Highly efficient sucrose phosphorylase and its use in the production of glycosylated glycerol

Abstract

The application discloses application of high-efficiency sucrose phosphorylase in production of glycerol glucoside or preparation of a glycerol glucoside product. A preparation method of the glycerol glucoside comprises generating glycerol glucoside from glycerol and sucrose by using sucrose phosphorylase for catalysis; and an amino acid sequence of the sucrose phosphorylase is shown as SEQ ID No. 1. The sucrose phosphorylase provided by the application has high catalytic activity, and a whole-cell biological catalyst can perform catalytic reaction to produce glycerol glucoside under the conditions of 30 DEG C to 50 DEG C and pH 4.5 to 9.0, the substrate conversion rate is high, the yield of GG can reach more than 400 g / L, the proportion of 2-alpha GG is high, and the sucrose phosphorylase has high industrial application potential.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of a highly efficient sucrose phosphorylase in the production of glycerol glucosides. Background Technology

[0002] Glyceryl glucosides (GG) are a class of compounds formed by the linkage of one molecule of glycerol and one molecule of glucose via a glycosidic bond. Based on their stereoconfiguration (α and β) and the position of the glycosidic bond, there are six different stereostructures. Among them, the α-configuration GG, especially 2-αGG... Figure 1 2-αGG has been extensively studied and has a wide range of applications. It exists in various plants, algae, and bacteria, and is an important active substance for combating extreme environments. As a multifunctional active compound, 2-αGG possesses strong physiological effects such as moisturizing, water-locking, anti-oxidation, and anti-aging. It is also a macromolecular stabilizer and food preservative / flavoring agent, and has significant effects in preventing tooth decay, reducing fat, and combating allergies. Therefore, 2-αGG has significant application value and broad market prospects in the cosmetics, food, health product, and pharmaceutical industries.

[0003] Currently, the main methods for producing 2-αGG include chemical synthesis, microbial synthesis, and enzyme-catalyzed synthesis. Compared to chemical synthesis and microbial extraction, enzyme synthesis has the advantages of being safe and environmentally friendly, low-cost, highly efficient, and highly specific, making it an ideal method for the industrial production of 2-αGG.

[0004] However, current methods for synthesizing 2-aGG based on enzyme catalysis also have some shortcomings. For example, α-glucosidase (EC 3.2.1.20) can catalyze the reaction of maltose and glycerol to synthesize GG, but its efficiency is low and the product configuration is complex, with 2-αGG accounting for only about 10%. Sucrose phosphorylase (EC 2.4.1.7), on the other hand, can catalyze the one-step reaction of inexpensive sucrose and glycerol to synthesize 2-αGG, with the advantage of a relatively simple product configuration, showing potential for industrial application. However, it also suffers from problems such as the still low catalytic efficiency and overall GG yield of existing sucrose phosphorylases, affecting its industrial competitiveness and limiting its industrial application to some extent. Therefore, developing efficient sucrose phosphorylases is of great significance for promoting the enzymatic industrial production of 2-αGG. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, the present invention provides a method for the rapid and specific production of 2-αGG by a highly efficient sucrose phosphorylase.

[0006] The method for preparing glycerol glucoside of the present invention includes the following steps: using sucrose phosphorylase to catalyze the reaction of glycerol and sucrose to produce glycerol glucoside.

[0007] Sucrose phosphorylase can be any of the following proteins:

[0008] A) A protein with the amino acid sequence shown in SEQ ID No. 1;

[0009] B) A protein derived from A) having a protein tag sequence or signal peptide sequence added to the end of the amino acid sequence of the protein described in A) and possessing sucrose phosphorylase activity;

[0010] C) Proteins that have 95%, 96%, 97%, 98%, or 99% or more homology with the amino acid sequence defined by A) or B) and are derived from Leuconostoc citreum;

[0011] D) Sucrose phosphorylase derived from Leuconostoc citreum.

[0012] "Homology" refers to the sequence similarity between sequences. Homology can be evaluated visually or using computer software. Using computer software, the homology between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the homology between related sequences.

[0013] A protein tag is a polypeptide or protein fused with a target protein using in vitro DNA recombination technology, which facilitates the expression, detection, tracing, and / or purification of the target protein. The protein tag can be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0014] In this preparation method, the catalytic reaction temperature can be 20℃-60℃, more preferably 28℃-52℃, even more preferably 30℃-50℃, and even more preferably 35℃-39℃.

[0015] As an example of the present invention, the temperature is 30°C, 37°C or 50°C.

[0016] In this preparation method, the pH of the catalytic reaction can be 4-10, preferably 4.5-9.0.

[0017] The inventors of this invention have discovered that the yield of GG and the substrate conversion rate can be further improved by adjusting the ratio of glycerol to sucrose. The preferred mass ratio of substrate glycerol to sucrose is ≥1:6, more preferably ≥1:2, even more preferably ≥1:1, even more preferably ≥1.5:1, or ≥2:1, or ≥3:1, or ≥4:1, or ≥6:1.

[0018] As some preferred embodiments of the present invention, in this preparation method, the mass ratio of glycerol to sucrose is 1:1-2:1, or 1:1-3:1, 1:6-6:1, 1:2-6:1, 1:1-6:1, 1.5:1-6:1, 2:1-6:1, 3:1-6:1, or 4:1-6:1. As some specific examples of the present invention, the mass ratio of substrate glycerol to sucrose is 1:6, 1:2, 1:1, 1.5:1, 2:1, 3:1, 4:1, 5:1, or 6:1.

[0019] In a preferred embodiment of the present invention, the concentrations of glycerol and sucrose in the reaction system are both greater than or equal to 100 g / L, preferably greater than or equal to 110 g / L, and more preferably greater than or equal to 150 g / L. Furthermore, this concentration is more suitable for situations where only water is added to the reaction system without a buffer solution.

[0020] In this method, the concentrations of glycerol and sucrose in the reaction system should not exceed their respective saturation concentrations.

[0021] In this method, the enzyme can participate in the reaction in any of the following forms: immobilized enzyme, non-immobilized enzyme, purified enzyme protein, microbial cells expressing the enzyme, fermentation broth of microbial cells expressing the enzyme, lysate of microbial cells expressing the enzyme, or enzyme expressed by unpurified microbial cells.

[0022] Among them, the microbial cells can be Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, yeast, and filamentous fungi such as Aspergillus niger and Aspergillus oryzae that have good expression effects.

[0023] The catalytic system of the present invention is suitable for both reaction systems with only buffer solution and reaction systems with only water but no buffer solution.

[0024] This method is particularly suitable for the preparation of 2-αGG, and mass spectrometry analysis shows that the proportion of 2-αGG in the product of this reaction is significantly improved compared with existing technologies.

[0025] To further increase GG yield and make it more suitable for large-scale production, the concentrations of glycerol and sucrose in the reaction system can be selected as greater than or equal to 300 g / L, 350 g / L, 400 g / L, 450 g / L, 500 g / L, 550 g / L, or 600 g / L.

[0026] Those skilled in the art should know that the substrates for such reactions should all be within their respective saturation concentration ranges.

[0027] As a specific example of the present invention, glycerol 450 g / L and sucrose 650 g / L are used.

[0028] In order to achieve better results, the method of feeding sugar in batches can be adopted in order to further increase GG production and make it more suitable for large-scale production.

[0029] A further preferred method is to feed the sucrose in batches every 4-12 hours. The amount of sucrose fed each time can be the same or different.

[0030] The sucrose phosphorylase provided by this invention has high catalytic activity. As a whole-cell biocatalyst, it can catalyze the production of glycerol glucoside at temperatures ranging from 30°C to 50°C and pH from 4.5 to 9.0. It exhibits high substrate conversion, with a GG yield of over 400 g / L and a high 2-αGG ratio, demonstrating significant potential for industrial application. Attached Figure Description

[0031] Figure 1 This is a schematic diagram of the 2-αGG structure.

[0032] Figure 2 Example 2: Determination of the catalytic rate of LcSP enzyme.

[0033] Figure 3 The time-course curves of LcSP whole-cell catalytic GG synthesis at different temperatures are shown.

[0034] Figure 4 The concentration-time curve for high GG production catalyzed by LcSP whole-cell catalysis.

[0035] Figure 5 Configurational analysis for the LcSP-catalyzed synthesis of GG. Detailed Implementation

[0036] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0038] pET28b(+) vector: Novagen

[0039] BL21(DE3): Transgen Biotech, product number CD601

[0040] Example 1: Cloning and expression of sucrose phosphorylase (LcSP)

[0041] Sucrose phosphorylase LcSP is derived from *Leuconostoc citreum* (strain KM20), and its amino acid sequence is shown in SEQ ID No. 1. The DNA sequence of the codon-optimized coding gene from *E. coli* is shown in SEQ ID No. 2. The optimized gene was constructed into the pET28b(+) vector via NdeI and XhoI restriction sites. Verification yielded the recombinant vector pET28b-LcSP, which contains the gene shown in SEQ ID No. 2.

[0042] The recombinant expression vector was transformed into Escherichia coli BL21(DE3), and the results were verified to yield a recombinant bacterium BL21(DE3)-pET28b-LcSP containing the encoding gene DNA (sequence shown in SEQ ID No. 2).

[0043] The recombinant bacteria were cultured to induce expression using IPTG. After induction, the supernatant was discarded by centrifugation, and the precipitate was collected as a bacterial cell slurry. This bacterial cell slurry can be used as a whole-cell biocatalyst.

[0044] Example 2: Detection of sucrose phosphorylase activity

[0045] (1) HPLC detection conditions

[0046] Column: Shodex Asahipak NH2P (4.6×250mm); Column temperature: 30℃; Mobile phase: 65% acetonitrile; Flow rate: 0.5ml / min; Injection volume: 10μl; Run time: 16 minutes; Detector: RID differential detector.

[0047] Qualitative and quantitative analyses were performed using GG standard (Qingdao Zhongke Lanzhi Biotechnology Development Co., Ltd., Spirulina GG, CAS: 22160-26-5) and sucrose standard (Sinopharm Group, catalog number 10021463). The peak retention times for GG and sucrose standards, measured by HPLC according to the above parameters, were 8.90 min and 11.79 min, respectively. A standard curve was established using the standards to correlate GG content with peak area: Y(Area) = 153.90804X(ppm) + 395.00728, R0. 2 =0.99954, Standard curve of sucrose content versus peak area: Y(Area) = 166.05635X(ppm) + 296.57099, R 2 =0.99954.

[0048] (2) Determination of enzyme activity of whole-cell biocatalysts

[0049] One enzyme activity unit (IU) is defined as the amount of enzyme required to catalyze the production of 1 μmol GG from sucrose and glycerol per minute at 37°C.

[0050] The bacterial slurry prepared in Example 1 was resuspended to a final concentration of 200 OD600 using 50 mM MES pH 6.5 buffer. This slurry was used as the whole-cell biocatalyst in this example, with 100 μL added to a 1 ml reaction system.

[0051] The final concentrations of each component in a 1 ml reaction system were as follows: 100 g / L glycerol, 100 g / L sucrose, 20 OD600 whole-cell biocatalyst, 50 mM MES, pH 6.5. The reaction was catalyzed at 37℃ and 220 rpm for 30 minutes, then terminated by heating at 85℃ for 15 minutes to obtain the enzyme reaction solution. The solution was centrifuged at 16000 g for 10 minutes to remove the precipitate. The supernatant from the centrifuged enzyme reaction solution was diluted with the HPLC mobile phase and filtered through a 0.2 μm syringe filter. HPLC analysis was performed to calculate the GG concentration, and subsequently, the whole-cell enzyme activity was calculated.

[0052] The elution times of GG and sucrose in the reaction solution were consistent with those of the standard, indicating that GG was synthesized in the enzyme reaction solution. The result showed that the enzyme activity of the whole cell at 20 OD600 was 5.5 IU.

[0053] (3) Determination of the catalytic rate of pure enzyme

[0054] The bacterial slurry obtained in Example 1 was thoroughly disrupted. The supernatant after centrifugation of the disrupted liquid was purified by Ni column affinity to obtain LcSP, which was used for enzyme activity assay. The control groups were sucrose phosphorylase LmSP from Leuconostocmesenteroides, sucrose phosphorylase BaSP-L341W mutant from Bifidobacterium adolescentis, sucrose phosphorylase LisSP from Listeria monocytogenes serotype, and sucrose phosphorylase PsSP from Pelomonas saccharophila.

[0055] The amino acid sequence information of LmSP was obtained from the uniprot database: Q59495 (November 1, 1996), and the literature J. Biotechnol. 129 (2007) 77-86.

[0056] The amino acid sequence information of BaSP-L341W comes from patent CN107858335B. The enzyme is described in the patent as BaSP / L341W, and its amino acid sequence is the amino acid sequence encoded by a DNA molecule in which the nucleotides at positions 1021-1023 of sequence 1 in the sequence listing are mutated from “CTC” to “UGG”.

[0057] The amino acid sequence information for LisSP was obtained from the uniprot database A0A0E1R3L4 (May 27, 2015), Listeria monocytogenes serotype 4b str.LL195.

[0058] The amino acid sequence information for PsSP was obtained from the uniprot database Q9XBR0 (November 1, 1999), Pelomonas saccharophila.

[0059] The enzymes from the control group were also synthesized with codon-optimized genes. The codon-optimized LmSP gene sequence is shown in SEQ ID No. 3, the codon-optimized BaSP-L341W gene sequence is shown in SEQ ID No. 4, the codon-optimized LisSP gene sequence is shown in SEQ ID No. 5, and the codon-optimized PsSP gene sequence is shown in SEQ ID No. 6. The codon-optimized gene was constructed into the pET28b(+) vector to obtain recombinant expression vectors pET28b-LmSP, pET28b-BaSP-L341W, pET28b-LisSP, and pET28b-PsSP expressing the corresponding enzymes. The subsequent transformation and expression steps were the same as in Example 1, and the corresponding bacterial slurry BL21(DE3)-pET28b-LmSP, BL21(DE3)-pET28b-BaSP-L341W, BL21(DE3)-pET28b-LisSP, and BL21(DE3)-pET28b-PsSP were obtained respectively.

[0060] The target protein was purified using a combination of ultrasonic disruption and nickel column affinity chromatography. After imidazole gradient elution, the protein was eluted with 22 mM HEPES pH 7.0, 300 mM NaCl, and 300 mM imidazole elution buffer. SDS-PAGE electrophoresis was used to detect the protein concentration, and Bradford reagent was used for protein concentration determination. The purified protein was then used for enzyme activity assays.

[0061] The target proteins obtained after SDS-PAGE electrophoresis of the bacterial cell slurry, BL21(DE3)-pET28b-LmSP bacterial cell slurry, BL21(DE3)-pET28b-BaSP-L341W bacterial cell slurry, BL21(DE3)-pET28b-LisSP bacterial cell slurry, and BL21(DE3)-pET28b-PsSP bacterial cell slurry obtained in Example 1 all contained bands of the same length as sucrose phosphorylase. SDS-PAGE electrophoresis results showed that BL21(DE3)-pET28b-LcSP, BL21(DE3)-pET28b-LmSP, BL21(DE3)-pET28b-BaSP-L341W, BL21(DE3)-pET28b-LisSP, and BL21(DE3)-pET28b-PsSP all expressed the corresponding sucrose phosphorylases. The target proteins obtained after cleavage and purification of their bacterial cell slurry were the corresponding sucrose phosphorylases LcSP, LmSP, BaSP-L341W, LisSP, and PsSP. Protein concentration analysis revealed that BL21(DE3)-pET28b-PsSP showed poor expression, with virtually no PsSP expression; the purified protein concentration was only 0.04 mg / ml, while the concentrations of the other proteins ranged from 1 to 3 mg / ml.

[0062] The purified enzyme was used for the GG catalytic reaction. The final concentrations of each component in the 5ml reaction system were as follows: 50ml MME S, pH 6.5, 100g / L glycerol, 100g / L sucrose, and 0.06mg / ml enzyme. The optimal reaction conditions for each component were as follows: LcSP, LmSP, LisSP, and PsSP were reacted at 30℃, and BaSP-L341W was reacted at 50℃, 220rpm, for 2h. The enzyme reaction was terminated by heating at 85℃ for 15 minutes and then centrifuged. The supernatant was diluted 100 times and analyzed by HPLC as in step (1). The enzyme activity was determined using the method in step (2). The catalytic rate of the enzyme was obtained by the formula: Catalytic rate = Enzyme activity / Reaction time.

[0063] The results are as follows Figure 2 The figure shows the relative enzyme catalytic rates after normalization of the catalytic rate of LmSP as the control. PsSP cannot be shown in the figure due to its low enzyme activity. The catalytic rate of LcSP for the synthesis of GG at 30℃ is 1.49 times that of LmSP, 1.51 times that of BaSP-L341W, and 4.37 times that of LisSP.

[0064] Example 3: Synthesis of GG at different temperatures

[0065] The whole cells of recombinant bacteria expressing LcSP obtained in Example 1 (i.e. the bacterial slurry prepared in Example 1) were used for the preparation of GG.

[0066] The methods for detecting the actual yield of GG and the residual sucrose content of the substrate are the same as those in Example 2.

[0067] GG yield calculation method: GG yield = actual output ÷ theoretical output × 100%.

[0068] Calculation method for substrate sucrose conversion rate: Sucrose conversion rate = (initial sucrose content - residual sucrose content) ÷ initial sucrose content × 100%.

[0069] The initial concentration (OD600) of the bacterial slurry prepared in Example 1 was 200, and the amount added to the reaction system was 2 ml.

[0070] The components and their final concentrations in the whole-cell reaction system are as follows: 50 mM MES, pH 6.5, 100 g / L glycerol, 100 g / L sucrose, and 20 OD600 whole-cell biocatalyst (containing approximately 5.5 IU / ml LcSP enzyme activity). The reaction solution was subjected to catalytic reactions at three temperatures: 30℃, 37℃, and 50℃. Samples were taken at different reaction times to terminate the reaction, and the product GG and substrate sucrose were quantitatively analyzed at these time points using HPLC. A GG synthesis-reaction time curve was plotted.

[0071] The relationship between GG synthesis and reaction time is shown in the curve. Figure 3 As shown, GG-30 represents the actual yield of GG produced by the catalytic reaction at 30℃, GG-37 represents the actual yield at 37℃, GG-50 represents the actual yield at 50℃, and Suc-30 represents the residual sucrose content at 30℃, 37℃, and 50 respectively. The results indicate that LcSP cells can catalyze the synthesis of GG within the temperature range of 30℃ to 50℃, with the highest catalytic efficiency observed at 37℃. After 48 hours of reaction, the substrate sucrose conversion reached 78.5%, producing 48.8 g / L of GG, resulting in a GG yield of 83.7%.

[0072] Example 4: Synthesis of GG at different pH values

[0073] The whole cells of the recombinant bacteria expressing sucrose phosphatase obtained in Example 1 (i.e. the bacterial slurry prepared in Example 1) were used for the preparation of 2-αGG.

[0074] The methods for calculating GG yield, detecting actual GG yield, calculating substrate sucrose conversion rate, and detecting residual sucrose content are the same as in Example 3.

[0075] The initial concentration (OD600) of the bacterial slurry prepared in Example 1 was 200, and the amount added to the reaction system was 0.365 ml.

[0076] The reaction system was as follows: 50 mM buffer solutions at different pH values, 200 g / L glycerol, 200 g / L sucrose, and a 7.3 OD600 whole-cell biocatalyst (containing approximately 2 IU / ml of LcSP enzyme activity). The specific components of the 50 mM pH buffers were: sodium citrate for pH 4.0, 4.5, and 5.0; MES for pH 5.5, 6.0, 6.5, and 7.0; and Tris-HCl for pH 7.5, 8.0, and 9.0. The reaction conditions were: 37℃, 220 rpm, and catalytic reaction for 20 h. After terminating the reaction, the product GG and substrate sucrose were quantitatively analyzed by HPLC to calculate the final GG yield and substrate sucrose conversion rate of the enzyme reaction system.

[0077] The results showed that at pH 4.0, after 20 hours of catalytic reaction, the sucrose conversion rate was only 29.5%, producing 34 g / L GG. At pH 4.5–9.0, after 20 hours of catalytic reaction, the sucrose conversion rate was 96–99.5%, producing 130–150 g / L GG. This indicates that the whole-cell biocatalyst has broad pH compatibility.

[0078] Example 5: Production of GG by whole-cell biocatalyst under different substrate glycerol:sucrose ratios

[0079] The whole cells of the recombinant bacteria expressing sucrose phosphatase obtained in Example 1 (i.e. the bacterial slurry prepared in Example 1) were used for the preparation of 2-αGG.

[0080] The methods for calculating GG yield, detecting actual GG yield, calculating substrate sucrose conversion rate, and detecting residual sucrose content are the same as in Example 3.

[0081] The initial concentration (OD600) of the bacterial slurry prepared in Example 1 was 200, and the amount added to the reaction system was 0.365 ml.

[0082] The 10ml reaction system is as follows: 50mM MES, pH 6.5, 7.3, whole-cell biocatalyst with OD600 (containing approximately 2 IU / ml LcSP enzyme activity), and substrates with different concentrations of glycerol / sucrose (glycerol:sucrose mass ratio 1:6 to 6:1, as shown in Table 1).

[0083] Table 1. Substrate concentration and mass ratio in the reaction system

[0084]

[0085]

[0086] In the table, "-" indicates that this group of experiments was not conducted.

[0087] The reaction conditions were set at 37℃, 220 rpm, and catalytic reaction for 24 h. After the reaction was terminated, the product GG and the substrate sucrose were quantitatively analyzed by HPLC, and the GG yield and substrate sucrose conversion rate of the final enzyme reaction system were calculated.

[0088] Comparing substrate conversion rates, when the glycerol:sucrose mass ratio was ≥1:2, the sucrose substrate maintained a high conversion rate, especially when the glycerol:sucrose mass ratio was ≥1:1, with sucrose conversion rates exceeding 99% after 24 hours. However, when the glycerol:sucrose mass ratio was <1:2, the substrate conversion rate decreased significantly. For example, when the glycerol:sucrose mass ratio was 1:6, the conversion rates of sucrose and glycerol after 24 hours were only 61% and 43%, respectively.

[0089] The GG yield results showed that the whole cells of LcSP could catalyze the synthesis of GG within the range of glycerol:sucrose mass ratio of 1:6 to 6:1. Moreover, the GG yield gradually increased from 36.6% to 86.8% as the glycerol:sucrose mass ratio in the reaction system gradually increased.

[0090] Example 6: Scale-up of GG production using LcSP whole-cell biocatalyst

[0091] The whole cells of the recombinant bacteria expressing sucrose phosphatase obtained in Example 1 (i.e., the bacterial slurry prepared in Example 1) were used for the preparation of GG.

[0092] The methods for calculating GG yield, detecting actual GG yield, calculating substrate sucrose conversion rate, and detecting residual sucrose content are the same as in Example 3.

[0093] (1) The 10L reaction system was as follows: 450g / L glycerol, 650g / L sucrose, and 21 OD600 whole-cell biocatalyst (containing approximately 5.8 IU / ml of LcSP enzyme activity). The sucrose was divided into three equal portions and added at 0, 12, and 24 hours of reaction, respectively. One reaction system was treated with 25mM MES at pH 6.5, while the other reaction system was treated with water but no buffer solution. The reaction was carried out at 37℃ and 220rpm for 120 hours. Samples were taken at different time points to terminate the reaction, and the actual yield of GG and the residual sucrose content at the above time points were detected by HPLC.

[0094] The results are as follows Figure 4 As shown, after 72 hours of catalytic reaction with buffer solution, the substrate sucrose conversion rate was >99%, producing 424 g / L GG, with a GG yield of 89%. After 72 hours of catalytic reaction with water, the substrate sucrose conversion rate was >99%, producing 425 g / L GG, with a GG yield of 89%.

[0095] (2) The 100L reaction system was as follows: 450g / L glycerol, 650g / L sucrose, and 21 OD600 whole-cell biocatalyst (containing approximately 5.8 IU / ml of LcSP enzyme activity). The sucrose was divided into three equal portions and added at 0, 4, and 8 hours of reaction, respectively. 25mM MES was added to one reaction system at pH 6.5, and water was added to the other reaction system without adding buffer. The reaction was carried out at 37℃ and 220rpm for 120h. Samples were taken at different time points to terminate the reaction, and the actual yield of GG and the residual sucrose content at the above time points were detected by HPLC.

[0096] As a result, after 72 hours of catalytic reaction with buffer solution, the sucrose conversion rate was >99%, producing 424 g / L GG, with a GG yield of 89%. After 72 hours of catalytic reaction with water, the sucrose conversion rate was >99%, producing 439 g / L GG, with a GG yield of 91%.

[0097] Example 7: Production of GG catalyzed by LcSP whole-cell lysis crude enzyme solution

[0098] The supernatant obtained from the whole-cell lysis of LcSP obtained in Example 2 was used for the synthesis of GG.

[0099] The methods for calculating GG yield, detecting actual GG yield, calculating substrate sucrose conversion rate, and detecting residual sucrose content are the same as in Example 3.

[0100] The 20ml reaction system consisted of: 50mM MES (pH 7.0), 100g / L glycerol, 100g / L sucrose, and 0.9mg / ml LcSP whole-cell lysis supernatant. The reaction was catalyzed at 30℃ and 220rpm for 18 hours, after which the reaction was terminated. The actual GG yield and residual sucrose content were determined by HPLC.

[0101] Results: After 18 hours of catalytic reaction, 97.8% of the sucrose was consumed, producing 25.6 g / L GG, indicating that the crude enzyme solution of whole cell lysis of LcSP can also efficiently catalyze the preparation of GG.

[0102] Example 8: Preparation of GG by LcSP purified enzyme catalysis

[0103] The purified LcSP enzyme obtained in Example 2 was used for the preparation of GG.

[0104] The methods for calculating GG yield, detecting actual GG yield, calculating substrate sucrose conversion rate, and detecting residual sucrose content are the same as in Example 3.

[0105] The reaction system was as follows: 50 mM MES, pH 6.5, 100 g / L glycerol, 100 g / L sucrose, and 0.06 mg / ml LcSP purified enzyme. The reaction was terminated after 2 h and 8 h at 30℃ and 220 rpm, respectively. The actual yield of GG and the residual sucrose content were determined by HPLC. Results: After 2 h of catalytic reaction, 14.6% of the sucrose was consumed, and the GG yield reached 66.8%. After 8 h of catalytic reaction, the substrate sucrose was completely consumed, producing 57.2 g / L GG, indicating that LcSP purified enzyme can also efficiently catalyze the preparation of GG.

[0106] The reaction system was as follows: 50 mM MES, pH 6.5, 250 g / L glycerol, 350 g / L sucrose, and 0.4 mg / ml LcSP purified enzyme. The reaction was terminated after 10 h at 30℃ and 220 rpm. HPLC was used to determine the actual yield of GG and the residual sucrose content. Results: After 10 h of catalytic reaction, the substrate sucrose was completely consumed, producing 235 g / L GG, indicating that LcSP purified enzyme can also efficiently catalyze the preparation of GG.

[0107] Example 9: Enzymatic synthesis of GG in water

[0108] (3) Reaction system: The final concentration of LcSP whole-cell catalyst was 2 IU / ml. The reaction was carried out at 30℃ for 24 h at six different substrate concentrations: 100 g / L, 110 g / L, 150 g / L, 200 g / L, 300 g / L, and 650 g / L. One reaction system was a 50 mM MES buffer solution with pH 6.5. The other reaction system was a solution without buffer solution but with water.

[0109] Results: At a substrate concentration of 100 g / L, the unbuffered system consumed 50% of the sucrose, and the whole-cell catalyst exhibited obvious precipitation and stratification. In the 50 mM MES pH 6.5 system, the sucrose was completely consumed, and the whole-cell catalyst maintained a homogeneous state at the start of the reaction. At a substrate concentration of 110 g / L, the unbuffered system consumed 75% of the sucrose, and the 50 mM MES pH 6.5 system consumed the sucrose completely. At substrate concentrations of 150 g / L and above, the conversion rate of sucrose was the same in both reaction systems, and the whole-cell catalyst remained stable throughout the entire reaction cycle.

[0110] Example 10: Identification of the GG configuration in LcSP-catalyzed synthesis

[0111] The configuration of GG samples synthesized by LcSP was analyzed according to the GC-MS method reported by Fumihito TAKENAKA et al. (Biosci. Biotechnol. Biochem., 64(2), 378-385, 2000).

[0112] (1) GC-MS detection conditions

[0113] Chromatographic column: Agilent J&W DB-5 (30m×0.25mm×0.25μm); carrier gas: 1ml / min helium; injection volume: 1μl, 200:1 split; initial column temperature 80℃, increased to 140℃ at 20℃ / min, then slowly increased to 200℃ at 2℃ / min, and after reaching 200℃, gradually increased to 300℃ at 30℃ / min and maintained at 300℃ for 10min.

[0114] (2) LcSP-catalyzed synthesis of GG

[0115] The bacterial cell slurry from Example 1 was used for the preparation of GG. The enzyme reaction system consisted of 50 mM MES at pH 6.5 (or water), 5 OD 600 whole-cell biocatalyst, 300 g / L sucrose, and 150 g / L glycerol. The reaction was carried out at 30°C for 24 h to obtain the enzyme reaction solution. After inactivation by heating at 85°C for 15 min, the enzyme was centrifuged, and the supernatant was used for HPLC peak collection of the product GG.

[0116] (3) Configurational analysis of the enzyme-catalyzed product GG

[0117] The above-mentioned GG preparation solution was derivatized with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA)-trimethylchlorosilane (TMCS) (99∶1) (Sigma, Cas: 25561-30-2) according to the method described in the literature (Biosci. Biotechnol. Biochem., 64(2), 378-385, 2000); finally, the derivatized sample was detected by GC-MS.

[0118] The results are as follows Figure 5 As shown, based on the GC-MS mass spectrometry identification results (reference: Biosci. Biotechnol. Biochem., 64(2), 378-385, 2000) and the peak area ratio based on the GC-MS chromatographic peaks, it can be seen that the GG synthesized by LcSP is mainly 2-αGG, of which the ratio of 2-αGG to 1-αGG (R configuration and S configuration) is 96.7%∶3.3%.

[0119] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims. sequence list <120> A Highly Efficient Sucrose Phosphorylase and Its Application in the Production of Glyceryl Glucosides <130> 212604 <160> 6 <170> SIPOSequenceListing 1.0 <210> 1 <211> 485 <212> PRT <213> Leuconostoc citreum <400> 1 Met Glu Ile Gln Asn Lys Ala Met Leu Ile Thr Tyr Ala Asp Ser Leu 1 5 10 15 Gly Ser Asn Ile Lys Glu Val His Gln Val Leu Lys Glu Asp Ile Gly 20 25 30 Asp Ala Ile Gly Gly Val His Leu Leu Pro Phe Phe Pro Ser Thr Gly 35 40 45 Asp Arg Gly Phe Ala Pro Ser Asp Tyr Thr Arg Val Asp Ala Thr Phe 50 55 60 Gly Asp Trp Asn Asp Val Glu Ala Leu Gly Gln Glu Tyr Tyr Leu Met 65 70 75 80 Phe Asp Phe Met Ile Asn His Ile Ser Arg Glu Ser Glu Met Tyr Gln 85 90 95 Asp Phe Lys Ala Asn His Asp Gln Ser Lys Tyr Arg Asp Phe Phe Ile 100 105 110 Arg Trp Glu Lys Phe Trp Gln Gln Ala Gly Pro Asp Arg Pro Thr Gln 115 120 125 Glu Asp Val Asp Leu Ile Tyr Lys Arg Lys Asp Lys Ala Pro Ile Gln 130 135 140 Glu Ile Thr Phe Ala Asp Gly Thr Thr Glu His Leu Trp Asn Thr Phe 145 150 155 160 Gly Glu Glu Gln Ile Asp Ile Asp Val Asn Ser Gln Ile Ala Lys Ala 165 170 175 Phe Ile Lys Ala Thr Leu Glu Asp Met Val Gln His Gly Ala Asn Leu 180 185 190 Ile Arg Leu Asp Ala Phe Ala Tyr Ala Val Lys Lys Val Gly Thr Asn 195 200 205 Asp Phe Phe Val Glu Pro Glu Ile Trp Ala Val Leu Asn Glu Val Arg 210 215 220 Asp Ile Leu Ala Pro Met His Ala Glu Ile Leu Pro Glu Ile His Glu 225 230 235 240 His Tyr Thr Ile Pro Gln Lys Ile Asn Ala His Gly Tyr Phe Thr Tyr 245 250 255 Asp Phe Ala Leu Pro Met Thr Val Leu Tyr Thr Leu Tyr Ser Gly Lys 260 265 270 Thr Asn Arg Leu Ala Asn Trp Leu Lys Gln Ser Pro Met Lys Gln Phe 275 280 285 Thr Thr Leu Asp Thr His Asp Gly Ile Gly Val Val Asp Ala Arg Asp 290 295 300 Ile Leu Thr Asp Glu Glu Ile Asp Tyr Ala Ser Glu Glu Leu Tyr Lys 305 310 315 320 Val Gly Ala Asn Val Lys Lys Thr Tyr Ser Ser Ala Ala Tyr Asn Asn 325 330 335 Leu Asp Ile Tyr Gln Ile Asn Ser Thr Tyr Tyr Ser Ala Leu Gly Asn 340 345 350 Asp Asp Ala Ala Tyr Leu Leu Ser Arg Val Phe Gln Val Phe Ala Pro 355 360 365 Gly Ile Pro Gln Ile Tyr Tyr Val Gly Leu Leu Ala Gly Glu Asn Asp 370 375 380 Ile Asp Leu Leu Glu Ser Ser Lys Glu Gly Arg Asn Ile Asn Arg His 385 390 395 400 Tyr Tyr Thr Arg Glu Glu Ile Lys Ser Ala Val Lys Arg Pro Val Val 405 410 415 Ala Asp Leu Leu Ala Leu Leu Ser Trp Arg Asn Gln Phe Ser Ala Phe 420 425 430 Ala Leu Asp Gly Thr Ile Thr Val Glu Thr Pro Ser Glu His Asp Ile 435 440 445 Lys Ile Thr Arg Thr Asp His Ser Gly Asp Asn Ile Ala Ile Leu Leu 450 455 460 Ala Asn Ala Lys Thr Arg Thr Phe Val Ile Thr Ala Asn Gly Lys Thr 465 470 475 480 Val Leu Gln Asn Lys 485 <210> 2 <211> 1458 <212> DNA <213> Artificial Sequence <400> 2 atggagatcc agaataaagc gatgctgatt acctatgcgg atagcctggg cagcaatatt 60 aaagaagtgc atcaggtgct gaaagaagac attggcgatg cgattggcgg cgtgcatctg 120 ctgccatttt ttccgagcac cggcgatcgc ggctttgcgc caagcgatta tacccgcgtt 180 gatgcgacct ttggcgattg gaatgatgtg gaagcgctgg gccaggaata ttatctgatg 240 tttgatttta tgatcaacca catcagccgc gagagcgaaa tgtatcagga ttttaaagcg 300 aatcacgacc agagcaaata ccgcgatttt ttcattcgct gggaaaagtt ttggcagcag 360 gcgggcccag atcgcccaac ccaagaagat gttgatctga tttataagcg caaagacaag 420 gcgccgattc aggaaattac ctttgcggat ggcaccaccg aacatctgtg gaataccttt 480 ggcgaagaac agattgatat tgacgtgaac agccagattg cgaaagcgtt tattaaagcg 540 accctggaag atatggtgca gcatggcgcg aatctgattc gcctggatgc gtttgcgtat 600 gcggtgaaaa aagtgggcac caatgatttt tttgtggagc cggaaatttg ggcggtgctg 660 aatgaagtgc gcgatattct ggcgccgatg catgcggaaa ttctgccgga aattcatgaa 720 cattatacca tcccgcagaa aatcaatgcg cacggctatt ttacctatga ctttgcgctg 780 ccgatgaccg tgctgtatac cctgtatagc ggcaaaacca atcgcctggc gaattggctg 840 aaacagagcc cgatgaaaca gtttaccacc ctggataccc atgatggcat tggcgtggtg 900 gatgcgcgcg atattctgac cgatgaagaa attgattatg cgagcgaaga actgtataag 960 gtgggcgcga atgtgaaaaa aacctatagc agcgcggcgt ataataatct ggatatttat 1020 cagatcaaca gcacctatta cagcgcgctg ggcaatgatg atgcggcgta tctgctgagc 1080 cgcgtgtttc aggtttttgc gccgggtatt ccgcagattt attatgtggg cctgctggcg 1140 ggcgaaaatg atattgatct gctggaaagc agcaaagagg gccgcaatat taatcgccat 1200 tattacaccc gcgaagagat taaaagcgcg gtgaaacgcc cggtggtggc ggatttattg 1260 gcgttattaa gctggcgtaa tcagtttagc gcgtttgcgc tggatggcac cattaccgtg 1320 gaaaccccga gcgaacatga tattaaaatt acccgcaccg atcacagcgg cgataatatt 1380 gcgattctgc tggcgaatgc gaaaacccgc acctttgtga ttaccgcgaa tggcaaaacc 1440 gtgctgcaga ataaataa 1458 <210> 3 <211> 1473 <212> DNA <213> Artificial Sequence <400> 3 atggagattc agaacaaagc gatgctgatt acctatgcgg atagcctggg caaaaatctg 60 aaagatgtgc atcaggtgct gaagaagat attggcgatg cgattggcgg cgtgcatctg 120 ctgccatttt ttccgagcac cggcgatcgc ggttttgcgc cggcggatta tactcgtgtt 180 gatgcggcat ttggcgattg ggcggatgtg gaagcgttag gcgaagaata ttatctgatg 240 tttgacttca tgatcaacca catcagccgc gagagcgtga tgtatcagga ttttaaaaaa 300 aaccacgacg acagcaaata caaggacttt ttcatccgct gggagaaatt ctgggcgaaa 360 gcgggcgaaa atcgcccgac ccaagcggat gttgatctga tttataaacg caaagacaag 420 gcgccgaccc aggaaattac ctttgatgat ggcaccaccg aaaatctgtg gaataccttt 480 ggcgaagaac agattgatat tgacgtgaac agcgcgattg cgaaagaatt tattaagacc 540 accctggagg atatggtgaa acacggcgcg aatctgattc gcctggatgc gtttgcgtat 600 gcggtgaaaa aagtggatac caatgatttc ttcgtggagc cggaaatttg ggataccctg 660 aatgaagtgc gcgaaattct gaccccgctg aaagcggaaa ttctgccgga aattcatgaa 720 cattatagca tcccgaaaaa gatcaacgat cacggctatt tcacctatga cttcgcgctg 780 ccgatgacca ccctgtatac cctgtatagc ggcaaaacca atcagctggc gaaatggctg 840 aaaatgagcc cgatgaaaca gtttaccacc ctggataccc atgatggcat tggcgtggtg 900 gatgcgcgcg atattctgac cgatgatgaa attgattatg cgagcgaaca gctgtataaa 960 gtgggcgcga atgtgaaaaa aacctatagc agcgcgagct ataataacct ggatatttat 1020 cagatcaaca gcacctacta tagcgcgctg ggcaatgatg atgcggcgta tctgctgagc 1080 cgcgtgtttc aggttttgc gccgggcatt ccgcagattt attatgtggg cctgctggcg 1140 ggcgaaaatg atattgcgct gctggaaagc accaaagaag gccgcaatat taatcgccat 1200 tattacaccc gcgaagaggt gaaaagcgaa gtgaaacgcc cggtggtggc gaatctgctg 1260 aaactgttga gctggcgcaa tgaaagcccg gcgtttgatc tggcgggcag cattactgtt 1320 gataccccga ccgataccac cattgtggtg acccgccaag atgaaaatgg ccagaataaa 1380 gcggtgctga ccgcggatgc ggcgaataaa acctttgaaa ttgtggaaaa tggccagacc 1440 gtgatgagca gcgataatct gacccagaat taa 1473 <210> 4 <211> 1515 <212> DNA <213> Artificial Sequence <400> 4 atgaaaaaca aggtgcagct gattacctac gcggatcgcc tgggcgatgg caccattaaa 60 agcatgaccg atattctgcg cacccgcttt gatggcgtgt atgatggcgt gcatattctg 120 ccgtttttta ccccgtttga tggcgcggat gcgggctttg atccgattga tcataccaaa 180 gtggatgagc gcctgggcag ctgggatgat gttgcggaat tgagcaaaac ccataatatt 240 atggtggacg cgattgtgaa tcacatgagc tgggaaagca aacagtttca ggatgtgctg 300 gcgaaaggcg aagaaagcga atattatccg atgtttctga ccatgagcag cgtgtttccg 360 aatggcgcga ccgaagaaga tctggcgggt atttatcgcc cgcgcccagg cttaccattt 420 acccattata aatttgcggg caagacccgc ctggtgtggg tgagctttac tccgcaacaa 480 gtggatattg ataccgatag cgataaaggc tgggaatatc tgatgagcat ttttgaccag 540 atggcggcga gccatgtgag ctatattcgc ctggatgcgg tgggctatgg cgcgaaagaa 600 gcgggcacca gctgttttat gaccccgaaa acctttaaac tgatcagccg cctgcgcgaa 660 gaaggcgtga aacgcggttt agaaattctg attgaagtgc acagctatta caaaaagcag 720 gtggaaatcg cgagcaaagt ggatcgcgtg tatgattttg cgctgccgcc gctgttactg 780 catgcgttga gcaccggtca tgttgaacca gttgcgcatt ggaccgatat tcgcccgaat 840 aatgcggtga ccgtgctgga tacccatgat ggcattggcg tgattgatat tggcagcgat 900 cagctggatc gcagcctgaa aggcctggtg ccagatgaag atgtggataa tctggtgaat 960 accattcacg cgaataccca tggcgaaagc caggcggcaa ccggtgcggc ggcgagcaat 1020 tgggatttgt atcaggtgaa tagcacctat tatagcgcgc tgggctgcaa tgatcagcat 1080 tatattgcgg cgcgcgcggt gcagtttttt ctgccgggtg ttccacaggt gtattatgtg 1140 ggcgcgctgg cgggtaaaaa tgatatggaa ctgctgcgca aaaccaataa tggccgcgat 1200 attaatcgcc attattacag caccgcggaa attgatgaaa atctgaaacg cccggtggtg 1260 aaagcgctga atgcgctggc gaaatttcgc aatgaactgg atgcgtttga tggcacctttt 1320 agctatacca ccgatgatga taccagcatt agctttacct ggcgcggcga aaccagccag 1380 gcaactttga cctttgaacc gaaacgcggc ctgggcgttg ataataccac cccggttgcg 1440 atgctggaat gggaagatag cgcgggcgat catcgcagcg atgatctgat tgcgaatccg 1500 ccggtggtgg cgtaa 1515 <210> 5 <211> 1443 <212> DNA <213> Artificial Sequence <400> 5 atgcagatca aaaacaaagc gatgctgatc acctatagcg atagcctggg caaaaacatg 60 gaagaactga gcaaagtgat ggagacctat tttgaggatg cggtgggcgg cattcatctg 120 ctgccttttt ttccgagcac cggcgatcgc ggctttgcgc ctagcgatta taccaccgtg 180 gatagcgatc tgggcagctg ggaaattatt gaaaaactgg gcgaaaaata ctacctgatg 240 tttgacttta tgatcaacca catcagccgc gaaagcctgt tttttcagga ttttaaaaag 300 gagcacctga acagcaagta taaggatatg tttatccgca tcaacgactt cttcccgccg 360 ggccgtccta atgaaaaaga tctggatctg atttataagc gcaaggacaa agcgccgttt 420 caggaagtgg aatttgcgga tggcggcacc gaactggtgt ggaatacctt tggcgaagaa 480 cagattgatc tggatgtgac cgcggaagtg accaaagaat ttattcgcca gaccattaaa 540 aacatggcgg cgcatggctg cagcattctg cgcttagatg cgtttgcgta tgcgattaaa 600 aaactggata ccaacgattt cttcgtggag ccggaaattt gggatctgct ggatgaagtg 660 aaagcggaag cggcgaaata tgatatggaa ctgctgccgg aaattcacga acattatagc 720 attcagatga aaatcgcgaa ccacgactac tacatttacg attttgcgct gccgatggtg 780 atgctgtata gcctgtatag cggccgcgtg gaacgcctgg cgaaatggtt agaaatgagc 840 ccgatgaaac agtttaccac cctggatacc catgatggca ttggcgtggt ggatgcgcgc 900 gatctgttaa ccgatgaaga actggattat accagcgcgg aactgtataa aattggcgcg 960 aacgtgaaaa aaatctacag cagcgaaaaa tacaacaacc tggacattta ccagatcaac 1020 agcacctact atagcgcgct gggcgatgat gataaaagct atctgctggc gcgcgtgatt 1080 cagtgctttg cgcctggtat tccgcagatt tattatgtgg gcctgctggc gggcaaaaac 1140 gatattgatc tgctggaaga aaccaaggag ggccgcaaca ttaaccgcca ttattatacc 1200 attgacgaga tcaagaacga ggtgaagcgc ccggtggtga aagcgttatg caatctgtta 1260 cgctttcgca acaccagcga agcgtttgat ctggaaggca gcattgaaat tgaaaccccg 1320 agcagcaacg aaattgtgat tattcgcaaa aacaagacca acaagatcac cgcgaccctg 1380 aaagcgaacc tgagcaccaa aacctttcag attagcgaaa acgaacgcaa cattctgatt 1440 tga 1443 <210> 6 <211> 1494 <212> DNA <213> Artificial Sequence <400> 6 atgaaaaatc aggtgcagct gattacctac gtggatcgcc tgggcggcca acgtgatggt 60 gcggatttag cgcgtttaaa aaaactgttg tgcgagccgg gccagccgct ggcagatgtt 120 tttggtggtg tgcatttgct gccgtttttt catgcgattg atggcgcgga tgcgggcttt 180 gatccgattg atcataccct ggtggatccg cgcctgggcg attggagtga tattaaagcg 240 ctgaccgaag gcctggaagt gatgggcgat gtgattgtga atcacatgag cagcgaaagc 300 ccgcagtttc aggattttag cgcgaaaggc cgcgaaagcg cgtatgatgg cttatttctg 360 acctggatg cggtgtttcc gaatggcgcg accgaacgcg atctgctgac tgttatcgc 420 ccgcgcccag gtccagcggc ggaattatgt gatgcggaag aacgccgtgc gcgcatttta 480 tggaccacct ttaccgcggc gcagattgat attgcggtgc atcatccgca gggccgcgca 540 tatttggaaa gtattttaca gacctttgcg gcgaatggca ttcgcatggt gcgcctggat 600 gcggtgggtt atgcgattaa aaaagcgggc gcgagctgct ttatgatgcc ggaaaccttt 660 ggctttattg cggaatttgc gcaggcggcg cgcgcgttag gcattgaagt tttagttgaa 720 attcatgcgt attaccagcg ccagattgaa attgcgagcc aggtggattg ggtgtatgat 780 tttgcgctgc cgccgctggt gctgcatgcg tttgaattta aaaccgcggg cgcgctgaaa 840 cgctggattg cggttcgcc aaccaatgcg ttaaccgtgt tagataccca tgatggcatt 900 ggcattattg atattggcgc ggatgcgagc gatcgcgcgg cgcatccagg tttagttcca 960 ccagaagaac tggatgcgtt ggtggaacgt attcatgcgg cgagccaggg ccaaagccgt 1020 aaaccaaccg gtgcggcggc aagcaatctg gatttgtatc aggtgaattg cagcttttt 1080 gacgcgatgg gccgcaatga aaccgcgtat ctgctggcgc gcgcaattca gtttttctg 1140 ccgggcgttc cgcaggtgta ttatgtgggc ttactggcgg gccataatga tatggaactg 1200 ctggcgcgca cccaggtggg tagagatatt aatcgccatt attatgacgc ggcggaaatt 1260 gcgagcgatt gcgaacgccc agttgtgcgt agattaattg aactgattcg cctgcgcaat 1320 cgccatccgg cgtttggcgg tgtatttagc gttgaagcgg gcggcgatga tgaactgcat 1380 ctgcgctggc aacagggcgg tgattggggt ccattacgcg ttaattttgc gagcctggat 1440 catgaactga gctgcagccg ccgcagacgc gatcgtgcgt ttgcatttag ctaa 1494

Claims

1. A method for preparing glycerol glucoside, characterized in that: This includes the use of sucrose phosphorylase to catalyze the reaction of glycerol and sucrose to produce glycerol glucoside; The sucrose phosphorylase is any of the following proteins: A) The amino acid sequence of the protein is shown in SEQ ID No. 1; B) The sucrose phosphorylase is a protein derived from A) by adding a protein tag sequence or signal peptide sequence to the end of the amino acid sequence of the protein described in A) and having sucrose phosphorylase activity. The glyceroglucoside is 2-αGG.

2. The preparation method according to claim 1, characterized in that: The catalytic reaction temperature is 20℃-60℃.

3. The preparation method according to claim 2, characterized in that: The catalytic reaction temperature is 28℃-52℃.

4. The preparation method according to claim 3, characterized in that: The catalytic reaction temperature is 30℃-50℃.

5. The preparation method according to claim 4, characterized in that: The catalytic reaction temperature is 35℃-39℃.

6. The preparation method according to claim 5, characterized in that: The catalytic reaction temperature is 37°C.

7. The preparation method according to any one of claims 1-6, characterized in that: The catalytic reaction is carried out at a pH of 4-10.

8. The preparation method according to claim 7, characterized in that: The catalytic reaction is carried out at a pH of 4.5-9.

0.

9. The preparation method according to any one of claims 1-6, characterized in that: In the catalytic reaction, the mass ratio of the substrate glycerol to sucrose is ≥1:

6.

10. The preparation method according to claim 9, characterized in that: In the catalytic reaction, the mass ratio of the substrate glycerol to sucrose is ≥1:

2.

11. The preparation method according to claim 10, characterized in that: In the catalytic reaction, the mass ratio of the substrate glycerol to sucrose is ≥1:

1.

12. The preparation method according to claim 11, characterized in that: In the catalytic reaction, the mass ratio of the substrate glycerol to sucrose is ≥1.5∶1.

13. The preparation method according to claim 12, characterized in that: In the catalytic reaction, the mass ratio of the substrate glycerol to sucrose is ≥2:

1.

14. The preparation method according to claim 13, characterized in that: In the catalytic reaction, the mass ratio of the substrate glycerol to sucrose is ≥3:

1.

15. The preparation method according to claim 14, characterized in that: In the catalytic reaction, the mass ratio of the substrate glycerol to sucrose is ≥4:

1.

16. The preparation method according to claim 15, characterized in that: In the catalytic reaction, the mass ratio of the substrate glycerol to sucrose is ≥6:

1.

17. The preparation method according to any one of claims 1-6, characterized in that: The concentrations of both glycerol and sucrose in the reaction system are greater than or equal to 100 g / L.

18. The preparation method according to claim 17, characterized in that: The concentrations of both glycerol and sucrose in the reaction system are greater than or equal to 110 g / L.

19. The preparation method according to claim 18, characterized in that: The concentrations of both glycerol and sucrose in the reaction system are greater than or equal to 150 g / L.

20. The preparation method according to any one of claims 1-6, characterized in that: The enzyme participates in the reaction in any of the following forms: immobilized enzyme, non-immobilized enzyme, purified enzyme protein, microbial cells expressing the enzyme, fermentation broth of microbial cells expressing the enzyme, lysate of microbial cells expressing the enzyme, or enzyme expressed by unpurified microbial cells.

21. The preparation method according to any one of claims 1-6, characterized in that: Buffer solution or water is added to the catalytic system.

22. The preparation method according to claim 19, characterized in that: The concentrations of glycerol and sucrose in the reaction system are both greater than or equal to 300 g / L.

23. The preparation method according to claim 22, characterized in that: Sugarcane is added in batches.

24. The preparation method according to claim 23, characterized in that: Feed the material every 4-12 hours.

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