A method for producing glucosylglycerol

Abstract

The present invention discloses a method for producing glucosylglycerol. This method uses glycerol and sucrose as substrates, and sucrose phosphorylase is used to catalyze the reaction of the substrates to obtain a product containing glucosylglycerol. The reaction system contains water but no buffer solution. This method does not require the participation of a buffer solution, and the reaction system is simpler, only containing three components: the substrate sucrose and glycerol, and the whole-cell catalyst, excluding the interference caused by the presence of buffer ions in the downstream separation and purification of GG, and to a certain extent simplifies the downstream separation and purification process of GG. The present invention further improves the catalytic efficiency of sucrose phosphorylase in synthesizing GG and shortens the reaction cycle by optimizing the number of batch feeding times and the feeding time of the reaction. It has great potential for industrial application.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a method for preparing glucosylglycerol by enzymatic method. Background Art

[0002] Glucosylglycerol (GG) is a type of glycoside compound formed by the connection of glycerol molecules and glucose molecules through glycosidic bonds. It has extremely strong physiological effects of "moisturizing, water-locking, and moisturizing", and can also activate the synthesis of collagen in human skin cells. It can be used as a functional raw material for cosmetics, playing the roles of skin moisturizing, repair, and anti-aging. In addition, glucosylglycerol is also a macromolecular stabilizer, which can stabilize the structures of biological macromolecules such as proteins under high-temperature or freeze-drying conditions, and can be used for the long-term preservation of protein drugs. More research shows that glucosylglycerol has a certain role in preventing dental caries, inhibiting the accumulation of neutral lipids in adipocytes, and anti-allergy. Therefore, glucosylglycerol has great application value and broad market prospects in the cosmetics, pharmaceutical, and food industries.

[0003] At present, there are two methods for the industrial production of glucosylglycerol. In China, the main method is direct extraction from algae, extracting glucosylglycerol from Spirulina, which is also an industrialization technology developed recently (ZL 2018 1 0908060.1, ZL2018 1 0907595.7, ZL 2013 10598111.2). However, this technology requires large-scale cultivation of Spirulina, and large-scale outdoor cultivation is severely affected by environmental factors such as weather, temperature, and site, affecting the process stability and having a high cost. Abroad, the main method is enzymatic catalytic synthesis. Through immobilized sucrose phosphorylase, sucrose and glycerol substrates are catalytically synthesized into GG in one step. Under the optimal reaction conditions, that is, at a final concentration of 20 IU / ml of immobilized enzyme, 30 degrees, and in a 50 mM MES pH 7.0 buffer solution, the final conversion rate of the substrate sucrose only reaches 88%, and the catalytic synthesis of GG is 165 g / L. This process has a high cost of enzyme immobilization, uses a relatively expensive MES buffer solution, and the conversion rate of the substrate sucrose still needs to be improved (Angew.Chem.Int.Ed.2008, 47, 10086-10089; US 10,683,525 B2). Summary of the Invention

[0004] The inventors of the present invention surprisingly found during the research process that when using sucrose phosphorylase to catalyze the reaction of glycerol and sucrose, it is also possible to well achieve the generation of glucosylglycerol from glycerol and sucrose by sucrose phosphorylase without adding a buffer solution and only adding water, and the effects such as conversion efficiency and yield are not lower than those of the buffer system. This discovery undoubtedly has important significance and practical application value in the field of enzymatic preparation of glucosylglycerol. It can not only save production costs but also provide convenience for subsequent purification.

[0005] Therefore, the present invention firstly aims to protect a method for producing glucosylglycerol. The method for producing glucosylglycerol comprises the following steps: using glycerol and sucrose as substrates, and subjecting the substrates to a catalytic reaction with sucrose phosphorylase to obtain a product containing glucosylglycerol, wherein the reaction system contains water but no buffer.

[0006] The fact that the reaction system does not contain any buffer means that from the start of formulating the reaction system until the end of the reaction, no buffer is added to the reaction system throughout the entire reaction process.

[0007] The present invention further improves the catalytic efficiency of sucrose phosphorylase in synthesizing GG and shortens the reaction cycle by optimizing the number of reaction batch feeding times and the feeding time. It has great potential for industrial application.

[0008] Through research, it is found that in this method, there is no particular limitation on sucrose phosphorylase.

[0009] The sequence of sucrose phosphorylase can be the sequence of sucrose phosphorylase derived from any microorganism, which can be from bacteria, such as Leuconostoc mesenteroides, Agrobacterium vitis, Bifidobacterium adolescentis, Bifidobacterium longum, Clostridium thermosaccharolyticum, Escherichia coli, Lactobacillus acidophilus, Lactobacillus delbrueckii, Listeria monocytogenes, Pseudomonas putrefaciens, Pseudomonas saccharophila, Rhodopirellula baltica, Shewanella baltica, Shewanella frigidimarina, Solibacterus itatus, Streptococcus mutans, Leuconostoc citreum, Lactobacillus gastricu, Bacillus macerans, Leuconostoc pseudomesenteroides, Lactobacillus reuteri, and / or Synechococcus sp.

[0010] Sucrose phosphorylase can be obtained from recombinant microorganisms expressing sucrose phosphorylase by means of genetic engineering, or can be obtained from natural microorganisms.

[0011] The sucrose phosphorylase can be a complete enzyme, a catalytically active fragment (which refers to an active protein fragment of sucrose phosphorylase having the same or substantially the same activity and substrate specificity as the native sucrose phosphorylase), or a fusion protein of the enzyme protein with other polypeptides.

[0012] The sucrose phosphorylase can be wild-type or a mutant. If it is a mutant, it is only required to be a sucrose phosphorylase mutant having the same or substantially the same activity and substrate specificity as the wild-type sucrose phosphorylase.

[0013] The sucrose phosphorylase can be in any form, can be a purified enzyme, an unpurified enzyme, whole cells expressing the enzyme, lysates of whole cells expressing the enzyme, fermentation broth of cells expressing the enzyme, or a suspension of cells expressing the enzyme.

[0014] The cells expressing the enzyme can be Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum or yeast, and the cells can be selected according to the techniques publicly available in the art.

[0015] In an actual reaction system, the sucrose phosphorylase can be a mixture of one or several sucrose phosphorylases from different sources.

[0016] The sucrose phosphorylase participating in the reaction also does not require immobilization.

[0017] The sucrose phosphorylase in the reaction system only needs to be present at an effective concentration, usually at a concentration of about 0.001 to about 100.0 IU / ml, preferably greater than or equal to 1 IU / ml, more preferably 1 to 50 IU / ml, and even more preferably 1 - 8 IU / ml. 1 IU (International Unit) is defined as the amount of enzyme required to catalyze the production of 1 μmol of GG from sucrose and glycerol per minute under the conditions of 30 °C, 100 g / L glycerol, 100 g / L sucrose, and a final concentration of 20 OD600 whole cell catalyst.

[0018] The concentrations of glycerol and sucrose in the reaction system can both be within their respective saturation concentrations. As a preferred embodiment, the concentrations of glycerol and sucrose in the reaction system can both be greater than 100 g / L, more preferably both greater than or equal to 110 g / L or both greater than or equal to 150 g / L.

[0019] As a preferred manner, it is better that the mass ratio of sucrose to glycerol is less than 2:1.

[0020] According to the common general knowledge in the art, the sucrose and glycerol can be fed in a one-time feeding manner. However, in order to obtain better effects, the inventors of the present invention further explored and finally found that it is better to add sucrose to the reaction system in a batch feeding manner. The number of feeding times can be determined according to the actual situation. As a preferred manner, the number of feeding times for batch feeding can be 2 - 10 times, preferably 2 - 3 times. Specifically, it can be 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times or 10 times.

[0021] The inventors further explored the feeding time and found that the interval time between two adjacent feedings is preferably 1 - 15 hours, more preferably 2 - 12 hours, and even more preferably 7 - 12 hours. Specifically, it can be 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, or 12 hours.

[0022] The reaction temperature can be determined by using conventional means in the art in combination with the common general knowledge in the art, such as determining the reaction temperature according to the suitable catalytic temperature of the sucrose phosphorylase used. The reaction temperature selected in the present invention is 20 - 65 °C, and 10 - 40 °C, 37 - 50 °C are also acceptable.

[0023] In order to further obtain pure glucosylglycerol, the reaction product can be separated and purified.

[0024] The reaction system established in the present invention, on the one hand, does not require the participation of a buffer solution, and the reaction system is simpler, only containing three components: the substrate sucrose, glycerol, and the whole cell catalyst, excluding the interference caused by the presence of buffer ions in the downstream separation and purification of GG, and to a certain extent simplifies the downstream separation and purification process of GG. On the other hand, the present invention adopts a production method of batch feeding of solid sucrose, which is simple in operation, and while maintaining a high conversion rate of the substrate sucrose (>99%) and a high yield of GG (94%), significantly shortens the reaction cycle. Brief Description of the Drawings

[0025] Figure 1 HPLC Detection of the Reaction Product Catalyzed by Sucrose Phosphorylase

[0026] Figure 2 Detection Results of GG Production by Different Forms of Enzyme Catalysts

[0027] Figure 3 Process Curve of GG Synthesis Catalyzed by Whole Cell Catalyst with or without MES Buffer System

[0028] Figure 4 Effect of Different Time Intervals of Batch Feeding of Sucrose on the Catalytic Reaction Efficiency Detailed Description of the Invention

[0029] The present invention will be further described in detail below in conjunction with specific embodiments. The provided embodiments are only for clarifying the present invention and not for limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements and do not constitute any limitation to the present invention in any way.

[0030] In the following embodiments, the experimental methods are conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified. Unless otherwise specified, in the following embodiments, the quantitative tests are all set with three repeated experiments, and the results are averaged.

[0031] Example 1: Preparation of sucrose phosphorylase using recombinant Escherichia coli

[0032] It can be achieved according to the conventional technical means of expressing foreign proteins by recombinant bacteria in this field.

[0033] The gene coding sequence of sucrose phosphorylase was cloned into the pET28b(+) expression vector (Novagen), and then introduced into Escherichia coli BL21(DE3) (Transgen biotech). It was induced to culture overnight with IPTG to prepare an Escherichia coli whole-cell catalyst expressing sucrose phosphorylase. The fermentation broth with an OD600 value of 30 was taken and reserved.

[0034] Example 2: Production of GG using different forms of enzyme catalysts

[0035] LmSP is derived from (Leuconostoc mesenteroides, Q59495), and is disclosed in Patent US 10,683,525 B2.

[0036] The fermentation broth with an OD600 value of 30 was taken, and the wet cells were collected by centrifugation for catalysis.

[0037] The fermentation broth with an OD600 value of 30 was directly used for catalysis.

[0038] The fermentation broth with an OD600 value of 30 was taken, the wet cells were collected by centrifugation, and then lysed with a lysis solution. The lysate was used for catalysis.

[0039] The catalytic results of the three forms of enzyme catalysts were detected under the same reaction conditions.

[0040] The three forms of enzyme catalysts were respectively added with glycerol at a final concentration of 250 g / L, sucrose at 350 g / L, and an aqueous phase for reaction. The enzyme dosage in the three different reaction systems was the same. The catalytic results were detected by HPLC method.

[0041] HPLC detection conditions: Chromatographic column: Shodex Asahipak NH2P(4.6×250mm); Column temperature: 30 °C; Mobile phase: 65% acetonitrile; Flow rate: 0.5 ml / min; Injection volume: 10 μl; Running time: 13 minutes; Detector: RID differential detector.

[0042] GG standard (Qingdao Zhongke Lanzhi Biotechnology Development Co., Ltd., Spirulina GG, CAS: 22160-26-5) and sucrose standard (Sinopharm Group, product number 10021463) were used for qualitative and quantitative analysis. The peak emergence times of GG in the enzymatic reaction solution sample and the standard were the same. Using the GG standard, a standard curve of GG content vs. peak area was established: Y(Area) = 153.90804X(ppm) + 395.00728, R 2 = 0.99954. The peak emergence times of the substrate sucrose and the corresponding standard were the same. Sucrose standard curve: Y(Area) = 166.05635X(ppm) + 296.57099, R 2 = 0.99954. ( Figure 1 )

[0043] GG yield calculation method: GG yield = actual yield ÷ theoretical yield × 100%.

[0044] Substrate sucrose conversion rate calculation method: Sucrose conversion rate = (initial sucrose content - residual sucrose content) ÷ initial sucrose content × 100%.

[0045] The results are as Figure 2 shown, where the lysate was the catalytic reaction using the cell lysate, the wet cells were the catalytic reaction using the wet cells, and the fermentation broth was the catalytic reaction using the fermentation broth. It shows that the catalytic effects of the three forms of catalysts, namely cell lysate, fermentation broth, and wet cells, are basically the same. The final sucrose conversion rate reached 99.7%, the GG concentration in the product was 256.5 g / L, and the GG yield reached 98.7%.

[0046] Example 3: Synthesis of GG

[0047] LmSP is derived from (Leuconostoc mesenteroides, Q59495), disclosed in patent US 10,683,525 B2.

[0048] Take the fermentation broth of the recombinant bacteria, centrifuge to collect the wet cells as the whole-cell catalyst for catalysis. Detect the contents of GG and sucrose in the enzyme reaction solution by the HPLC method listed in Example 2.

[0049] 1. 100 g / L of each substrate

[0050] Reaction system: 100 g / L glycerol, 100 g / L sucrose, 4 IU / ml LmSP whole-cell catalyst. In the MES buffer system, the balance is 25 mM MES pH 6.5 buffer solution. In the absence of the MES buffer system, the balance is water. React at 30 °C for 24 h.

[0051] Results: Without the MES buffer system, after reacting for 24 h, 53.9% of the substrate sucrose was consumed, and approximately 22.5 g / L of GG was produced. With the MES buffer system, the substrate sucrose completely reacted, and the reaction solution remained homogeneous at the start of the reaction until 24 h.

[0052] 2. 110 g / L of each substrate

[0053] Reaction system: 110 g / L glycerol, 110 g / L sucrose, 4 IU / ml LmSP whole-cell catalyst. In the MES buffer system, the balance is 25 mM MES pH 6.5 buffer solution. In the absence of the MES buffer system, the balance is water. React at 30 °C for 24 h.

[0054] Results: Without the MES buffer system, after reacting for 24 h, the catalytic efficiency of the whole-cell catalyst was slightly lower than that with the MES buffer system. For the former, 88% of the substrate sucrose was consumed, producing 54 g / L of GG; for the latter, the substrate sucrose completely reacted, producing 60 g / L of GG. However, in both reaction systems, the whole-cell catalyst remained stable throughout the reaction cycle.

[0055] 3. 150 g / L of each substrate

[0056] Reaction system: 150 g / L glycerol, 150 g / L sucrose, 4 IU / ml LmSP whole-cell catalyst. In the MES buffer system, the balance is 25 mM MES pH 6.5 buffer solution. In the absence of the MES buffer system, the balance is water. React at 30 °C for 24 h.

[0057] Results: With or without the MES buffer system, there was no significant difference in the catalytic efficiency of the whole-cell catalyst. After reacting for 24 h, it was detected that the sucrose was completely consumed, producing an average of 89 g / L of GG, and the whole-cell catalyst remained stable throughout the reaction cycle.

[0058] 4. 300 g / L of each substrate

[0059] Reaction system: 300 g / L glycerol, 300 g / L sucrose, 4 IU / ml LmSP whole-cell catalyst. In the MES buffer system, the balance is 25 mM MES pH 6.5 buffer solution. In the absence of the MES buffer system, the balance is water. React at 30 °C for 24 h.

[0060] The results are as Figure 3As shown, with or without the MES buffer system, there is no significant difference in the catalytic efficiency of the whole-cell catalyst. After 24 hours of reaction, it was detected that the sucrose was completely consumed, and an average of 200 g / L of GG was produced. At a substrate concentration of 300 g / L, the whole-cell catalyst in the reaction system without the MES buffer system remained stable throughout the reaction cycle, just like in the system with the MES buffer system.

[0061] 5. 650 g / L of each substrate

[0062] Reaction system: 650 g / L of glycerol, 650 g / L of sucrose, 4 IU / ml of LmSP whole-cell catalyst. In the system with the MES buffer system, the balance is 25 mM MES buffer at pH 6.5. In the system without the MES buffer system, the balance is water. React at 30 °C for 24 hours.

[0063] Results: When reaching the saturation solubility concentration of the substrate sucrose at 30 °C, with or without the MES buffer system, there is no significant difference in the catalytic efficiency of the whole-cell catalyst. After 24 hours of reaction, it was detected that 75% of the sucrose was consumed in both cases, and an average of 326 g / L of GG was produced. At a substrate concentration of 650 g / L, the whole-cell catalyst in the reaction system without the MES buffer system remained stable throughout the reaction cycle, just like in the system with the MES buffer system.

[0064] Example 4: Optimize the substrate feeding amount and shorten the reaction cycle

[0065] LmSP is derived from (Leuconostoc mesenteroides, Q59495), which is disclosed in Patent US 10,683,525 B2.

[0066] Taking the LmSP enzyme as an example, the whole-cell catalyst is the wet bacterial cells collected by centrifuging the LmSP fermentation broth prepared in Example 1 at 4 °C, 11,500 g for 10 minutes. The fixed substrate usage amounts are 90 g of glycerol and 130 g of sucrose, and the optimization of the substrate feeding method is carried out. The enzymatic reaction conditions are 30 °C and 220 rpm. The same method as in Example 2 is used to detect GG and sucrose.

[0067] 1. Batch feeding

[0068] Reaction conditions: Fix 90 g of glycerol, 1 IU / ml of LmSP whole-cell catalyst, and add 130 g of sucrose to the reaction system in four ways: one-time feeding (130-1-Suc), two-time feeding (130-2-Suc), three-time feeding (130-3-Suc), and ten-time feeding (130-10-Suc) for reaction. The time when 90 g of glycerol, 1 IU / ml of LmSP whole-cell catalyst are added and the first feeding of sucrose is completed is recorded as reaction 0 h.

[0069] 130-1-Suc: At 0 h of reaction, 130 g of sucrose was added to the reaction system all at once.

[0070] 130-2-Suc: At 0 h and 4 h of reaction, with a feeding interval of 4 h, 130 g of sucrose was added to the reaction solution in two portions of 65 g each.

[0071] 130-3-Suc: At 0 h, 2.5 h, and 5 h of reaction, with a feeding interval of 2.5 h, 130 g of sucrose was added to the reaction solution in three portions of 44 g, 44 g, and 42 g in sequence.

[0072] 130-10-Suc: At 0 h and at a frequency of once every 2 h from 2 h to 18 h of reaction, a total of 10 times of feeding, with a fixed amount of 13 g each time, 130 g of sucrose was added to the reaction solution.

[0073] Results: When 130 g of sucrose was added all at once and the catalytic reaction was carried out for 184 h, 5.2% of sucrose remained unreacted and was not completely consumed; when added in two portions, the catalytic reaction was carried out for 112 h, and the substrate sucrose was basically completely consumed, and the reaction time was shortened by 3 days compared with adding all at once; when added in three portions, the catalytic reaction was carried out for 88 h, and the substrate sucrose was basically completely consumed, and the reaction time was shortened by 4 days compared with adding all at once; when added in ten portions, the catalytic reaction was carried out for 88 h, and the substrate sucrose was basically completely consumed, and the reaction time was the same as that of adding in three portions. For the four feeding methods, the yields of GG were 91%, 92%, 94%, and 92% respectively, with no significant difference, but the reaction cycles showed obvious differences. It shows that the use of multiple feeding methods can significantly shorten the reaction cycle.

[0074] 2. Optimize the feeding time interval in batches and increase the amount of whole-cell catalyst

[0075] Reaction conditions: Fix 90 g of glycerol, and use two concentrations of LmSP at 1 IU / ml (i.e., 4 g of LmSP whole-cell catalyst) and 2 IU / ml (i.e., 8 g of LmSP whole-cell catalyst). 130 g of sucrose was added to the reaction system in two feeding methods, and the feeding intervals were combinations of 0 h - 3 h and 0 h - 7 h respectively. The time when 90 g of glycerol, 4 g or 8 g of LmSP whole-cell catalyst was added and the first feeding of sucrose was completed was recorded as 0 h of reaction. The specific feeding methods are as follows.

[0076] 4g-3h-Suc: 4 g of whole-cell catalyst, at 0 h and 3 h of reaction, 130 g of sucrose was added to the reaction system in two portions of 65 g each.

[0077] 4g-7h-Suc: 4 g of whole-cell catalyst, at 0 h and 7 h of reaction, 130 g of sucrose was added to the reaction system in two portions of 65 g each.

[0078] 8g-3h-Suc: 8g whole-cell catalyst. At 0 h and 3 h of the reaction, 130 g of sucrose was added to the reaction system in two portions at 65 g per portion.

[0079] 8g-7h-Suc: 8g whole-cell catalyst. At 0 h and 7 h of the reaction, 130 g of sucrose was added to the reaction system in two portions at 65 g per portion.

[0080] The results are as Figure 4 shown. For the 4g LmSP whole-cell catalyst, when the feeding interval was extended from 3 h to 7 h, the reaction time was significantly shortened. Combining with the result that 130-2-Suc completely consumed sucrose in 112 h, it can be seen that as the feeding interval gradually extended, the reaction time showed a gradually shortening phenomenon, and the effect was the best when the interval was 7 h, and sucrose could be completely converted in 96 h. For the 8g LmSP whole-cell catalyst, when the feeding interval was extended from 3 h to 7 h, the reaction time was also shortened. Secondary feeding at 7 h could shorten the reaction time to 3 days, greatly improving the catalytic efficiency. The yields of GG in the above four groups of reactions were also maintained between 90-92%, with no significant difference.

[0081] The above exploration results show that: (1) Extending the interval time of multiple feedings can shorten the reaction cycle; (2) Increasing the amount of whole-cell catalyst used in the reaction system can also shorten the reaction cycle; (3) Extending the feeding interval time and increasing the amount of whole-cell catalyst can have a superimposed effect, further shortening the reaction cycle.

[0082] 3. Scale-up production of batch feeding + buffer-free catalytic system

[0083] Reaction conditions: In a 100 L reaction system, 40 kg of glycerol was fixed, the LmSP whole-cell catalyst was 7 IU / ml, and 75 kg of sucrose was added to the reaction system in a feeding mode of 25 kg per portion × 3 times, with a feeding interval of about 12 h to ensure that the final concentration of the substrate sucrose was lower than the saturation concentration upper limit of 650 g / L throughout the reaction cycle. As a result, at 30 °C and 220 rpm, the catalytic reaction was carried out for 72 h, and sucrose completely reacted to produce 523 g / L of GG, and the GG yield was 94%.

[0084] Example 5. Preparation of GG by different sucrose phosphorylases

[0085] TtSP is derived from (Thermoanaerobacterium thermosaccharolyticum (ATCC 7956), as disclosed in CN109576239 A. Reaction system: The final concentration of the TtSP whole-cell catalyst is 2 IU / ml. At 50 °C, the catalytic reaction is carried out for 24 h at five substrate concentrations of 100 g / L, 110 g / L, 150 g / L, 300 g / L, and 650 g / L respectively. One reaction system is a 50 mM trisodium citrate buffer system with a pH of 4.0. Another reaction system does not contain a buffer system and contains water. Results: At a substrate concentration of 100 g / L, 50% of the substrate sucrose is consumed in the reaction system without a buffer, and the whole-cell catalyst shows obvious precipitation and stratification. In the 50 mM trisodium citrate buffer pH 4.0 system, the substrate sucrose is completely consumed, and the whole-cell catalyst always maintains the homogeneous state at the start of the reaction; at 110 g / L, 90% of the substrate sucrose is consumed in the reaction system without a buffer, and a little precipitation occurs in the whole-cell catalyst until the end of the reaction. In the 50 mM trisodium citrate buffer pH 4.0 system, the substrate sucrose is completely consumed, and the whole-cell catalyst always maintains the homogeneous state at the start of the reaction; when the substrate concentration is 150 g / L and above, the substrate sucrose in both reaction systems is completely consumed, and the whole-cell catalyst remains stable throughout the reaction cycle.

[0086] LmSP is derived from (Leuconostoc mesenteroides, Q59495), as disclosed in patent US 10,683,525 B2. Reaction system: The final concentration of the LmSP whole-cell catalyst is 4 IU / ml. At 30 °C, the catalytic reaction is carried out for 24 h at five substrate concentrations of 100 g / L, 110 g / L, 150 g / L, 300 g / L, and 650 g / L respectively. One reaction system is a 50 mM MES buffer with a pH of 7.0. Another reaction system is a buffer-free system containing water. Results: At a substrate concentration of 100 g / L, 53.9% of the substrate sucrose is consumed in the reaction system without a buffer, and the whole-cell catalyst shows obvious precipitation and stratification. In the 50 mM MES pH 7.0 system, the substrate sucrose is completely consumed, and the whole-cell catalyst always maintains the homogeneous state at the start of the reaction; at 110 g / L, 88% of the substrate sucrose is consumed in the reaction system without a buffer, and the substrate sucrose in the 50 mM MES pH 7.0 system is completely consumed; when the substrate concentration is 150 g / L and above, the substrate sucrose in both reaction systems is completely consumed, and the whole-cell catalyst remains stable throughout the reaction cycle.

[0087] LrSP is derived from (Lactobacillus reuteri, Q1KMT7) and is disclosed in Patent CN111172127A. Reaction system: The final concentration of the LrSP whole-cell catalyst is 4 IU / ml. At 37°C, the catalytic reaction is carried out for 24 h at five substrate concentrations of 100 g / L, 110 g / L, 150 g / L, 300 g / L, and 650 g / L respectively. One reaction system is 50 mM glycine-sodium hydroxide buffer, pH 8.5. Another reaction system is a buffer-free system containing water. Results: At a substrate concentration of 100 g / L, in the buffer-free system, 47% of the substrate sucrose is consumed, and the whole-cell catalyst shows obvious precipitation stratification. In the 50 mM glycine-sodium hydroxide pH 8.5 system, the substrate sucrose is completely consumed, and the whole-cell catalyst always maintains the homogeneous state at the start of the reaction. At 110 g / L, 85% of the substrate sucrose is consumed in the buffer-free system, and the substrate sucrose is completely consumed in the 50 mM glycine-sodium hydroxide pH 8.5 system. When the substrate concentration is 150 g / L and above, the substrate sucrose in both reaction systems is completely consumed, and the whole-cell catalyst also remains stable throughout the reaction cycle.

[0088] The present invention has been described in detail above. For those skilled in the art, without departing from the gist and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any changes, uses, or improvements to the present invention, including those that depart from the scope disclosed in this application but are made by using conventional techniques known in the art. Some basic features can be applied within the scope of the following appended claims.

Claims

1. A method for producing glucosylglycerol, using glycerol and sucrose as substrates, and subjecting the substrates to a catalytic reaction with sucrose phosphorylase to obtain a product containing glucosylglycerol, characterized in that: The reaction system contains water but no buffer solution; The concentrations of the glycerol and the sucrose in the reaction system are both 150 - 650 g / L; The mass ratio of the sucrose to the glycerol is less than 2:

1.

2. The method according to claim 1, characterized in that: The sucrose phosphorylase is added to the reaction system in the form of pure sucrose phosphorylase enzyme, recombinant cells expressing sucrose phosphorylase, fermentation broth of recombinant cells expressing sucrose phosphorylase, lysate of recombinant cells expressing sucrose phosphorylase, or suspension of recombinant cells expressing sucrose phosphorylase.

3. The method according to claim 1 or 2, characterized in that: The sucrose is added to the reaction system in a batch feeding manner.

4. The method according to claim 2, wherein: The number of batch feeding times is 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.

5. The method according to claim 3, characterized in that: The interval time between two adjacent feedings is 1 - 15 hours.

6. The method according to claim 5, wherein: The interval time between two adjacent feedings is 2 - 12 hours.

7. The method according to claim 6, characterized in that: The interval time between two adjacent feedings is 7 - 12 hours.

8. The method according to claim 6, characterized in that: The interval time between two adjacent feedings is 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, or 12 hours.

9. The method according to claim 1 or 2, characterized in that: After the catalytic reaction is completed, it further includes the step of separating and purifying the reaction product to obtain glucosyl glycerol.

Images