacttcgat 30

<210> 16

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> reverse primer MAT-F81AR

<400> 16

cgcaaaatta ttaccgagaa aaatgttata 30

<210> 17

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer MAT-N83AF

<400> 17

aataattttt tcgccgcgtt cgattgcgtg 30

<210> 18

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> reverse primer MAT-N83AR

<400> 18

gcacagttat caccgatgcg aataggg 27

<210> 19

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer MAT-M101AF

<400> 19

atcggtgata actgtgcgtt ggcacca 27

<210> 20

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> reverse primer MAT-M101AR

<400> 20

gcacagttat caccgatgcg aataggg 27

<210> 21

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer MAT-W137AF

<400> 21

atcggtaata acgtcgcgat tggcgga 27

<210> 22

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> reverse primer MAT-W137AR

<400> 22

gcgacgttat taccgatggt gacgggt 27

<210> 23

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer MAT-E125AF

<400> 23

gtaatagcgg tgctgcgctg gggaaac 27

<210> 24

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> reverse primer MAT-E125AR

<400> 24

cgcagcaccg ctattacgtg ctacagg 27

<210> 25

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer MAT-E125FF

<400> 25

cgtaatagcg gtgcttttct ggggaaac 28

<210> 26

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> reverse primer MAT-E125FR

<400> 26

aaaagcaccg ctattacgtg ctacaggg 28

<210> 27

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer MAT-E125YF

<400> 27

cgtaatagcg gtgcttatct ggggaaac 28

<210> 28

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> reverse primer MAT-E125YR

<400> 28

ataagcaccg ctattacgtg ctacaggg 28

<210> 29

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer MAT-E125WF

<400> 29

cgtaatagcg gtgcttggct ggggaaac 28

<210> 30

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> reverse primer MAT-E125WR

<400> 30

ccaagcaccg ctattacgtg ctacaggg 28

<210> 31

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer MAT-E125SF

<400> 31

cgtaatagcg gtgctagcct ggggaaac 28

<210> 32

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> reverse primer MAT-E125SR

<400> 32

gctagcaccg ctattacgtg ctacaggg 28

<210> 33

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer MAT-E125TF

<400> 33

cgtaatagcg gtgctaccct ggggaaac 28

<210> 34

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> reverse primer MAT-E125TR

<400> 34

ggtagcaccg ctattacgtg ctacaggg 28

<210> 35

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer MAT-E125CF

<400> 35

cgtaatagcg gtgcttgcct ggggaaac 28

<210> 36

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> reverse primer MAT-E125CR

<400> 36

gcaagcaccg ctattacgtg ctacaggg 28

<210> 37

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer MAT-E125MF

<400> 37

cgtaatagcg gtgctatgct ggggaaac 28

<210> 38

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> reverse primer MAT-E125MR

<400> 38

catagcaccg ctattacgtg ctacaggg 28

<210> 39

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer MAT-E125NF

<400> 39

cgtaatagcg gtgctaacct ggggaaac 28

<210> 40

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> reverse primer MAT-E125NR

<400> 40

gttagcaccg ctattacgtg ctacaggg 28

<210> 41

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer MAT-E125QF

<400> 41

cgtaatagcg gtgctcagct ggggaaac 28

<210> 42

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> reverse primer MAT-E125QR

<400> 42

ctgagcaccg ctattacgtg ctacaggg 28

<210> 43

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer MAT-E125DF

<400> 43

taatagcggt gctgatctgg ggaaac 26

<210> 44

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> reverse primer MAT-E125DR

<400> 44

atcagcaccg ctattacgtg ctacag 26

<210> 45

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer MAT-E125KF

<400> 45

cgtaatagcg gtgctaaact ggggaa 26

<210> 46

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> reverse primer MAT-E125KR

<400> 46

tagcaccgct attacgtgct acaggg 26

<210> 47

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer MAT-E125RF

<400> 47

cgtaatagcg gtgctcgcct ggggaaac 28

<210> 48

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> reverse primer MAT-E125RR

<400> 48

gcgagcaccg ctattacgtg ctacaggg 28

<210> 49

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer MAT-E125HF

<400> 49

cgtaatagcg gtgctcatct ggggaaac 28

<210> 50

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> reverse primer MAT-E125HR

<400> 50

atgagcaccg ctattacgtg ctacaggg 28

<210> 51

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer MAT-E125GF

<400> 51

gtaatagcgg tgctggcctg gggaaac 27

<210> 52

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> reverse primer MAT-E125GR

<400> 52

gccagcaccg ctattacgtg ctacagg 27

<210> 53

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer MAT-E125VF

<400> 53

gtaatagcgg tgctgtgctg gggaaac 27

<210> 54

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> reverse primer MAT-E125VR

<400> 54

cacagcaccg ctattacgtg ctacagg 27

<210> 55

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer MAT-E125LF

<400> 55

cgtaatagcg gtgctctgct ggggaaac 28

<210> 56

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> reverse primer MAT-E125LR

<400> 56

cagagcaccg ctattacgtg ctacaggg 28

<210> 57

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer MAT-E125IF

<400> 57

cgtaatagcg gtgctattct ggggaaac 28

<210> 58

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> reverse primer MAT-E125IR

<400> 58

aatagcaccg ctattacgtg ctacaggg 28

<210> 59

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer MAT-E125PF

<400> 59

cgtaatagcg gtgctccgct ggggaaac 28

<210> 60

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> reverse primer MAT-E125PR

<400> 60

cggagcaccg ctattacgtg ctacaggg 28

Claims (13)

1. A method for enzymatic synthesis of puerarin 6' -O-acetate by using diacerein as acyl donor is characterized in that,

a) maltose-O-acyltransferase MAT or its mutant MAT-E125V, MAT-E125M, MAT-E125R is biocatalyst;

b) Puerarin is a receptor substrate;

c) Diacerein is used as a donor substrate;

d) MAT or its mutant MAT-E125V, MAT-E125M, MAT-E125R catalyze puerarin and diacerein to synthesize puerarin 6' -O-acetate.

2. The method of claim 1, wherein the molecular structural formula of the donor substrate diacerein is as follows:

3. a method for enzymatic synthesis of puerarin 6' -O-acetate by using acetyl coenzyme A as acyl donor is characterized in that,

a) maltose-O-acyltransferase MAT or its mutant MAT-E125N, MAT-E125T and MAT-E125P are biocatalysts;

b) Puerarin is used as a receptor substrate;

c) Acetyl coenzyme A is used as a donor substrate;

d) MAT or its mutant MAT-E125N, MAT-E125T, MAT-E125P catalyzes puerarin and acetyl coenzyme A to synthesize puerarin 6' -O-acetate.

4. The method of claim 1 or 3, wherein puerarin 6 "-O-acetate has the following molecular formula:

5. the method according to claim 1 or 3, wherein said maltose-O-acyltransferase MAT has an amino acid sequence selected from the group consisting of SEQ ID No.1 and a nucleotide sequence selected from the group consisting of SEQ ID No.2.

6. The method according to claim 1, wherein said maltose-O-acyltransferase mutant MAT-E125V, MAT-E125M, MAT-E125R, the amino acid sequence thereof is selected from SEQ ID No.3, SEQ ID No.5, SEQ ID No.7, the nucleotide sequence thereof is selected from SEQ ID No.4, SEQ ID No.6, SEQ ID No.8.

7. The method according to claim 3, wherein said maltose-O-acyltransferase mutant MAT-E125N, MAT-E125T, MAT-E125P, the amino acid sequence thereof is selected from SEQ ID No.9, SEQ ID No.11, SEQ ID No.13, the nucleotide sequence thereof is selected from SEQ ID No.10, SEQ ID No.12, SEQ ID No.14.

8. The method of claim 1, wherein the synthesis of puerarin 6 "-O-acetate is performed in an enzymatic system, diacerein and puerarin are donor substrates, respectively; the reaction temperature is 20-80 ℃; the reaction pH is 3-9.

9. The method of claim 3, wherein the synthesis of puerarin 6 "-O-acetate is carried out in an enzyme-catalyzed system, acetyl-CoA and puerarin are the donor substrates, respectively; the reaction temperature is 20-80 ℃; the reaction pH is 3-7.

10. The method as claimed in claim 1 or 3, wherein the biocatalyst involved in the synthesis of puerarin 6 "-O-acetate is capable of existing in the form of crude enzyme, pure enzyme, immobilized and whole cell.

11. The method of claim 10, wherein the whole cell is selected from the group consisting of E.coli.

12. An expression vector comprising the nucleotide sequence of any one of claims 5-7.

13. The expression vector of claim 12, wherein the host cell of the expression vector is selected from the group consisting of E.

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