A kind of sucrose synthase and its application in food

Abstract

The present invention provides a kind of sucrose synthase and its application in food, the preparation method of sucrose synthase comprises the following steps: (1) strain activation: methyl bacterium is inoculated into beef extract peptone medium, cultured, and seed liquid is obtained; methyl bacterium deposit number is SHBCC D10457, Shanghai Collection Microorganism Center; (2) fermentation: seed liquid is inoculated into fermentation medium, fermentation culture is carried out in fermenter, and fermentation liquid is obtained; (3) purification; (4) sucrose synthase in eluate is ultrafiltration displacement, and is replaced in 20mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution with pH of 4.6 to obtain sucrose synthase liquid, and the enzyme activity in sucrose synthase liquid is 500U / μL, and sucrose synthase liquid and protective liquid are mixed in equal volumes. The enzyme activity of the sucrose synthase of the present invention is high, the stability is strong, and it is convenient for storage, long-distance transportation and promotion.

Description

Technical Field

[0001] The invention belongs to the technical field of food, and in particular relates to a sucrose synthase and application thereof in food. Background Art

[0002] Stevioside, also known as stevioside and stevioside, is a new type of natural sweetener extracted from the leaves of Stevia rebaudiana, a herbaceous plant of the Asteraceae family. Stevia is native to South America, and for centuries, locals have used its leaves as a natural sweetener and medicinal herb. Stevioside has attracted much attention due to its high sweetness, low calories, and the fact that it does not cause a significant increase in blood sugar. It is especially suitable for diabetic patients and people who pursue a healthy diet.

[0003] In industry, the use of enzymatic conversion to produce steviol glycosides is an efficient and environmentally friendly method. This method uses specific enzymes to catalyze the conversion of steviol glycoside precursors (such as steviol) in stevia plants into steviol glycosides with higher sweetness and better taste. Patent 202110853028.X discloses a sucrose synthase and its application, which combines glucosyltransferase with sucrose synthase to catalyze the synthesis of rebaudioside A, rebaudioside D or rebaudioside M to achieve a cascade reaction; it solves the problem of expensive glycosyl donors UDPG and ADPG.

[0004] In industrial production, sucrose synthase can be extracted from Methylobacterium, but the enzyme activity of the fermentation product and the storage stability of the final preparation are not ideal, resulting in low production efficiency, unstable product quality, poor storage stability of the preparation, and the product is more likely to deteriorate during transportation and sales, increasing the risk of returns and losses, while limiting the market distribution of the product. Summary of the invention

[0005] The purpose of the present invention is to provide a sucrose synthase and its application in food. The sucrose synthase has high enzyme activity and strong stability and is convenient for storage, long-distance transportation and promotion and use.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a sucrose synthase, and a preparation method thereof comprises the following steps:

[0008] (1) Strain activation: inoculate methyl bacterium into beef extract peptone medium at an inoculum rate of 2-4% of the volume percentage of beef extract peptone medium, and culture at 40-45° C. and 160-180 r / min for 50-60 h to obtain a seed solution; the deposit number of methyl bacterium is SHBCC D10457, Shanghai Collection Center of Microorganisms;

[0009] (2) Fermentation: The seed liquid is inoculated into a fermentation medium at an inoculum rate of 2-4% by volume of the fermentation medium, wherein the fermentation medium comprises the following components: 3-5 g / L cellobiose, 7-9 g / L corn steep liquor, 6-8 g / L peptone, 2-4 g / L soybean powder, 8-11 g / L glucose, 14-16 g / L glycerol, 4-6 g / L sodium glutamate, 3-5 g / L tripotassium citrate, 1-2 g / L potassium dihydrogen phosphate, 2-4 g / L sodium bicarbonate, 1-3 g / L ammonium chloride, urea. 4-6g / L, yeast extract 1-2g / L, calcium chloride dihydrate 0.6-0.9g / L, magnesium sulfate 1.3-1.7g / L, aminoadipic acid 0.006-0.009g / L and betaine 0.005-0.008g / L; fermentation culture is carried out in a fermenter at a fermentation temperature of 40-45°C, a tank pressure of 0.02-0.03MPa, a ventilation volume of 1:1v / v / min, a stirring speed of 350-400 rpm, and a fermentation time of 50-60h to obtain a fermentation liquid;

[0010] (3) Purification: The fermentation broth is subjected to solid-liquid separation to remove the bacterial cells, and the filtrate is concentrated under reduced pressure at 55-60° C. to 20-25% of the volume of the filtrate to obtain a concentrated solution; a 35 wt % aqueous solution of ammonium sulfate is added to the concentrated solution and allowed to stand for 4-5 h to remove the precipitate, and then a 65 wt % aqueous solution of ammonium sulfate is added and allowed to stand for 7-8 h, and the supernatant is removed to obtain a crude sucrose synthase, which is purified using a gel chromatography column to obtain an eluate;

[0011] (4) The sucrose synthase in the eluate is replaced by ultrafiltration in a 20 mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer having a pH of 4.6 to obtain a sucrose synthase solution having an enzyme activity of 500 U / μL. The sucrose synthase solution and the protective solution are mixed.

[0012] Furthermore, the volume ratio of the sucrose synthase solution to the protective solution is 1:1.

[0013] The high enzyme activity of the fermentation liquid in the enzyme production process can save manpower and material resources and has high economic value. However, the activity of sucrose synthase in the fermentation liquid prepared by using methylbacterium fermentation in the prior art is not ideal, resulting in high production costs. The present invention optimizes the fermentation process of methylbacterium, optimizes the culture medium, fermentation temperature and time, and the three work together to greatly improve the enzyme activity of sucrose synthase in the fermentation liquid.

[0014] Furthermore, the protective solution comprises the following components: 200 mM dextran, 500 mM xylitol, 300 mM lysine, 60 mM lactose, 20 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer with a pH of 4.6, 100 mM potassium chloride, 10 mM magnesium chloride and 0.4 wt % surfactant.

[0015] Furthermore, the surfactant is selected from one or more of polydimethylsiloxane, polyethylene glycol PEG400, and polyethylene glycol sorbitan fatty acid ester.

[0016] Furthermore, the surfactant is a compound of polydimethylsiloxane, polyethylene glycol PEG400 and polyethylene glycol sorbitan fatty acid ester in a mass ratio of 1:1.3-1.6:0.4-0.7.

[0017] Further, the volume of the added 35 wt % ammonium sulfate aqueous solution is the same as that of the concentrated solution.

[0018] Further, the volume of the added 65 wt % ammonium sulfate aqueous solution is the same as that of the concentrated solution.

[0019] Furthermore, the purification steps are as follows: firstly, the gel chromatography column is balanced with distilled water, then the crude sucrose synthase is passed through the column, and then eluted with 3 times column volume of 0.25 mol / L phosphate buffer to obtain an eluate.

[0020] Furthermore, the gel chromatography column is cross-linked dextran Sephadex G-100.

[0021] As a biologically active substance, the activity of enzyme preparation is the key point. In the prior art, the purified sucrase is generally dried directly, packaged and stored, and the product has poor temperature resistance, poor storage stability, and is easily deteriorated during long-term transportation or storage. The present invention adds a protective liquid during the preparation process to improve the storage stability of sucrose synthase at room temperature and low temperature in the product. The analysis is that the protective liquid system of the present invention can provide a stable and mild microenvironment for sucrose synthase, which helps to maintain the structural integrity of the enzyme molecule. The various components act together through various mechanisms to provide a relatively stable storage environment for sucrose synthase, reduce the denaturation, degradation and activity loss of the enzyme, thereby extending its effective storage period. The inventors found that the use of a specific surfactant in the protective liquid can improve the storage stability of sucrose synthase under high temperature, high humidity and illumination. The analysis is that the surfactant of the present invention can better form a protective film on the surface of the enzyme molecule, and the surfactant can optimize the solvation shell around the enzyme, ensuring that the enzyme molecule is in a more stable and more adaptable microenvironment, slowing down the structural damage caused by high temperature.

[0022] The invention provides application of the sucrose synthase in food.

[0023] Furthermore, the sucrose synthase is used to prepare steviol glycosides.

[0024] The ventilation rate is 1:1v / v / min, which means that the ratio of the volume of gas introduced into the fermentation system per minute to the volume of the fermentation liquid is 1:1, that is, the amount of gas introduced per minute is equal to the volume of the fermentation liquid.

[0025] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0026] 1. The present invention provides a sucrose synthase and its application in food. The sucrose synthase has high enzyme activity and strong stability, and is convenient for storage, long-distance transportation and promotion and use.

[0027] 2. The present invention optimizes the fermentation process of Methylobacterium, the culture medium, the fermentation temperature and the time, and the three work together to greatly improve the enzyme activity of sucrose synthase in the fermentation broth.

[0028] 3. The present invention adds a protective liquid during the preparation process, thereby improving the storage stability of sucrose synthase at room temperature and low temperature.

[0029] 4. The use of a specific surfactant in the protective solution of the present invention can improve the storage stability of sucrose synthase under high temperature, high humidity and light. DETAILED DESCRIPTION

[0030] 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.

[0031] Example 1

[0032] This embodiment provides a sucrose synthase, and the preparation method thereof comprises the following steps:

[0033] (1) Strain activation: Methylobacterium was inoculated into beef extract peptone medium at an inoculum rate of 3% by volume of beef extract peptone medium, and cultured at 43° C. and 170 rpm for 55 h to obtain a seed solution; the Methylobacterium deposit number is SHBCC D10457, Shanghai Collection of Microorganisms Center;

[0034] (2) Fermentation: The seed liquid is inoculated into a fermentation medium at an inoculum amount of 3% by volume of the fermentation medium, wherein the fermentation medium comprises the following components: 4 g / L of cellobiose, 8 g / L of corn steep liquor, 7 g / L of peptone, 3 g / L of soybean powder, 9 g / L of glucose, 15 g / L of glycerol, 5 g / L of sodium glutamate, 4 g / L of tripotassium citrate, 1.3 g / L of potassium dihydrogen phosphate, 3 g / L of sodium bicarbonate, 2 g / L of ammonium chloride, 5 g / L of urea, 1.2 g / L of yeast extract, 0.7 g / L of calcium chloride dihydrate, 1.5 g / L of magnesium sulfate, 0.007 g / L of aminoadipic acid and 0.006 g / L of betaine, and the fermentation is carried out in a fermenter at a fermentation temperature of 42° C., a tank pressure of 0.02 MPa, an aeration rate of 1:1 v / v / min, a stirring speed of 350 rpm, and a fermentation time of 50 h to obtain a fermentation liquid;

[0035] (3) Purification: The fermentation broth was subjected to solid-liquid separation to remove the bacterial cells, and the filtrate was concentrated under reduced pressure at 57°C to 22% of the volume of the filtrate to obtain a concentrate; a 35 wt% aqueous solution of ammonium sulfate was added to the concentrate and allowed to stand for 4.5 h, the volume of the 35 wt% aqueous solution of ammonium sulfate added being the same as that of the concentrate; the precipitate was removed, and a 65 wt% aqueous solution of ammonium sulfate was added, the volume of the 65 wt% aqueous solution of ammonium sulfate added being the same as that of the concentrate; the precipitate was removed, and a 65 wt% aqueous solution of ammonium sulfate was added, the volume of the 65 wt% aqueous solution of ammonium sulfate added being the same as that of the concentrate; the precipitate was allowed to stand for 7.5 h, and the supernatant was removed to obtain a crude sucrose synthase, which was purified using a gel chromatography column, and the purification steps were as follows: first, the gel chromatography column was equilibrated with distilled water, and then the crude sucrose synthase was passed through the column, and eluted with 3 times the column volume of 0.25 mol / L phosphate buffer, the gel chromatography column was cross-linked dextran Sephadex G-100, and an eluate was obtained;

[0036] (4) The sucrose synthase in the eluate is replaced by ultrafiltration in a 20 mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer having a pH of 4.6 to obtain a sucrose synthase solution having an enzyme activity of 500 U / μL. The sucrose synthase solution and the protective solution are mixed in equal volumes.

[0037] Beef extract peptone medium, Beijing Hongrun Baoshun Technology Co., Ltd., catalog number: HRBS-Y006A.

[0038] The protective solution comprises the following components: prepared with water, 200 mM dextran, 500 mM xylitol, 300 mM lysine, 60 mM lactose, 20 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer with a pH of 4.6, 100 mM potassium chloride, 10 mM magnesium chloride and 0.4 wt % surfactant.

[0039] The surfactant is a compound of polydimethylsiloxane (Foshan Guirunbao Biotechnology Co., Ltd.), polyethylene glycol PEG400 (Xi'an Jinxiang Pharmaceutical Excipients Co., Ltd.) and Tween 60 (Hebei Pengyu Biotechnology Co., Ltd.) in a mass ratio of 1:1.5:0.6.

[0040] Example 2

[0041] This embodiment provides a sucrose synthase, and the preparation method thereof comprises the following steps:

[0042] (1) Strain activation: Methylobacterium was inoculated into beef extract peptone medium at an inoculum rate of 2% by volume of beef extract peptone medium, and cultured at 45° C. and 160 rpm for 60 h to obtain a seed solution; the Methylobacterium deposit number is SHBCC D10457, Shanghai Collection Center of Microorganisms;

[0043] (2) Fermentation: The seed liquid is inoculated into a fermentation medium at an inoculum amount of 2% by volume of the fermentation medium, wherein the fermentation medium comprises the following components: 5 g / L of cellobiose, 7 g / L of corn steep liquor, 8 g / L of peptone, 2 g / L of soybean powder, 11 g / L of glucose, 14 g / L of glycerol, 6 g / L of sodium glutamate, 3 g / L of tripotassium citrate, 2 g / L of potassium dihydrogen phosphate, 2 g / L of sodium bicarbonate, 3 g / L of ammonium chloride, 4 g / L of urea, 2 g / L of yeast extract, 0.6 g / L of calcium chloride dihydrate, 1.7 g / L of magnesium sulfate, 0.006 g / L of aminoadipic acid and 0.008 g / L of betaine, and the fermentation is carried out in a fermenter at a fermentation temperature of 40° C., a tank pressure of 0.03 MPa, an aeration rate of 1:1 v / v / min, a stirring speed of 350 rpm, and a fermentation time of 50 h to obtain a fermentation liquid;

[0044] (3) Purification: The fermentation broth is subjected to solid-liquid separation to remove the bacterial cells, and the filtrate is concentrated under reduced pressure at 56°C to 25% of the volume of the filtrate to obtain a concentrate; a 35 wt% aqueous solution of ammonium sulfate is added to the concentrate and allowed to stand for 4 hours, the volume of the 35 wt% aqueous solution of ammonium sulfate added being the same as that of the concentrate; the precipitate is removed, and then a 65 wt% aqueous solution of ammonium sulfate is added, the volume of the 65 wt% aqueous solution of ammonium sulfate added being the same as that of the concentrate; the precipitate is allowed to stand for 8 hours, and the supernatant is removed to obtain a crude sucrose synthase, which is purified using a gel chromatography column, the purification steps being as follows: first balancing the gel chromatography column with distilled water, then passing the crude sucrose synthase through the column, and eluting it with 3 times the column volume of 0.25 mol / L phosphate buffer, the gel chromatography column is cross-linked dextran Sephadex G-100, and obtaining an eluate;

[0045] (4) The sucrose synthase in the eluate is replaced by ultrafiltration in a 20 mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer having a pH of 4.6 to obtain a sucrose synthase solution having an enzyme activity of 500 U / μL. The sucrose synthase solution and the protective solution are mixed in equal volumes.

[0046] The protective solution comprises the following components: prepared with water, 200 mM dextran, 500 mM xylitol, 300 mM lysine, 60 mM lactose, 20 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer with a pH of 4.6, 100 mM potassium chloride, 10 mM magnesium chloride and 0.4 wt % surfactant.

[0047] The surfactant is a compound of polydimethylsiloxane (Foshan Guirunbao Biotechnology Co., Ltd.), polyethylene glycol PEG400 (Xi'an Jinxiang Pharmaceutical Excipients Co., Ltd.) and Tween 60 (Hebei Pengyu Biotechnology Co., Ltd.) in a mass ratio of 1:1.3:0.7.

[0048] Comparative Example 1

[0049] The difference between this comparative example and Example 1 is that the fermentation medium includes the following components: 10 g / L of cellobiose, 2 g / L of corn steep liquor, 2 g / L of peptone, 13 g / L of soybean powder, 5 g / L of glucose, 11 g / L of glycerol, 9 g / L of sodium glutamate, 1 g / L of tripotassium citrate, 4 g / L of potassium dihydrogen phosphate, 7 g / L of sodium bicarbonate, 1 g / L of ammonium chloride, 1 g / L of urea, 2 g / L of yeast extract, 1.6 g / L of calcium chloride dihydrate, 0.7 g / L of magnesium sulfate, 0.010 g / L of aminoadipic acid and 0.004 g / L of betaine, and is prepared with water.

[0050] Comparative Example 2

[0051] The difference between this comparative example and Example 1 is that the fermentation temperature in step (2) is 30°C.

[0052] Comparative Example 3

[0053] The difference between this comparative example and Example 1 is that the fermentation temperature in step (2) is 55°C.

[0054] Comparative Example 4

[0055] The difference between this comparative example and Example 1 is that the fermentation time in step (2) is 40 h.

[0056] Comparative Example 5

[0057] The difference between this comparative example and Example 1 is that the fermentation time in step (2) is 80 h.

[0058] Comparative Example 6

[0059] The difference between this comparative example and Example 1 is that in step (2), the fermentation medium is a commercially available product, Shandong Weiduofeng Biotechnology Co., Ltd., product number 201902.

[0060] Comparative Example 7

[0061] The difference between this comparative example and Example 1 is that the protective solution includes the following components: prepared with water, 500mM dextran, 200mM xylitol, 60mM lysine, 300mM lactose, 20mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer with a pH of 4.6, 10mM potassium chloride, 100mM magnesium chloride and 0.2wt% surfactant.

[0062] Comparative Example 8

[0063] The difference between this comparative example and Example 1 is that the surfactant is a compound of polydimethylsiloxane (Foshan Guirunbao Biotechnology Co., Ltd.), polyethylene glycol PEG400 (Xi'an Jinxiang Pharmaceutical Excipients Co., Ltd.) and Tween 60 (Hebei Pengyu Biotechnology Co., Ltd.) in a mass ratio of 1:1:1.

[0064] Comparative Example 9

[0065] The difference between this comparative example and Example 1 is that the surfactant is a compound of Tween 20, Tween 80 and Triton X-100 in a mass ratio of 1:1.5:0.6.

[0066] Performance Testing

[0067] 1. Determine the enzyme activity of the fermentation broth obtained in step (2) of Example 1-2 and Comparative Example 1-6. Take 50 mg of filter paper, add 1 mL of 0.1 mol / L citric acid buffer at pH 6, add 0.5 ml of appropriately diluted enzyme solution, react at 50°C for 1 hour, and add 3 ml of DNS reagent to measure sugar. The above enzyme activities are calculated after deducting the reducing sugar in the fermentation broth. The enzyme activity is in international units, which is defined as 1 activity unit for the amount of enzyme that hydrolyzes 1 μmol glucose per min. The results are shown in Table 1.

[0068] Table 1 Enzyme activity determination results

[0069]

[0070] The results show that the enzyme activity test results of Examples 1 to 2 are higher than those of Comparative Examples 1 to 6, indicating that the enzyme activity decreases to varying degrees when the fermentation medium and fermentation conditions are changed.

[0071] 2. The final product sucrose synthase of Examples 1-2 and Comparative Examples 7-9 was sealed and packaged. Storage stability test was performed to determine the enzyme activity preservation rate (%), which refers to the percentage of residual enzyme activity after the enzyme treatment process. That is, after the enzyme was treated, the ratio of the enzyme activity after treatment to the enzyme activity before treatment was measured. The results are shown in Table 2.

[0072] (1) Normal temperature experiment: relative humidity 60%, 25°C, for 30 days.

[0073] (2) Low temperature experiment: relative humidity 60%, 4°C for 1 year.

[0074] (3) High temperature test: Place at 60℃ for 12h.

[0075] (4) Lighting experiment: 4500Lx illumination, placed for 10 days.

[0076] Table 2 Enzyme activity preservation rate test results (%)

[0077]

[0078]

[0079] The results show that the sucrose synthase of Example 1-2 of the present invention has high storage stability by adding a specific protective solution.

[0080] 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 sucrose synthase, characterized in that The preparation method of sucrose synthase comprises the following steps: (1) Strain activation: inoculate methyl bacterium into beef extract peptone medium at an inoculum rate of 2-4% of the volume percentage of beef extract peptone medium, and culture at 40-45°C and 160-180 r / min for 50-60 h to obtain a seed solution; the Methyl bacterium deposit number is SHBCC D10457; (2) Fermentation: The seed liquid is inoculated into the fermentation medium at an inoculation rate of 2-4% by volume of the fermentation medium, wherein the fermentation medium comprises the following components: 3-5 g / L cellobiose, 7-9 g / L corn steep liquor, 6-8 g / L peptone, 2-4 g / L soybean powder, 8-11 g / L glucose, 14-16 g / L glycerol, 4-6 g / L sodium glutamate, 3-5 g / L tripotassium citrate, 1-2 g / L potassium dihydrogen phosphate, 2-4 g / L sodium bicarbonate, 1-3 g / L ammonium chloride, and urea. 4-6g / L, yeast extract 1-2g / L, calcium chloride dihydrate 0.6-0.9g / L, magnesium sulfate 1.3-1.7g / L, aminoadipic acid 0.006-0.009g / L and betaine 0.005-0.008g / L; fermentation culture is carried out in a fermenter at a fermentation temperature of 40-45°C, a tank pressure of 0.02-0.03MPa, a ventilation volume of 1:1v / v / min, a stirring speed of 350-400 rpm, and a fermentation time of 50-60h to obtain a fermentation liquid; (3) Purification: The fermentation broth is subjected to solid-liquid separation to remove the bacterial cells, and the filtrate is concentrated under reduced pressure at 55-60°C to 20-25% of the filtrate volume to obtain a concentrated solution; a 35 wt% ammonium sulfate aqueous solution is added to the concentrated solution and allowed to stand for 4-5 hours to remove the precipitate, and then a 65 wt% ammonium sulfate aqueous solution is added and allowed to stand for 7-8 hours, and the supernatant is removed to obtain a crude sucrose synthase, which is purified using a gel chromatography column to obtain an eluent; (4) The sucrose synthase in the eluate is replaced by ultrafiltration in a 20 mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer having a pH of 4.6 to obtain a sucrose synthase solution having an enzyme activity of 500 U / μL. The sucrose synthase solution and the protective solution are mixed to obtain sucrose synthase; The protective solution comprises the following components: 200 mM dextran, 500 mM xylitol, 300 mM lysine, 60 mM lactose, 20 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer with a pH of 4.6, 100 mM potassium chloride, 10 mM magnesium chloride and 0.4 wt % surfactant; The surfactant is selected from one or more of polydimethylsiloxane, polyethylene glycol PEG400, and polyethylene glycol sorbitan fatty acid ester.

2. A sucrose synthase according to claim 1, characterized in that: The surfactant is a compound of polydimethylsiloxane, polyethylene glycol PEG400 and polyethylene glycol sorbitan fatty acid ester in a mass ratio of 1:1.3-1.6:0.4-0.

7.

3. A sucrose synthase according to claim 1, characterized in that: The volume of the added 35 wt % ammonium sulfate aqueous solution was the same as that of the concentrate.

4. A sucrose synthase according to claim 1, characterized in that: The volume of the added 65 wt % ammonium sulfate aqueous solution was the same as that of the concentrate.

5. A sucrose synthase according to claim 1, characterized in that: The purification steps are as follows: firstly, the gel chromatography column is balanced with distilled water, then the crude sucrose synthase is passed through the column, and then eluted with 3 times the column volume of 0.25 mol / L phosphate buffer to obtain an eluate.

6. A sucrose synthase according to claim 5, characterized in that: The gel chromatography column was cross-linked dextran Sephadex G-100.

7. Use of the sucrose synthase according to any one of claims 1 to 6 in food, characterized in that: The sucrose synthase is used for preparing steviol glycosides.

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