A method for preparing N-acetamidoglucose based on enzymatic hydrolysis technology

Abstract

The present invention provides a method for preparing N-acetamido glucose based on enzymolysis technology, which belongs to the field of biochemical technology. The method comprises the following steps: (1) chitin and water in a mass ratio of 1:8-10 are mixed, chitinase is added, and enzymolysis is performed. After the enzymolysis is completed, the enzyme is inactivated to obtain a first enzymolysis product; (2) chitosanase is added to the first enzymolysis product, and enzymolysis is performed. After the enzymolysis is completed, the enzyme is inactivated to obtain a second enzymolysis product; (3) β-glucosidase is added to the second enzymolysis product, and after the enzymolysis is completed, the enzyme is inactivated to obtain a third enzymolysis product; (4) the third enzymolysis product is purified and dried to obtain N-acetamido glucose. The N-acetamido glucose prepared by the present invention has high purity and yield, good batch stability, low cost, and is convenient for industrial production.

Description

Technical Field

[0001] The invention belongs to the field of biochemical technology, and specifically relates to a method for preparing N-acetamidoglucose based on enzymatic hydrolysis technology. Background Art

[0002] N-acetylglucosamine, also known as N-Acetyl-D-glucosamine or GlcNAc for short, is an important monosaccharide and a type of hexosamine. N-acetylglucosamine has important applications in the fields of medicine and health care. For example, it can be used as a raw material for repairing joint cartilage and for treating osteoarthritis; it can also be used as an immunomodulator to improve the body's immunity; it can also be used as an ingredient in skin care products with moisturizing and anti-aging effects.

[0003] The existing methods for preparing N-acetylglucosamine mainly include direct enzymatic hydrolysis of chitin. Direct enzymatic hydrolysis means that N-acetylglucosamine can be produced by directly enzymatic hydrolysis of chitin with a single enzyme, which has low efficiency, high actual large-scale industrial production costs, and poor benefits. In order to improve efficiency, the existing technology uses composite enzymes for enzymatic hydrolysis, which can shorten the reaction time and improve the efficiency of enzymatic hydrolysis.

[0004] However, currently, complex enzymes (such as chitinase, β-glucosidase, etc.) are used to enzymatically hydrolyze chitin for the production of N-acetylglucosamine, but there are the following technical problems: (1) Enzyme compatibility and synergy: The synergistic effect between different enzymes may not always be ideal, and the optimal conditions of the enzymes (such as pH, temperature, etc.) are different, which may affect the overall activity and stability of the enzymes. (2) Byproduct formation: The complex enzyme system may lead to the formation of a variety of byproducts, especially when the enzyme ratio is inappropriate or the reaction conditions are not strictly controlled. This will reduce the purity and yield of N-acetylglucosamine. (3) Reaction kinetics: The kinetics of the enzymatic hydrolysis reaction may be complex and require fine control to optimize the conversion rate and reaction rate. This may involve the optimization of multiple parameters such as reaction time, substrate concentration, and enzyme concentration. The use of complex enzymes makes the parameters of the reaction kinetics difficult to control. (4) Enzyme inhibition and inactivation: The product of one enzyme in the complex enzyme may have an inhibitory effect on another enzyme, resulting in a decrease in enzyme activity.

[0005] The above problems lead to low purity and yield, long time consumption, low stability of product batches, which is not conducive to industrial production and cannot meet the needs of the existing market.

[0006] Therefore, there is an urgent need for a method for preparing N-acetamidoglucose based on enzymatic hydrolysis technology. Summary of the invention

[0007] The purpose of the present invention is to provide a method for preparing N-acetamidoglucose based on enzymolysis technology.

[0008] In order to achieve the above object, the present invention provides the following technical solutions:

[0009] A method for preparing N-acetamidoglucose based on enzymatic hydrolysis technology, the method comprising the following steps:

[0010] (1) chitosan and water are mixed in a mass ratio of 1:8-10, chitinase is added, and enzymolysis is performed at a temperature of 30-33°C and a pH of 6.8-7 for 2-3 hours. After the enzymolysis is completed, the enzyme is inactivated to obtain a first enzymolysis product;

[0011] (2) adding chitosanase to the first enzymatic hydrolysis product, performing enzymatic hydrolysis for 4-5 hours at a temperature of 38-40° C. and a pH of 7.3-7.6, and after the enzymatic hydrolysis is completed, inactivating the enzyme to obtain a second enzymatic hydrolysis product;

[0012] (3) adding β-glucosidase to the second enzymatic hydrolysis product, performing enzymatic hydrolysis for 3-4 hours at a temperature of 43-45° C. and a pH of 5.5-6, and after the enzymatic hydrolysis is completed, inactivating the enzyme to obtain a third enzymatic hydrolysis product;

[0013] (4) treating the third enzymatic hydrolysis product through a ceramic membrane filter to remove incompletely enzymatically hydrolyzed macromolecular suspended matter to obtain a filtrate;

[0014] (5) The filtrate is subjected to a first ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 250 Da to obtain a permeate;

[0015] (6) The permeate is subjected to a second ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 200 Da to obtain a concentrate with a molecular weight between 200 and 250 Da;

[0016] (7) Drying the concentrate to obtain N-acetamidoglucose.

[0017] The present invention selects three specific enzymes for segmented enzymolysis, improves the purity and yield of the product, and ensures batch stability. Each enzyme can play a role under optimal conditions, shorten the enzymolysis time, and avoid the inhibitory effect of different enzymolysis products on other enzymes in the enzymolysis process of the composite enzyme. At the same time, the inventor finds that the enzymolysis order of the three enzymes selected by the present invention also affects the yield and purity of the product. Analysis is due to the three enzymes having their specific substrate recognition sites and action modes, and the correct order can ensure that each enzyme can play the maximum effect on its most suitable substrate.

[0018] Furthermore, the dosage of the chitinase and the water in step (1) is in the ratio of (15-20) U: 1 mL.

[0019] Furthermore, the dosage relationship of the chitosanase and the first enzymatic hydrolysis product is (8-12) U: 1 mL.

[0020] Furthermore, the dosage relationship of the β-glucosidase and the second enzymatic hydrolysis product is (5-10) U: 1 mL.

[0021] The present invention selects the concentration of enzymes to keep the product with high yield and ensure low cost at the same time. When the concentration of enzyme is too high, the reaction can be accelerated, but the production cost will also be increased. The concentration of the present invention controls the cost input of enzyme while ensuring reasonable reaction rate and product yield. At the same time, the products of the enzymolysis before and after the three enzymes also have different requirements for the concentration of enzymes. The concentration of the three enzymes of the present invention can maximize the enzymolysis efficiency.

[0022] Furthermore, the enzyme inactivation condition is heating at 120-125° C. for 15-20 min.

[0023] Furthermore, the total duration of the enzymatic hydrolysis in steps (1), (2) and (3) is less than 12 hours.

[0024] Furthermore, in step (5), the filtrate flow rate is 5-8 L / h, and the membrane element pressure is 0.2-0.5 MPa.

[0025] Furthermore, in step (6), the filtrate flow rate is 2-4 L / h, and the membrane element pressure is 0.6-0.8 MPa.

[0026] Due to the type of enzyme used in the present invention and the specificity of the enzymatic hydrolysis method, the impurities in the enzymatic hydrolysis product are not ideal using existing methods. The purification step of the present invention can further improve the purity of the product.

[0027] Furthermore, the yield of N-acetamido glucose is greater than 88% and the purity is greater than 95%.

[0028] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0029] 1. The present invention provides a method for preparing N-acetamido glucose based on enzymatic hydrolysis technology. The method of the present invention is time-saving, the prepared N-acetamido glucose has high yield and purity, high batch stability, is convenient for industrial production, and is conducive to reducing costs.

[0030] 2. The present invention uses three specific enzymes for segmented enzymolysis, which improves the purity and yield of the product while ensuring batch stability.

[0031] 3. The concentration of the enzyme selected in the present invention can maintain a high yield of the product while ensuring a low cost. The concentration of the present invention controls the cost input of the enzyme while ensuring a reasonable reaction rate and product yield.

[0032] 4. Due to the type of enzyme used in the present invention and the specificity of the enzymatic hydrolysis method, it is difficult to remove impurities in the enzymatic hydrolysis product. The purification step of the present invention can further improve the purity of the product. DETAILED DESCRIPTION

[0033] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. 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.

[0034] The enzymes used in the present invention were purchased from the following manufacturers:

[0035] Chitosanase, Shanghai Yuanye Biotechnology Co., Ltd., catalog number: S25847.

[0036] Chitinase, Shanghai Yuanye Biotechnology Co., Ltd., catalog number: S10178.

[0037] β-Glucosidase, Shanghai Yuanye Biotechnology Co., Ltd., catalog number: S24786.

[0038] β-N-acetylglucosaminidase, Shanghai Yuanye Biotechnology Co., Ltd., catalog number: S10237.

[0039] Example 1

[0040] This embodiment provides a method for preparing N-acetamidoglucose based on enzymatic hydrolysis technology, the method comprising the following steps:

[0041] (1) Chitosan and water are mixed in a mass ratio of 1:9, chitinase is added, and enzymolysis is carried out at a temperature of 32° C. and a pH of 6.8 for 2.5 hours. After the enzymolysis is completed, the enzyme is inactivated to obtain a first enzymolysis product; the amount of the chitinase and the water in step (1) is 18 U: 1 mL.

[0042] (2) adding chitosanase to the first enzymatic hydrolysis product, performing enzymatic hydrolysis for 4.5 h at a temperature of 39° C. and a pH of 7.5, and then inactivating the enzyme to obtain a second enzymatic hydrolysis product; the amount of the chitosanase used to the first enzymatic hydrolysis product is in the ratio of 10 U:1 mL.

[0043] (3) adding β-glucosidase to the second enzymatic hydrolysis product, performing enzymatic hydrolysis for 3.5 h at a temperature of 44° C. and a pH of 5.7, and then inactivating the enzyme to obtain a third enzymatic hydrolysis product; the dosage of the β-glucosidase to the second enzymatic hydrolysis product is in a ratio of 8 U:1 mL.

[0044] (4) treating the third enzymatic hydrolysis product through a ceramic membrane filter to remove incompletely enzymatically hydrolyzed macromolecular suspended matter to obtain a filtrate;

[0045] (5) The filtrate is subjected to a first ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 250 Da to obtain a permeate;

[0046] (6) The permeate is subjected to a second ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 200 Da to obtain a concentrate with a molecular weight between 200 and 250 Da;

[0047] (7) Drying the concentrate to obtain N-acetamidoglucose.

[0048] The enzyme inactivation condition is heating at 121° C. for 15 min.

[0049] In step (5), the filtrate flow rate is 6 L / h and the membrane element pressure is 0.3 MPa.

[0050] In step (6), the filtrate flow rate is 3 L / h and the membrane element pressure is 0.7 MPa.

[0051] Example 2

[0052] This embodiment provides a method for preparing N-acetamido glucose based on enzymatic hydrolysis technology, the method comprising the following steps:

[0053] (1) Chitosan and water are mixed in a mass ratio of 1:10, chitinase is added, and enzymolysis is carried out at a temperature of 30° C. and a pH of 7 for 2 h. After the enzymolysis is completed, the enzyme is inactivated to obtain a first enzymolysis product; the amount of the chitinase and the water in step (1) is 15 U: 1 mL.

[0054] (2) adding chitosanase to the first enzymatic hydrolysis product, performing enzymatic hydrolysis for 5 h at a temperature of 40° C. and a pH of 7.3, and then inactivating the enzyme to obtain a second enzymatic hydrolysis product; the amount of the chitosanase used to the first enzymatic hydrolysis product is in the ratio of 12 U:1 mL.

[0055] (3) adding β-glucosidase to the second enzymatic hydrolysis product, performing enzymatic hydrolysis for 3 h at a temperature of 45° C. and a pH of 5.5, and then inactivating the enzyme to obtain a third enzymatic hydrolysis product; the dosage of the β-glucosidase to the second enzymatic hydrolysis product is in a ratio of 10 U:1 mL.

[0056] (4) treating the third enzymatic hydrolysis product through a ceramic membrane filter to remove incompletely enzymatically hydrolyzed macromolecular suspended matter to obtain a filtrate;

[0057] (5) The filtrate is subjected to a first ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 250 Da to obtain a permeate;

[0058] (6) The permeate is subjected to a second ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 200 Da to obtain a concentrate with a molecular weight between 200 and 250 Da;

[0059] (7) Drying the concentrate to obtain N-acetamidoglucose.

[0060] The enzyme inactivation condition is heating at 121° C. for 15 min.

[0061] In step (5), the filtrate flow rate is 5 L / h and the membrane element pressure is 0.2 MPa.

[0062] In step (6), the filtrate flow rate is 2 L / h and the membrane element pressure is 0.6 MPa.

[0063] Comparative Example 1

[0064] The difference between this comparative example and Example 1 is that the order of enzymolysis is different.

[0065] A method for preparing N-acetamidoglucose based on enzymatic hydrolysis technology, the method comprising the following steps:

[0066] (1) Chitosan and water are mixed in a mass ratio of 1:9, chitinase is added, and enzymolysis is carried out at a temperature of 39° C. and a pH of 7.5 for 4.5 hours. After the enzymolysis is completed, the enzyme is inactivated to obtain a first enzymolysis product; the amount of the chitinase and the water in step (1) is 18 U: 1 mL.

[0067] (2) adding β-glucosidase to the first enzymatic hydrolysis product, performing enzymatic hydrolysis for 3.5 h at a temperature of 44° C. and a pH of 5.7, and then inactivating the enzyme to obtain a second enzymatic hydrolysis product; the dosage of the β-glucosidase and the second enzymatic hydrolysis product is in a ratio of 10 U:1 mL.

[0068] (3) adding chitosanase to the second enzymatic hydrolysis product, performing enzymatic hydrolysis for 2.5 h at a temperature of 32° C. and a pH of 6.8, and then inactivating the enzyme to obtain a third enzymatic hydrolysis product; the amount of the chitosanase used to the second enzymatic hydrolysis product is in the ratio of 8 U:1 mL.

[0069] (4) treating the third enzymatic hydrolysis product through a ceramic membrane filter to remove incompletely enzymatically hydrolyzed macromolecular suspended matter to obtain a filtrate;

[0070] (5) The filtrate is subjected to a first ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 250 Da to obtain a permeate;

[0071] (6) The permeate is subjected to a second ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 200 Da to obtain a concentrate with a molecular weight between 200 and 250 Da;

[0072] (7) Drying the concentrate to obtain N-acetamidoglucose.

[0073] The enzyme inactivation condition is heating at 121° C. for 15 min.

[0074] In step (5), the filtrate flow rate is 6 L / h and the membrane element pressure is 0.3 MPa.

[0075] In step (6), the filtrate flow rate is 3 L / h and the membrane element pressure is 0.7 MPa.

[0076] Comparative Example 2

[0077] The difference between this comparative example and Example 1 is that the concentration of the enzyme is different.

[0078] A method for preparing N-acetamidoglucose based on enzymatic hydrolysis technology, the method comprising the following steps:

[0079] (1) Chitosan and water in a mass ratio of 1:9 are mixed, chitinase is added, and enzymolysis is carried out at a temperature of 32° C. and a pH of 6.8 for 2.5 hours. After the enzymolysis is completed, the enzyme is inactivated to obtain a first enzymolysis product; the amount of the chitinase and the water in step (1) is in a ratio of 8 U:1 mL.

[0080] (2) adding chitosanase to the first enzymatic hydrolysis product, performing enzymatic hydrolysis for 4.5 h at a temperature of 39° C. and a pH of 7.5, and then inactivating the enzyme to obtain a second enzymatic hydrolysis product; the amount of the chitosanase used to the first enzymatic hydrolysis product is in the ratio of 18 U:1 mL.

[0081] (3) adding β-glucosidase to the second enzymatic hydrolysis product, performing enzymatic hydrolysis for 3.5 h at a temperature of 44° C. and a pH of 5.7, and then inactivating the enzyme to obtain a third enzymatic hydrolysis product; the dosage of the β-glucosidase to the second enzymatic hydrolysis product is in a ratio of 10 U:1 mL.

[0082] (4) treating the third enzymatic hydrolysis product through a ceramic membrane filter to remove incompletely enzymatically hydrolyzed macromolecular suspended matter to obtain a filtrate;

[0083] (5) The filtrate is subjected to a first ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 250 Da to obtain a permeate;

[0084] (6) The permeate is subjected to a second ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 200 Da to obtain a concentrate with a molecular weight between 200 and 250 Da;

[0085] (7) Drying the concentrate to obtain N-acetamidoglucose.

[0086] The enzyme inactivation condition is heating at 121° C. for 15 min.

[0087] In step (5), the filtrate flow rate is 6 L / h and the membrane element pressure is 0.3 MPa.

[0088] In step (6), the filtrate flow rate is 3 L / h and the membrane element pressure is 0.7 MPa.

[0089] Comparative Example 3

[0090] The difference between this comparative example and Example 1 is that the types of enzymes are different.

[0091] A method for preparing N-acetamidoglucose based on enzymatic hydrolysis technology, the method comprising the following steps:

[0092] (1) Chitosan and water in a mass ratio of 1:9 are mixed, β-glucosidase is added, and enzymolysis is carried out at a temperature of 32° C. and a pH of 6.8 for 2.5 hours. After the enzymolysis is completed, the enzyme is inactivated to obtain a first enzymolysis product; the concentration of the β-glucosidase in water is 18 U / mL.

[0093] (2) adding chitosanase to the first enzymatic hydrolysis product, performing enzymatic hydrolysis for 4.5 h at a temperature of 39° C. and a pH of 7.5, and then inactivating the enzyme to obtain a second enzymatic hydrolysis product; the amount of the chitosanase used to the first enzymatic hydrolysis product is in the ratio of 10 U:1 mL.

[0094] (3) adding β-N-acetylglucosaminidase to the second enzymatic hydrolysis product, performing enzymatic hydrolysis for 3.5 h at a temperature of 44° C. and a pH of 5.7, and then inactivating the enzyme to obtain a third enzymatic hydrolysis product; the dosage of the β-N-acetylglucosaminidase to the second enzymatic hydrolysis product is in a ratio of 8 U:1 mL.

[0095] (4) treating the third enzymatic hydrolysis product through a ceramic membrane filter to remove incompletely enzymatically hydrolyzed macromolecular suspended matter to obtain a filtrate;

[0096] (5) The filtrate is subjected to a first ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 250 Da to obtain a permeate;

[0097] (6) The permeate is subjected to a second ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 200 Da to obtain a concentrate with a molecular weight between 200 and 250 Da;

[0098] (7) Drying the concentrate to obtain N-acetamidoglucose.

[0099] The enzyme inactivation condition is heating at 121° C. for 15 min.

[0100] In step (5), the filtrate flow rate is 6 L / h and the membrane element pressure is 0.3 MPa.

[0101] In step (6), the filtrate flow rate is 3 L / h and the membrane element pressure is 0.7 MPa.

[0102] Comparative Example 4

[0103] The difference between this comparative example and Example 1 is that a composite enzyme is used for preparation.

[0104] A method for preparing N-acetamidoglucose based on enzymatic hydrolysis technology, the method comprising the following steps:

[0105] (1) Chitosan and water are mixed in a mass ratio of 1:9, and a composite enzyme is added. The mixture is enzymolyzed at a temperature of 35°C and a pH of 7 for 12 hours. After the enzymolysis is completed, the enzyme is inactivated to obtain an enzymolysis product; the concentration of the composite enzyme in water is 30 U / mL. The composite enzyme is a mixture of chitinase, β-glucosidase, and β-N-acetylglucosaminidase in a mass ratio of 2:1:1.

[0106] (2) The enzymatic hydrolysis product is treated through a ceramic membrane filter to remove the macromolecular suspended matter that is not completely enzymatically hydrolyzed, thereby obtaining a filtrate;

[0107] (3) The filtrate is subjected to a first ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 250 Da to obtain a permeate;

[0108] (4) The permeate is subjected to a second ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 200 Da to obtain a concentrate with a molecular weight between 200 and 250 Da;

[0109] (5) Drying the concentrate to obtain N-acetamidoglucose.

[0110] The enzyme inactivation condition is heating at 121° C. for 15 min.

[0111] In step (3), the filtrate flow rate is 6 L / h and the membrane element pressure is 0.3 MPa.

[0112] In step (4), the filtrate flow rate is 3 L / h and the membrane element pressure is 0.7 MPa.

[0113] Comparative Example 5

[0114] The difference between this comparative example and Example 1 is that a composite enzyme is used for preparation.

[0115] A method for preparing N-acetamidoglucose based on enzymatic hydrolysis technology, the method comprising the following steps:

[0116] (1) Chitosan and water are mixed in a mass ratio of 1:9, and a composite enzyme is added. The mixture is enzymolyzed at a temperature of 35°C and a pH of 7 for 12 hours. After the enzymolysis is completed, the enzyme is inactivated to obtain an enzymolysis product; the concentration of the composite enzyme in water is 30 U / mL. The composite enzyme is a mixture of chitinase, β-glucosidase, and chitosanase in a mass ratio of 2:1:1.

[0117] (2) The enzymatic hydrolysis product is treated through a ceramic membrane filter to remove the macromolecular suspended matter that is not completely enzymatically hydrolyzed, thereby obtaining a filtrate;

[0118] (3) The filtrate is subjected to a first ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 250 Da to obtain a permeate;

[0119] (4) The permeate is subjected to a second ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 200 Da to obtain a concentrate with a molecular weight between 200 and 250 Da;

[0120] (5) Drying the concentrate to obtain N-acetamidoglucose.

[0121] The enzyme inactivation condition is heating at 121° C. for 15 min.

[0122] In step (3), the filtrate flow rate is 6 L / h and the membrane element pressure is 0.3 MPa.

[0123] In step (4), the filtrate flow rate is 3 L / h and the membrane element pressure is 0.7 MPa.

[0124] Comparative Example 6

[0125] The difference between this comparative example and Example 1 is that the purification method is different.

[0126] A method for preparing N-acetamidoglucose based on enzymatic hydrolysis technology, the method comprising the following steps:

[0127] (1) Chitosan and water are mixed in a mass ratio of 1:9, chitinase is added, and enzymolysis is carried out at a temperature of 32° C. and a pH of 6.8 for 2.5 hours. After the enzymolysis is completed, the enzyme is inactivated to obtain a first enzymolysis product; the amount of the chitinase and the water in step (1) is 18 U: 1 mL.

[0128] (2) adding chitosanase to the first enzymatic hydrolysis product, performing enzymatic hydrolysis for 4.5 h at a temperature of 39° C. and a pH of 7.5, and then inactivating the enzyme to obtain a second enzymatic hydrolysis product; the amount of the chitosanase used to the first enzymatic hydrolysis product is in the ratio of 10 U:1 mL.

[0129] (3) adding β-glucosidase to the second enzymatic hydrolysis product, performing enzymatic hydrolysis for 3.5 h at a temperature of 44° C. and a pH of 5.7, and then inactivating the enzyme to obtain a third enzymatic hydrolysis product; the dosage of the β-glucosidase to the second enzymatic hydrolysis product is in a ratio of 8 U:1 mL.

[0130] (4) The third enzymatic hydrolysis product is precipitated to obtain a supernatant, the supernatant is passed through a 717# anion resin, the effluent is passed through a 732# cation resin, the effluent of the cation resin is filtered, the filtrate is concentrated, and spray-dried to obtain N-acetamidoglucose.

[0131] The enzyme inactivation condition is heating at 121° C. for 15 min.

[0132] Performance Testing

[0133] Three batches of N-acetamido glucose were produced using the methods of Examples 1-2 and Comparative Examples 1-6, the purity and yield of N-acetamido glucose were determined, and the reaction time for each reaction was calculated.

[0134] Yield = actual mass of N-acetamido glucose obtained / theoretical mass of N-acetamido glucose*100%.

[0135] Purity = actual mass of N-acetamidoglucose obtained / mass of product obtained*100%.

[0136] Time = the sum of the enzymatic hydrolysis times of step (1), step (2), and step (3).

[0137] Table 1 First Batch Test Results

[0138] Time / h Yield / % purity / % Example 1 10.5 89.3 95.7 Example 2 10 89.0 95.6 Comparative Example 1 10.5 85.2 95.3 Comparative Example 2 10.5 86.7 95.5 Comparative Example 3 10.5 86.2 95.4 Comparative Example 4 12 83.4 95.2 Comparative Example 5 12 83.8 95.3 Comparative Example 6 10.5 87.8 94.4

[0139] Table 2 Second batch test results

[0140] Time / h Yield / % purity / % Example 1 10.5 89.4 95.7 Example 2 10 89.2 95.7 Comparative Example 1 10.5 85.1 95.4 Comparative Example 2 10.5 86.5 95.6 Comparative Example 3 10.5 86.3 95.6 Comparative Example 4 12 86.4 95.5 Comparative Example 5 12 81.2 95.0 Comparative Example 6 10.5 87.9 94.6

[0141] Table 3 The third batch test results

[0142] Time / h Yield / % purity / % Example 1 10.5 89.4 95.8 Example 2 10 89.4 95.7 Comparative Example 1 10.5 85.0 95.4 Comparative Example 2 10.5 86.5 95.4 Comparative Example 3 10.5 86.5 95.2 Comparative Example 4 12 81.2 95.0 Comparative Example 5 12 85.3 95.6 Comparative Example 6 10.5 87.2 94.5

[0143] From the above performance test results, it can be seen that the products of Examples 1-2 have high purity, yield and batch stability, especially the comprehensive performance of Example 1 is the most outstanding. This is mainly because the enzymatic hydrolysis process and the purification effect performed by a specific type of enzyme work synergistically, resulting in a more excellent effect.

[0144] The comparative example does not adopt the necessary technical scheme, which causes it to be significantly worse than the embodiment in the corresponding performance test. In comparative examples 4-5, the complex enzyme is used for enzymolysis, and it can be seen that the purity and yield effect decrease, and the stability is poor. The scheme of enzymolysis without using enzymes of specific order and concentration in comparative examples 1-3 can be seen from the results that the purity and yield of the product are affected. The purification method of comparative example 6 is different, which has an impact on purity and yield. The above experimental results further prove the importance of the technical scheme defined in the present invention for its technical effect.

[0145] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing N-acetamidoglucose based on enzymatic hydrolysis technology, characterized in that: The method comprises the following steps: (1) chitosan and water are mixed in a mass ratio of 1:8-10, chitinase is added, and enzymolysis is performed at a temperature of 30-33°C and a pH of 6.8-7 for 2-3 hours. After the enzymolysis is completed, the enzyme is inactivated to obtain a first enzymolysis product; (2) adding chitosanase to the first enzymatic hydrolysis product, performing enzymatic hydrolysis for 4-5 hours at a temperature of 38-40° C. and a pH of 7.3-7.6, and after the enzymatic hydrolysis is completed, inactivating the enzyme to obtain a second enzymatic hydrolysis product; (3) adding β-glucosidase to the second enzymatic hydrolysis product, performing enzymatic hydrolysis for 3-4 hours at a temperature of 43-45° C. and a pH of 5.5-6, and after the enzymatic hydrolysis is completed, inactivating the enzyme to obtain a third enzymatic hydrolysis product; (4) treating the third enzymatic hydrolysis product through a ceramic membrane filter to remove incompletely enzymatically hydrolyzed macromolecular suspended matter to obtain a filtrate; (5) The filtrate is subjected to a first ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 250 Da to obtain a permeate; (6) The permeate is subjected to a second ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 200 Da to obtain a concentrate with a molecular weight between 200 and 250 Da; (7) drying the concentrate to obtain N-acetamidoglucose; The dosage of the chitinase and the water in step (1) is (15-20) U: 1 mL; The dosage relationship of the chitosanase and the first enzymatic hydrolysis product is (8-12) U: 1 mL; The dosage relationship of the β-glucosidase and the second enzymatic hydrolysis product is (5-10) U: 1 mL; The total duration of the enzymatic hydrolysis in steps (1), (2) and (3) is less than 12 hours.

2. The method for preparing N-acetamidoglucose based on enzymatic hydrolysis technology according to claim 1, characterized in that: The enzyme inactivation condition is heating at 120-125° C. for 15-20 min.

3. The method for preparing N-acetamidoglucose based on enzymatic hydrolysis technology according to claim 1, characterized in that: In step (5), the filtrate flow rate is 5-8 L / h, and the membrane element pressure is 0.2-0.5 MPa.

4. The method for preparing N-acetamidoglucose based on enzymatic hydrolysis technology according to claim 1, characterized in that: In step (6), the filtrate flow rate is 2-4 L / h, and the membrane element pressure is 0.6-0.8 MPa.

5. The method for preparing N-acetamidoglucose based on enzymatic hydrolysis technology according to claim 1, characterized in that: The yield of N-acetamidoglucose is greater than 88%.

6. The method for preparing N-acetamidoglucose based on enzymatic hydrolysis technology according to claim 1, characterized in that: The purity of N-acetamidoglucose is greater than 95%.