Method for synthesizing peracetylated sugar through enzyme catalysis
Through the enzyme catalytic reaction of immobilized penicillin G acylase in organic solvents, environmental and equipment problems during the acetylation of sugars are solved, and efficient and environmentally friendly production of all acetyl sugars is achieved, and it is suitable for food, medicine and other fields.
Patent Information
- Application Number
- CN202411491725.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-24
- Publication Date
- 2024-12-27
AI Technical Summary
The prior art has problems such as environmental toxicity, unpleasant odor, equipment corrosion and by-product formation in the acetylation process of sugars, making it difficult to achieve an enzyme catalytic synthesis of all acetyl sugars with mild process conditions and suitable for large-scale production.
Immobilized penicillin G acylase is used to mix with sugar and acid anhydride in an organic solvent, and undergo enzyme catalytic reaction, followed by filtration and recrystallization to obtain whole acetyl sugar, and the enzyme is fixed using carriers such as agarose and calcium alginate to optimize the reaction temperature and Time, select the appropriate solvent and anhydride ratio.
The process conditions for enzyme-catalyzed synthesis of all acetyl sugars are mild, environmentally friendly, suitable for large-scale production, reducing production costs, and improving the utilization rate of enzymes and product purity.
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Figure CN119193734A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biotechnology, and more specifically to a method for synthesizing full acetyl sugar by enzyme catalysis. Background Art
[0002] Peracetylated sugars are important glycoside intermediates that can be used to synthesize a series of glycosides and oligosaccharides, and have important applications in the fields of biomedicine, food, and cosmetics. Peracetylation of sugars provides an effective means to protect the hydroxyl groups of sugars, and is also the key to synthesizing oligosaccharides with important biological significance and many biologically active natural products. Therefore, peracetylated sugars are an important research and application direction for the preparation of functional sugar compounds and oligosaccharides.
[0003] Conventional sugar acetylation processes use a large excess of carboxylic acid or anhydride as an acylating agent and a solvent in an organic solvent such as pyridine under the action of an acidic or alkaline catalyst. Although these catalysts can be used to prepare fully acetylated sugars, many of them have several significant disadvantages, such as environmental toxicity, unpleasant odor, equipment corrosion, formation of unwanted byproducts, and incompatibility with carbohydrate derivatives containing acid-labile functional groups.
[0004] Therefore, there is an urgent need in the art to develop a method for the enzyme-catalyzed synthesis of peracetylated sugars with mild process conditions, environmental friendliness, and suitability for large-scale production. Summary of the invention
[0005] In view of the above problems, the object of the present invention is to provide a method for enzymatically synthesizing peracetylated sugars with mild process conditions, environmental friendliness and suitability for large-scale production.
[0006] In a first aspect of the present invention, a method for synthesizing peracetyl sugar using penicillin G acylase catalyzed by penicillin G acylase is provided, comprising the following steps:
[0007] S1: adding a substrate, a carboxylic acid or anhydride into a reaction solvent and mixing them to obtain a mixture, wherein the substrate is a monosaccharide or a polysaccharide;
[0008] S2: adding immobilized penicillin G acylase to the mixture in step S1 to perform a catalytic reaction to obtain a fully acetylated sugar suspension;
[0009] S3: filtering and recrystallizing the fully acetylated sugar suspension obtained in step S2 to obtain fully acetylated sugar.
[0010] In another preferred embodiment, in step S1, the reaction solvent is selected from the following group: dimethylformamide, dichloromethane or ethanol, preferably dimethylformamide.
[0011] In another preferred embodiment, the substrate is selected from the group consisting of glucose, fructose, galactose, sucrose, lactose, chitosan, dextran, trehalose or hyaluronic acid.
[0012] In another preferred embodiment, the acid anhydride is selected from acetic anhydride.
[0013] In another preferred embodiment, step S1 further comprises: in a reaction solvent, mixing the substrate and the acid anhydride at a molar ratio of 1:5-10 (preferably 1:8) to obtain a mixture; the volume ratio of the acid anhydride to the reaction solvent is 1:2-3, preferably 1:2.5.
[0014] In another preferred embodiment, in step S2, the reaction temperature of the catalytic reaction is 46°C-55°C, preferably 50°C; the reaction time is 12-18 hours, preferably 14-16 hours, and more preferably 15 hours.
[0015] In another preferred embodiment, in step S2, the mass volume ratio of the immobilized penicillin G acylase to the mixture is: 1 g: 10-20 mL; preferably 1 g: 14-15 mL.
[0016] In another preferred embodiment, step S2 further comprises step S2.1: immobilizing penicillin G acylase via a carrier to obtain immobilized penicillin G acylase; the carrier is selected from the group consisting of agarose, calcium alginate, divinylbenzene resin, styrene resin or acrylic resin.
[0017] In another preferred embodiment, the step S2.1 further comprises: uniformly stirring the penicillin G acylase and the carrier, dropping them into a calcium chloride solution, and placing them at 4°C for solidification for 20-40 minutes (preferably 30 minutes) to obtain immobilized penicillin G acylase; the mass ratio of the penicillin G acylase to the carrier is 1:1-4 (preferably 1:2.4).
[0018] In another preferred embodiment, the weight percentage of the calcium chloride solution is 1%-5%, preferably 3%.
[0019] In another preferred embodiment, the penicillin G acylase is selected from the group consisting of Bacillus megaterium penicillin G acylase, Escherichia coli penicillin G acylase, Alcaligenes faecalis penicillin G acylase, Arthrobacter viscosus penicillin G acylase, Providencia rettgeri penicillin G acylase or Kluyvera penicillin G acylase.
[0020] In another preferred embodiment, in step S2, when the substrate is a monosaccharide, the immobilized penicillin G acylase is immobilized by agarose or calcium alginate embedding method.
[0021] In another preferred embodiment, in step S2, when the substrate is a polysaccharide, the immobilized penicillin G acylase is obtained by digital immobilization.
[0022] In another preferred embodiment, in step S3, the filtration refers to filtering to remove the immobilized penicillin G acylase.
[0023] In another preferred embodiment, in step S3, the recrystallization method comprises: adding a certain amount of deionized water to the filtrate obtained after filtration and stirring, then adding an equal volume of anhydrous ethanol, and slowly stirring at a temperature of 0°C-4°C to recrystallize the fully acetylated sugar suspension to obtain fully acetylated sugar; the amount of deionized water added is 0.5-1 times, preferably 0.8 times, the amount of the acid anhydride added.
[0024] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.
[0025] Compared with the prior art, the present invention has the following technical effects:
[0026] (1) The present invention uses the corresponding immobilized penicillin G acylase to efficiently synthesize fully acetylated sugars by enzyme catalysis for sugars with different molecular weight ranges. Compared with the chemical synthesis method, the reaction is milder and the enzyme can be reused, thereby reducing the production cost of fully acetylated sugars and being environmentally friendly.
[0027] (2) The process for preparing fully acetylated sugars according to the present invention is simple, suitable for industrial scale-up, and can be widely used in the fields of food, medicine, biology, etc. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] Figure 1 A diagram showing the results of optimizing the catalytic reaction conditions in one embodiment of the present invention, wherein Figure A shows different reaction solvent pairs.
[0029] Figure 2 Displays a partial spectrum analyzed by a gas chromatography-mass spectrometer in one embodiment of the present invention; Figure 2 Figure A is a partial mass spectrum of the prepared full acetylated glucose, and Figure B is a partial mass spectrum of the full acetylated glucose standard.
[0030] Figure 3 The carbon NMR spectrum of the acylation product, the all-acetyl sugar, in one embodiment of the present invention is shown.
[0031] Figure 4 The infrared spectrum analysis chart of the acylation reaction product, full acetyl sugar, in one embodiment of the present invention is shown, wherein the blue line represents the infrared spectrum of full acetyl sugar, and the red line represents the infrared spectrum of glucose. DETAILED DESCRIPTION
[0032] In order to make the purpose, technical solution, beneficial effects and significant progress of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, all the embodiments described are only partial embodiments of the present invention, not all embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0033] the term
[0034] As used herein, the terms "peracetylated sugar" and "peracetylglucose" are used interchangeably.
[0035] Penicillin G acylase
[0036] With the rapid development of genetic engineering and enzymology, the use of recombinant DNA technology to efficiently express enzymes in various types of cells such as microorganisms has improved the production efficiency of enzymes and provided more possibilities for their production and utilization. In recent years, penicillin G acylase has been put into commercial use. In-depth research on the structure of penicillin G acylase and research on substrate-enzyme interactions have shown that penicillin G acylase is a multifunctional enzyme that can catalyze a variety of chemical reactions, and its active site can also catalyze one or more types of enzymatic reactions. This undoubtedly provides a new catalytic pathway for the synthesis of more types of bioactive substances. In addition, the use of this type of enzyme for acylation has great application prospects due to its mild reaction conditions, strong selectivity, and high product quality. However, the process conditions and product yields of this type of enzyme for acylation reactions of different substrates need to be further improved.
[0037] Although penicillin G acylase has many applications and is well studied in enzyme-catalyzed hydrolysis and semi-synthetic antibiotics, there are few reports on its catalytic preparation of fully acetylated sugars. The study of the multifunctional properties of penicillin G acylase has deepened the understanding of its catalytic nature, expanded the application range of biocatalysts, and provided a new efficient and green way to synthesize complex molecules with biological activity.
[0038] The present invention aims at the shortcomings of the current organic synthesis method for preparing full acetyl sugar, adopts immobilized penicillin G acylase, and prepares full acetyl sugar by enzyme catalysis. The method has mild process conditions, is environmentally friendly, and is suitable for large-scale production.
[0039] The present invention provides a method for preparing fully acetyl sugar by catalysis using immobilized penicillin G acylase, comprising the following steps:
[0040] Step 1: Immobilizing penicillin G acylase;
[0041] Step 2: adding substrate monosaccharide or polysaccharide, acid, and immobilized penicillin G acylase to an organic solvent for catalytic reaction;
[0042] Step 3: After the catalytic reaction is terminated, the immobilized penicillin G acylase is removed, and the fully acetylated sugar is recrystallized and the crystals are collected to obtain the fully acetylated sugar.
[0043] Preferably, in step 1, the immobilized penicillin G acylase is a carrier-immobilized Bacillus megaterium penicillin G acylase, Escherichia coli penicillin G acylase, Alcaligenes faecalis penicillin G acylase, Arthrobacter viscosus penicillin G acylase, Providencia rettgeri penicillin G acylase, or Kluyvera penicillin G acylase.
[0044] Preferably, the carrier of the immobilized enzyme in step 1 is agarose, calcium alginate, divinylbenzene resin, styrene resin, acrylic resin, etc.
[0045] Preferably, in step 1, the penicillin G acylase used for monosaccharide or oligosaccharide acylation is immobilized by agarose or calcium alginate embedding method, and the penicillin G acylase used for polysaccharide acylation is immobilized by macroporous resin. The embedding method has good protection for the enzyme, and the enzyme molecules are not easy to fall off. The molecules such as monosaccharides or oligosaccharides are small and can pass through the pores of the embedding material to catalyze the reaction with the enzyme. The enzyme immobilized by the macroporous resin adsorption method can fully contact with the large molecular weight substrate, realize the continuous catalytic reaction of the enzyme, and maximize the utilization rate of the enzyme.
[0046] Preferably, in step 2, the organic solvent is dichloromethane, dimethylformamide, or ethanol; the substrate is glucose, fructose, galactose, sucrose, lactose, chitosan, dextran, trehalose, or hyaluronic acid; and the acid is a carboxylic acid or anhydride.
[0047] Preferably, in the enzymatic reaction system of step 2, the substrate concentration is 10-80 g / L, the acid addition ratio is 10%-40%, the reaction temperature is 40-60° C., and the reaction time is 5-24 hours.
[0048] Preferably, in step 3, the immobilized penicillin G acylase is removed by centrifugation or filtration, and deionized water is added in a volume of 0.5-1.2 times the volume of the acid, and after stirring evenly, an equal volume of anhydrous ethanol is added and stirred at low temperature for recrystallization.
[0049] Example 1
[0050] The immobilization of penicillin G acylase comprises the following steps:
[0051] (1) Weigh 0.6 g of sodium alginate and dissolve it in 10 mL of water to prepare a 6% sodium alginate solution. Stir and heat continuously to dissolve.
[0052] (2) Dissolve 0.25 g of penicillin G acylase (purchased from Merck, catalog number 76427) in 10 mL of PBS buffer. Pour the dissolved enzyme solution quickly into the sodium alginate solution and stir evenly. Use a 20 mL syringe to add it dropwise into the 3% calcium chloride solution. The purpose of calcium chloride is to form solid calcium alginate with sodium alginate, and in the process, penicillin G acylase is embedded in it; place it at 4°C to solidify for 30 minutes.
[0053] (3) Filter to obtain white spheres, wash with distilled water, and air-dry. The obtained microspheres are immobilized penicillin G acylase. The immobilized penicillin G acylase is repeatedly washed with PBS buffer to remove the unencapsulated free enzyme, thereby obtaining immobilized penicillin G acylase.
[0054] Example 2
[0055] Based on the immobilized penicillin G acylase obtained in Example 1, the conditions for the acylation reaction of glucose catalyzed by the immobilized penicillin G acylase were optimized, and the specific implementation included the following steps:
[0056] (1) Add 1.44 g of substrate glucose and different volumes of acetic anhydride to 20 mL of different reaction solvents.
[0057] (2) Add 2 g of the immobilized penicillin G acylase obtained in Example 1 to the above solution and react at different reaction temperatures for 15 hours. After the reaction is completed, cool to room temperature to obtain full acetylglucose. Filter and separate the immobilized penicillin G acylase using filter paper.
[0058] (3) Add deionized water equivalent to 0.8 times the volume of acetic anhydride (8*0.8=6.4 mL) to the filtrate, add an equal volume of anhydrous ethanol, and slowly stir at 0°C to recrystallize the peracetylated glucose. Collect the peracetylated glucose crystals and recover the remaining acetic anhydride.
[0059] Based on the above experimental process, this embodiment optimizes the following conditions respectively:
[0060] (a) Optimization of reaction solvents: Chloroform, cyclohexane, dimethylformamide (DMF), pyridine and dimethyl sulfoxide (DMSO) were used as reaction solvents, and the effects of different solvents on the acylation of glucose catalyzed by penicillin G acylase were detected by thin layer chromatography.
[0061] The results showed that the immobilized penicillin G acylase had a high catalytic reaction efficiency in dimethylformamide (DMF) and pyridine solvents, and the yield of all acetylglucose was over 85%. However, pyridine has a strong pungent odor and is not environmentally friendly, so dimethylformamide (DMF) was selected as the solvent ( Figure 1 (Figure A in the middle).
[0062] (2) Optimization of the amount of acetic anhydride: 3.2, 4.0, 5.6, 6.4, 7.2, 8.0, and 8.8 mL of acetic anhydride were added to 20 mL of dimethyl diamide solvent, respectively, and the effect of the amount of acetic anhydride on the reaction was detected by thin layer chromatography.
[0063] The results showed that with the increase of acetic anhydride dosage, the yield of all-acetylglucose gradually increased. When the dosage of acetic anhydride was 6.4 mL, that is, the molar ratio of glucose to acetic anhydride was 1:8, a better degree of substitution could be obtained. Therefore, the optimal dosage of acetic anhydride was 6.4 mL ( Figure 1 (Figure B).
[0064] (3) Optimization of reaction temperature: 1.44 g of glucose and 6.4 mL of acetic anhydride were added to 20 mL of dimethyl diamide solvent, and immobilized penicillin G acylase was added. The reaction temperature was set to 40°C, 45°C, 50°C, 55°C, and 60°C. The effect of temperature on the reaction was detected by thin layer chromatography.
[0065] The results show that as the reaction temperature increases, the yield of fully acetylated glucose increases first and then decreases. When the temperature is 50°C, glucose can be completely dissolved, and the yield of fully acetylated sugar is the highest (about 30%) and the purity of fully acetylated sugar is the highest (about 99%). Therefore, the reaction temperature is selected to be 50°C ( Figure 1 (Figure C).
[0066] Example 3
[0067] Penicillin G acylase catalyzes the acylation reaction of glucose, and the specific implementation includes the following steps:
[0068] (1) Add 1.44 g of substrate glucose and 8 mL of acetic anhydride to 20 mL of dimethylformamide.
[0069] (2) Add 2 g of the immobilized penicillin G acylase obtained in Example 1 to the above solution and react at 50°C for 15 hours. After the reaction is completed, cool to room temperature to obtain full acetylglucose. Filter and separate the immobilized penicillin G acylase using filter paper.
[0070] (3) Add deionized water equivalent to 0.8 times the volume of acetic anhydride (8*0.8=6.4 mL) to the filtrate, add an equal volume of anhydrous ethanol, and slowly stir at 0°C to recrystallize the peracetylated glucose. Collect the peracetylated glucose crystals and recover the remaining acetic anhydride.
[0071] The purity of the fully acetylated glucose product obtained by this method is 98.9%±0.97%.
[0072] Example 4
[0073] Characterization of all-acetylglucose products
[0074] (1) Gas chromatography-mass spectrometry analysis of acylation reaction
[0075] The experimental conditions for the analysis of peracetylated sugars by gas chromatography-mass spectrometry were as follows: the chromatographic column model was DB-5MS; the temperature program was 80°C for 4 min, then increased to 150°C at a rate of 5°C / min, and then increased to 240°C at a rate of 20°C / min and maintained for 5 min; the split ratio was 30:1, the injection volume was 1 µL; the vaporization chamber temperature was 250°C, and the carrier gas flow rate was 1.0 mL / min.
[0076] Figure 2 Figure A is a partial mass spectrum of the prepared all-acetylated glucose, and Figure B is a partial mass spectrum of the all-acetylated glucose standard. Mass spectrometry analysis found a characteristic peak at a retention time of 23.53 min, a relative retention time of 0.34 min, a mass-to-charge ratio (m / z) of 43.10, and an abundance of 100%. This result is consistent with the mass spectrum data of the all-acetylated sugar standard. Partial spectrum ( Figure 2 ) can clearly identify signals consistent with the unique mass spectrometric fragmentation pattern of peracetylated sugar, so the product is peracetylated sugar.
[0077] (2) NMR spectroscopy analysis of acylation reaction
[0078] The NMR carbon spectrum experiment of peracetylated sugar uses CDCl3 as solvent, TMD as internal standard, chemical shift is expressed in δ (ppm), and is detected on a Bruker 400MHz NMR spectrometer. By comparing the standard spectrum, the characteristic absorption peaks of peracetylated sugar are identified, and then its structural information is determined.
[0079] The results show that in 13 C-NMR spectrum ( Figure 3 ) Two peaks with high δ values were observed: 173.46 ppm and 173.44 ppm. These peaks correspond to ester or acyl groups (C=O). Generally, the chemical shift of acyl groups ranges from 170-180 ppm, while the chemical shift of ester groups is around 170 ppm, proving the presence of ester or acyl groups (C=O) in the product. A series of peaks were observed between 93-166 ppm, the positions of which represent the chemical bonds of sugar rings, glycosidic bonds, and other carbon atoms, which are consistent with the positions of the standard peaks. Compared with the standard, the peak positions and peak intensities are similar, indicating that the catalytic product is consistent with the expected fully acetylated sugar structure.
[0080] (3) Infrared spectroscopy analysis of acylation reaction
[0081] The catalytic products, peracetylated sugar and KBr, were ground into powders respectively. The peracetylated sugar sample and KBr were mixed evenly at a ratio of about 1:100 (mass ratio). The mixture was placed in a tablet press to make transparent or translucent sheets. The tablets were placed in the sample chamber of the Thermo Scientific Nicolet iS20 infrared spectrometer and the scanning wavelength was 4000-400 cm -1 , select appropriate resolution and scanning times according to actual needs. After scanning, identify the characteristic absorption peaks of peracetylated sugars by comparing with the standard spectrum, and then determine the structural information.
[0082] Infrared spectroscopy analysis ( Figure 4 ) shows that in fully acetylated sugars, 2931 cm -1 、2867 cm -1 Corresponding to CH stretching vibration, 1722 cm -1 、1653 cm -1 Corresponding to C=O stretching vibration, 1385 cm -1 Corresponding to CH bending vibration, 1253 cm -1 Corresponding to CO stretching vibration, 1092 cm -1 Corresponding to COC stretching vibration, 660 cm -1 Corresponding to CH bending vibration, these absorption peaks are the absorption peaks of sugar, and the peak position correspondence is consistent with that of glucose; the acetyl group will introduce new chemical bonds such as CH and C=O. In the infrared spectrum of the sample, 1753 cm -1 Corresponding to the C=O stretching vibration of the acetyl group, 1222 cm -1 The corresponding CH bending vibration in the acetyl group; in addition, the original fully acetylated sugar has a 1653 cm -1 The peak position of C=O in glucose is 1646 cm -1 The above results indicate that fully acetylated sugars can be prepared by immobilizing penicillin G acylase.
[0083] The above describes the preferred embodiments of the present invention. It should be understood that the present invention is not limited to the above-mentioned specific embodiments, and the devices and structures that are not described in detail should be understood to be implemented in a common manner in the art; any technician familiar with the art can use the above-disclosed methods and technical contents to make many possible changes and modifications to the technical solutions of the present invention without departing from the scope of the technical solutions of the present invention, or modify them into equivalent embodiments of equivalent changes, which does not affect the essential content of the present invention. Therefore, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solutions of the present invention are still within the scope of protection of the technical solutions of the present invention.
Claims
1. A method for synthesizing fully acetyl sugars using penicillin G acylase, characterized in that: The steps include: S1: adding a substrate, a carboxylic acid or anhydride into a reaction solvent and mixing them to obtain a mixture, wherein the substrate is a monosaccharide or a polysaccharide; S2: adding immobilized penicillin G acylase to the mixture in step S1 to perform a catalytic reaction to obtain a fully acetylated sugar suspension; S3: filtering and recrystallizing the fully acetylated sugar suspension obtained in step S2 to obtain fully acetylated sugar.
2. The method according to claim 1, characterized in that In step S1, the reaction solvent is selected from the following group: dimethylformamide, dichloromethane or ethanol, preferably dimethylformamide.
3. The method according to claim 1, characterized in that The substrate is selected from the group consisting of glucose, fructose, galactose, sucrose, lactose, chitosan, dextran, trehalose or hyaluronic acid.
4. The method according to claim 1, characterized in that The acid anhydride is selected from acetic anhydride.
5. The method according to claim 1, characterized in that Step S1 further comprises: in a reaction solvent, mixing the substrate and the acid anhydride at a molar ratio of 1:5-10 (preferably 1:8) to obtain a mixture; the volume ratio of the acid anhydride to the reaction solvent is 1:2-3, preferably 1:2.
5.
6. The method according to claim 1, characterized in that In step S2, the reaction temperature of the catalytic reaction is 46°C-55°C, preferably 50°C; the reaction time is 12-18 hours, preferably 14-16 hours, and more preferably 15 hours.
7. The method according to claim 1, characterized in that In step S2, the mass volume ratio of the immobilized penicillin G acylase to the mixture is: 1 g: 10-20 mL; preferably 1 g: 14-15 mL.
8. The method according to claim 1, characterized in that Step S2 further comprises step S2.1: immobilizing penicillin G acylase via a carrier to obtain immobilized penicillin G acylase; the carrier is selected from the group consisting of agarose, calcium alginate, divinylbenzene resin, styrene resin or acrylic resin.
9. The method according to claim 8, characterized in that The step S2.1 further comprises: uniformly stirring the penicillin G acylase and the carrier, dropping them into a calcium chloride solution, and placing them at 4°C for solidification for 20-40 minutes (preferably 30 minutes) to obtain immobilized penicillin G acylase; the mass ratio of the penicillin G acylase to the carrier is 1:1-4 (preferably 1:2.4).
10. The method according to claim 1, characterized in that In step S3, the recrystallization method comprises: adding a certain amount of deionized water to the filtrate obtained after filtration and stirring, then adding an equal volume of anhydrous ethanol, and slowly stirring at a temperature of 0°C-4°C to recrystallize the fully acetylated sugar suspension to obtain fully acetylated sugar; the amount of deionized water added is 0.5-1 times the amount of the acid anhydride added, preferably 0.8 times.