A sucrose phosphorylase for efficient production of glyceroglucoside and application thereof

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 can be used for catalyzing production of glycerol glucoside, catalysis reaction can be carried out under conditions of 30 DEG C to 50 DEG C and pH 4.5 to 9.0, the enzyme activity is high, the substrate conversion rate is high, the generated GG yield can reach more than 400 g / L, and the 2-alpha GG proportion in the catalysis synthesis product is high.

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] 2-O-α-D-glyceroglucoside (2-αGG) is an important chemical substance with multiple functions and has been applied in various fields, such as: as a functional ingredient in cosmetics, providing skin moisturizing, repairing, and anti-aging effects; and as a macromolecular stabilizer and food preservative and flavoring additive, used in preventing tooth decay, inhibiting the accumulation of neutral lipids in adipocytes, and anti-allergy applications, etc. The chemical structure of 2-αGG is shown below. Figure 1 .

[0003] Currently, the main methods for producing 2-αGG include direct extraction from plants or algae, chemical synthesis, and enzyme-catalyzed synthesis. Enzyme-catalyzed synthesis is the ideal method for industrial-scale production of 2-αGG due to its minimal environmental interference, low site requirements, mild and easily controllable catalytic conditions.

[0004] However, current methods for synthesizing 2-αGG based on enzyme catalysis also have many shortcomings, such as low enzyme activity, low yield, low 2-αGG ratio, and complex subsequent purification processes, making it difficult to achieve industrial production. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, the first objective of this invention is to provide a highly efficient sucrose phosphorylase for the rapid and specific production of 2-αGG.

[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 Streptococcus mutans.

[0011] D) Sucrose phosphorylase derived from Streptococcus mutans.

[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 some specific examples 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] Even at low substrate concentrations, the enzyme of this invention can still achieve high GG yields (higher GG yields than other enzymes at the same substrate concentration), for example, when the concentration of glycerol or sucrose is in the range of 50 g / L-300 g / L, 100 g / L-300 g / L, or 200 g / L-300 g / L, which is also what this invention claims. Specifically, in the preparation method of this invention, the substrate concentration can be selected as 50 g / L-300 g / L, 100 g / L-300 g / L, or 200 g / L-300 g / L for both glycerol and sucrose, specifically 50 g / L, 100 g / L, 200 g / L, or 300 g / L.

[0031] The sucrose phosphorylase of the present invention can be used to catalyze the production of glycerol glucoside. It can carry out the catalytic reaction under conditions of 30℃~50℃ and pH 4.5~9.0. It has high enzyme activity, high substrate conversion rate, and the yield of GG can reach more than 400g / L. The catalytic synthesis product has a high proportion of 2-αGG. Attached Figure Description

[0032] Figure 1 This is a schematic diagram of the 2-αGG structure.

[0033] Figure 2 Example 2: Determination of the catalytic rate of SmSP enzyme.

[0034] Figure 3 The time-course curves of SmSP whole-cell catalytic GG synthesis at different temperatures are shown.

[0035] Figure 4 pH compatibility for whole-cell catalytic GG synthesis by SmSP.

[0036] Figure 5 Configurational analysis for the synthesis of GG catalyzed by SmSP. Detailed Implementation

[0037] 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.

[0038] 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.

[0039] pET28b(+) vector: Novagen

[0040] BL21(DE3): Transgen Biotech, product number CD601

[0041] Example 1: Cloning and expression of sucrose phosphorylase (SmSP)

[0042] SmSP was derived from *Streptococcus mutans* strain ATCC 700610 / UA159, and its amino acid sequence is shown in SEQ ID No. 1. The DNA sequence of the codon-optimized coding gene from *Escherichia 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-SmSP, which contains the gene shown in SEQ ID No. 2.

[0043] The recombinant expression vector was transformed into Escherichia coli BL21(DE3), and the results were verified to yield a recombinant bacterium BL21(DE3)-pET28b-SmSP containing the encoding gene DNA (sequence shown in SEQ ID No. 2).

[0044] 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.

[0045] Example 2: Detection of sucrose phosphorylase activity

[0046] (1) HPLC detection conditions

[0047] 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.

[0048] 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.

[0049] (2) Determination of enzyme activity of whole-cell biocatalysts

[0050] 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.

[0051] In Example 1, the bacterial slurry prepared in step (2) was resuspended in 50mM MES pH6.5 buffer to a final concentration of 200OD600 as the whole-cell biocatalyst for this example. The amount added to 1ml of the reaction system was 100ul.

[0052] 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 50℃ 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.

[0053] 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 5.6 IU.

[0054] (3) Determination of the catalytic rate of pure enzyme

[0055] The bacterial slurry obtained in step (2) of Example 1 was thoroughly disrupted. The supernatant after centrifugation of the disrupted liquid was purified by Ni column affinity to obtain SmSP, 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.

[0056] 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.

[0057] 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”.

[0058] The amino acid sequence information for LisSP was obtained from the uniprot database A0A0E1R3L4 (May 27, 2015), Listeria monocytogenes serotype 4b str.LL195.

[0059] The amino acid sequence information for PsSP was obtained from the uniprot database Q9XBR0 (November 1, 1999), Pelomonas saccharophila.

[0060] 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 using NdeI-XhoI 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.

[0061] 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.

[0062] The target protein obtained after SDS-PAGE electrophoresis of the bacterial slurry obtained in step (2) of Example 1, the bacterial slurry of BL21(DE3)-pET28b-LmSP, the bacterial slurry of BL21(DE3)-pET28b-BaSP-L341W, the bacterial slurry of BL21(DE3)-pET28b-LisSP, and the bacterial slurry of BL21(DE3)-pET28b-PsSP all contained a band of the same length as sucrose phosphorylase. SDS-PAGE electrophoresis results showed that BL21(DE3)-pET28b-SmSP, 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 SmSP, 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.

[0063] 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: SmSP reaction temperature 37℃, LmSP, LisSP, and PsSP reaction temperature 30℃, and BaSP-L341W reaction temperature 50℃, 220rpm, for 2h. After terminating the enzyme reaction by heating at 85℃ for 15 minutes, the enzyme was 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.

[0064] The results are as follows Figure 2 The figure shows the relative enzyme catalytic rate after normalization of the catalytic rate of LmSP control. PsSP cannot be shown in the figure due to its low enzyme activity. The catalytic rate of SmSP for the synthesis of GG at 37℃ is 2.01 times that of LmSP control, 2.04 times that of BaSP-L341W control, and 5.89 times that of LisSP control.

[0065] Example 3: Preparation of GG at different temperatures

[0066] The whole cells of recombinant bacteria expressing SmSP obtained in step (2) of Example 1 (i.e. the bacterial slurry prepared in Example 1) were used for the preparation of 2-αGG.

[0067] 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.

[0068] GG yield calculation method: GG yield = actual output ÷ theoretical output × 100%.

[0069] Calculation method for substrate sucrose conversion rate: Sucrose conversion rate = (initial sucrose content - residual sucrose content) ÷ initial sucrose content × 100%.

[0070] 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.

[0071] The components and their final concentrations in the 20 ml whole-cell reaction system were as follows: 50 mM MES pH 6.5, 100 g / L glycerol, 100 g / L sucrose, and whole-cell OD600 (approximately 26 g / L of the bacterial slurry harvested in Example 1, containing 5.6 IU / ml of SmSP enzyme). The above reaction solution was subjected to catalytic reactions at three temperatures: 30°C, 37°C, and 50°C. 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.

[0072] 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 SmSP cells can catalyze the synthesis of GG at temperatures ranging from 30℃ to 50℃, but the catalytic efficiency is highest at 37℃. After 48 hours of reaction, the substrate sucrose conversion rate reaches 98.3%, producing 68.5 g / L of GG, with a GG yield of 93.8%.

[0073] Example 4: Preparation of GG under different pH conditions

[0074] The whole cells of recombinant bacteria expressing sucrose phosphatase obtained in step (2) of Example 1 (i.e. the bacterial slurry prepared in Example 1) were used for the preparation of 2-αGG.

[0075] 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.

[0076] The initial concentration (OD600) of the bacterial slurry prepared in Example 1 was 200, and the amount added to the reaction system was 0.357 ml.

[0077] The reaction system was as follows: 50 mM buffer solutions at different pH values, 200 g / L glycerol, 200 g / L sucrose, and 7OD600 whole-cell biocatalyst SmSP (containing approximately 2 IU / mL of SmSP 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.

[0078] The results are as follows Figure 4 As shown, GG represents the actual yield of GG, and Suc represents the residual sucrose content. At pH 4.0, after 20 hours of catalytic reaction, the substrate sucrose conversion rate was 28%, producing 32.7 g / L GG. At pH 4.5–9.0, after 20 hours of catalytic reaction, the substrate sucrose conversion rate was 99.7–99.9%, producing 136–144 g / L GG, with a GG yield of 91.5%–96.9%. This indicates that the whole-cell biocatalyst has broad pH compatibility.

[0079] Example 5: Preparation of GG under different substrate glycerol:sucrose ratios

[0080] 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.

[0081] 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.

[0082] The initial concentration (OD600) of the bacterial slurry prepared in Example 1 was 200, and the amount added to the reaction system was 0.375 ml.

[0083] The reaction system is as follows: 50mM MES pH6.5, 7OD600 whole-cell biocatalyst (containing approximately 2 IU / ml SmSP enzyme activity), and substrates with different concentrations of glycerol / sucrose (glycerol:sucrose mass ratio 1:6 to 6:1, as shown in Table 1).

[0084] Table 1. Substrate concentration and mass ratio in the reaction system

[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 the sucrose conversion rate being >99% after 24 hours. However, when the glycerol:sucrose mass ratio was <1:2, the sucrose 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 about 54% and 36%, respectively.

[0089] The GG yield results showed that SmSP cells could catalyze the synthesis of GG within the glycerol:sucrose mass ratio range of 1:6 to 6:1. Furthermore, the GG yield gradually increased from 25% to 96% as the glycerol:sucrose mass ratio in the reaction system gradually increased. For example, when the sucrose concentration was 100 g / L, the GG yield was 61.7% with a glycerol:sucrose mass ratio of 1:2, 81% with a glycerol:sucrose mass ratio of 1:1, and 88.6% with a glycerol:sucrose mass ratio of 2:1. When the sucrose concentration was 300 g / L, the GG yield was 71.5% with a glycerol:sucrose mass ratio of 1:3, 85.3% with a glycerol:sucrose mass ratio of 2:3, and 93.4% with a glycerol:sucrose mass ratio of 1:1.

[0090] Example 6: Production of GG by Whole-Cell Biocatalyst Catalysis

[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: 25mM MES pH 6.5, 450g / L glycerol, 650g / L sucrose, and whole-cell OD600 (containing approximately 4.3 IU / ml SmSP enzyme activity). The sucrose was divided into three equal portions and fed at 0, 12, and 24 hours of reaction, respectively. After catalytic reaction at 37℃ and 220rpm for 136 hours, the actual yield of GG and the residual sucrose content were detected by HPLC.

[0094] Results: 428 g / L of GG was produced, with a GG yield of 88%.

[0095] The 10L reaction system was as follows: H2O buffer system, 450 g / L glycerol, 650 g / L sucrose, and whole-cell 15 OD600 (containing approximately 4.3 IU / ml SmSP enzyme activity). The sucrose was divided into three equal portions and added at 0, 4, and 8 hours of reaction, respectively. After catalytic reaction at 37℃ and 220 rpm for 136 hours, the actual yield of GG and the residual sucrose content were determined by HPLC.

[0096] Results: 432 g / L of GG was produced, with a GG yield of 89%.

[0097] (2) The 100L reaction system was as follows: 450g / L glycerol, 650g / L sucrose, and whole-cell 18OD600 (containing approximately 5IU / ml SmSP enzyme activity). The sucrose was divided into three equal portions and added at 0, 4, and 8 hours of reaction, respectively. 25ml MMES was added to one system to adjust the pH to 6.5, while water was added to the other system without buffer. The reaction was catalyzed at 50℃ and 220rpm. 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.

[0098] Results: After 120 h of catalytic reaction with buffer solution, the substrate sucrose was completely converted, producing 439 g / L GG, with a GG yield of 91%. After 120 h of catalytic reaction with water, the substrate sucrose was completely converted, producing 451 g / L GG, with a GG yield of 93%.

[0099] Example 7: Preparation of GG using different forms of SmSP catalysts

[0100] I. Preparation of SmSP Catalyst

[0101] (a) Take 22 ml of the fermentation broth obtained in step (2) of Example 1 (OD600 value of 30), 12000 g, centrifuge for 10 minutes and discard the supernatant. After resuspending the precipitate in 3.75 ml of 1M MES pH6.5 buffer, add water to make up to 25.75 ml. This catalyst solution is a cell catalyst.

[0102] (b) Take 22 ml of the fermentation broth obtained in step (2) of Example 1 (OD600 value is 30), add 3.75 ml of 1M MES pH6.5 buffer directly to the fermentation broth to obtain 25.75 ml of bacterial catalyst;

[0103] (c) Take 22 ml of the fermentation broth obtained in step (2) of Example 1 (OD600 value of 30), centrifuge at 12000 g for 10 minutes and discard the supernatant. Then, resuspend the precipitate in 3.75 ml of 1M MES pH6.5 buffer. Add 60 mg of lysozyme (Solarbio, CAS: 12650-88-3) to the above bacterial suspension and add water to make up to 25.75 ml. After the bacterial suspension is lysed at 30°C for 1 h, the lysate catalyst is obtained.

[0104] II. Production of GG using different enzyme catalysts

[0105] 25.75 ml of equal amounts of three forms of SmSP catalyst were added to enzyme catalysis systems with a final concentration of 250 g / L glycerol, 350 g / L sucrose, and 25 mM MES at pH 6.5 (total volume 30 ml). The catalytic reaction was carried out at 37°C and 220 rpm for 64 h. During the catalytic reaction, samples were taken at regular intervals, and the content of glycerol and sucrose in the enzyme reaction solution was determined by HPLC.

[0106] 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.

[0107] The results showed that the three forms of SmSP catalyst had basically the same catalytic effect, and the final sucrose conversion rate reached 99.7%, producing 256.5 g / L GG, with a GG yield of 98.7%.

[0108] Example 8: Preparation of GG using SmSP purified enzyme

[0109] The purified SmSP enzyme obtained in Example 2 was used for the preparation of GG.

[0110] 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.

[0111] The 20ml reaction system consisted of: 50mM MES (pH 6.5), 100g / L glycerol, 100g / L sucrose, and 0.06mg / ml SmSP purified enzyme. The reaction was catalyzed at 37℃ and 220rpm for 10 hours, after which the reaction was terminated. The actual yield of GG and the residual sucrose content were determined by HPLC.

[0112] Results: After 10 hours of catalytic reaction, the substrate sucrose was completely consumed, producing 68 g / L GG, with a GG yield of 91.6%, indicating that the SmSP pure enzyme can also efficiently catalyze the preparation of GG.

[0113] The 20ml reaction system consisted of: 50mM MES (pH 6.5), 300g / L glycerol, 300g / L sucrose, and 0.4mg / ml SmSP purified enzyme. The reaction was catalyzed at 37℃ and 220rpm for 8 hours, after which the reaction was terminated. The actual yield of GG and the residual sucrose content were determined by HPLC.

[0114] Results: After 8 hours of catalytic reaction, the substrate sucrose was completely consumed, producing 221.6 g / L GG, indicating that the SmSP pure enzyme can also efficiently catalyze the preparation of GG.

[0115] Example 9: Comparison of GG yields under low substrate concentration conditions

[0116] Whole cells of recombinant bacteria expressing various sucrose phosphatases obtained in Examples 1 and 2 were used. A 10 ml reaction system was prepared containing different whole-cell biocatalysts (i.e., control LmSP whole cells or SmSP whole cells) with 50 mM MES, pH 6.5 (or water), and an enzyme activity of approximately 2 IU / ml at 10 OD600. The reactions were carried out at 37°C and 220 rpm for 24 h under substrate glycerol and sucrose concentrations of 50 g / L, 100 g / L, 200 g / L, and 300 g / L, respectively. After terminating the reaction, the GG yield was quantitatively analyzed by HPLC. The results are shown in Table 2: Even under lower substrate concentrations, SmSP maintained a higher GG yield, significantly higher than the control enzyme LmSP.

[0117] Table 2 Comparison of GG yield under low substrate concentration conditions

[0118]

[0119] Example 10: Enzymatic synthesis of GG in water

[0120] Reaction system: A final concentration of 2 IU / ml SmSP whole-cell catalyst was used. The reaction was carried out at 37℃ for 24 h at six substrate concentrations: 100 g / L, 110 g / L, 150 g / L, 200 g / L, 300 g / L, and 650 g / L. One reaction system consisted of 50 ml MME S buffer at pH 6.5. The other reaction system consisted of water without buffer.

[0121] Results: At a substrate concentration of 100 g / L, 58% of the substrate sucrose was consumed in the unbuffered system, and the whole-cell catalyst showed obvious precipitation and stratification. In the 50 mM MES pH 6.5 system, the substrate sucrose was completely consumed, and the whole-cell catalyst maintained the homogeneous state at the start of the reaction. At a substrate concentration of 110 g / L, 83% of the substrate sucrose was consumed in the unbuffered system, and the substrate sucrose was completely consumed in the 50 mM MES pH 6.5 system. At substrate concentrations of 150 g / L and above, the conversion rate of substrate sucrose was the same in both reaction systems, and the whole-cell catalyst remained stable throughout the entire reaction cycle.

[0122] Example 11: Identification of the GG configuration in the SmSP-catalyzed synthesis

[0123] The configuration of GG samples synthesized by SmSP was analyzed according to the GC-MS method reported by Fumihito TAKENAKA et al. (Biosci. Biotechnol. Biochem., 64(2), 378-385, 2000).

[0124] (1) GC-MS detection conditions

[0125] 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.

[0126] (2) SmSP-catalyzed synthesis of GG

[0127] The bacterial cell slurry from Example 1 was used for the preparation of GG. A 20 ml volume enzyme reaction system was prepared: 50 mM MES pH 6.5 (or water), 5 OD600 whole-cell biocatalyst, 300 g / L sucrose, 150 g / L glycerol. The reaction was carried out at 37°C for 24 h to obtain the enzyme reaction solution. After inactivation by heating at 85°C for 15 min, the mixture was centrifuged, and the supernatant was used for HPLC peak collection of the product GG.

[0128] (3) Configurational analysis of the enzyme-catalyzed product GG

[0129] 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.

[0130] 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 GC-MS chromatographic peaks, it can be seen that the GG synthesized by SmSP is mainly 2-αGG, of which the ratio of 2-αGG to 1-αGG (R configuration and S configuration) is 95.7%∶4.3%, which is significantly higher than that of the patented sucrose phosphorylase BaSP. In the GG synthesized by wild-type BaSP, the ratio of 2-αGG∶1-αGG=1.5∶1 (refer to patent CN107858335A).

[0131] 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 for the production of glycerol glucosides and its application <130> 212606 <160> 6 <170> SIPOSequenceListing 1.0 <210> 1 <211> 481 <212> PRT <213> Streptococcus mutans <400> 1 Met Pro Ile Ile Asn Lys Thr Met Leu Ile Thr Tyr Ala Asp Ser Leu 1 5 10 15 Gly Lys Asn Leu Lys Glu Leu Asn Glu Asn Ile Glu Asn Tyr Phe Gly 20 25 30 Asp Ala Val Gly Gly Val His Leu Leu Pro Phe Phe Pro Ser Thr Gly 35 40 45 Asp Arg Gly Phe Ala Pro Ile Asp Tyr His Glu Val Asp Ser Ala Phe 50 55 60 Gly Asp Trp Asp Asp Val Lys Cys Leu Gly Glu Lys Tyr Tyr Leu Met 65 70 75 80 Phe Asp Phe Met Ile Asn His Ile Ser Arg Gln Ser Lys Tyr Tyr Lys 85 90 95 Asp Tyr Gln Glu Lys His Glu Ala Ser Ala Tyr Lys Asp Leu Phe Leu 100 105 110 Asn Trp Asp Lys Phe Trp Pro Lys Asn Arg Pro Thr Gln Glu Asp Val 115 120 125 Asp Leu Ile Tyr Lys Arg Lys Asp Arg Ala Pro Lys Gln Glu Ile Gln 130 135 140 Phe Ala Asp Gly Ser Val Glu His Leu Trp Asn Thr Phe Gly Glu Glu 145 150 155 160 Gln Ile Asp Leu Asp Val Thr Lys Glu Val Thr Met Asp Phe Ile Arg 165 170 175 Ser Thr Ile Glu Asn Leu Ala Ala Asn Gly Cys Asp Leu Ile Arg Leu 180 185 190 Asp Ala Phe Ala Tyr Ala Val Lys Lys Leu Asp Thr Asn Asp Phe Phe 195 200 205 Val Glu Pro Glu Ile Trp Thr Leu Leu Asp Lys Val Arg Asp Ile Ala 210 215 220 Ala Val Ser Gly Ala Glu Ile Leu Pro Glu Ile His Glu His Tyr Thr 225 230 235 240 Ile Gln Phe Lys Ile Ala Asp His Asp Tyr Tyr Val Tyr Asp Phe Ala 245 250 255 Leu Pro Met Val Thr Leu Tyr Ser Leu Tyr Ser Ser Lys Val Asp Arg 260 265 270 Leu Ala Lys Trp Leu Lys Met Ser Pro Met Lys Gln Phe Thr Thr Leu 275 280 285 Asp Thr His Asp Gly Ile Gly Val Val Asp Val Lys Asp Ile Leu Thr 290 295 300 Asp Glu Glu Ile Thr Tyr Thr Ser Asn Glu Leu Tyr Lys Val Gly Ala 305 310 315 320 Asn Val Asn Arg Lys Tyr Ser Thr Ala Glu Tyr Asn Asn Leu Asp Ile 325 330 335 Tyr Gln Ile Asn Ser Thr Tyr Tyr Ser Ala Leu Gly Asp Asp Asp Gln 340 345 350 Lys Tyr Phe Leu Ala Arg Leu Ile Gln Ala Phe Ala Pro Gly Ile Pro 355 360 365 Gln Val Tyr Tyr Val Gly Phe Leu Ala Gly Lys Asn Asp Leu Glu Leu 370 375 380 Leu Glu Ser Thr Lys Glu Gly Arg Asn Ile Asn Arg His Tyr Tyr Ser 385 390 395 400 Ser Glu Glu Ile Ala Lys Glu Val Lys Arg Pro Val Val Lys Ala Leu 405 410 415 Leu Asn Leu Phe Thr Tyr Arg Asn Gln Ser Ala Ala Phe Asp Leu Asp 420 425 430 Gly Arg Ile Glu Val Glu Thr Pro Asn Glu Ala Thr Ile Val Ile Glu 435 440 445 Arg Gln Asn Lys Asp Gly Ser His Ile Ala Lys Ala Glu Ile Asn Leu 450 455 460 Gln Asp Met Thr Tyr Arg Val Thr Glu Asn Asp Gln Thr Ile Ser Phe 465 470 475 480 Glu <210> 2 <211> 1446 <212> DNA <213> Artificial Sequence <400> 2 atgccgatta ttaacaagac catgctgatc acctatgcgg atagcctggg caaaaacctg 60 aaagaactga acgaaaacat tgagaactac tttggcgatg cggtgggcgg cgttcatctg 120 ttaccttttt ttccgagcac cggcgatcgc ggctttgcgc ctattgatta tcatgaagtg 180 gatagcgcgt ttggcgattg ggatgatgtg aaatgcctgg gcgaaaaata ttatctgatg 240 ttcgatttca tgatcaacca catcagccgc cagagcaaat attacaaaga ttatcaggag 300 aagcacgagg cgagcgcgta taaagatctg tttctgaact gggataaatt ctggccgaaa 360 aaccgcccga cccaggaaga tgtggatctg atttataaac gcaaagaccg cgcgccgaaa 420 caggaaattc agtttgcgga tggcagcgtg gaacatctgt ggaacacctt tggcgaagaa 480 cagattgatc tggatgtgac caaagaagtg accatggatt ttattcgcag caccattgaa 540 aacctggcgg cgaacggctg cgatctgatt cgcttagatg cgtttgcgta tgcggtgaaa 600 aaactggata ccaacgattt ttttgtggag ccggaaattt ggaccctgct ggataaagtg 660 cgcgatattg cggcggtgag cggcgcggaa attctgcctg aaattcatga acattatacc 720 atccagttca agatcgcgga ccatgattac tatgtgtacg attttgcgct gccgatggtg 780 accctgtata gcctgtatag cagcaaagtg gatcgcctgg cgaaatggct gaaaatgagc 840 ccgatgaaac agtttaccac cctggatacc catgatggca ttggcgtggt ggatgtgaaa 900 gatattctga ccgatgaaga aatcacctac accagcaacg aactgtataa ggtgggcgcg 960 aacgtgaacc gcaaatatag caccgcggaa tataacaacc tggatattta tcagatcaac 1020 agcacctatt acagcgcgct gggcgatgat gatcagaaat attttctggc gcgcctgatt 1080 caggcgtttg cgccgggcat tcctcaggtt tattatgtgg gctttctggc gggcaaaaac 1140 gatctggaac tgctggaaag caccaaagaa ggccgcaaca ttaaccgcca ttattatagc 1200 agcgaagaga tcgcgaaaga agtgaaacgc ccggtggtga aagcgctgct gaatctgttt 1260 acctatcgca accagagcgc ggcgtttgat ctggatggcc gcattgaagt ggaaaccccg 1320 aacgaagcga ccattgtgat tgaacgccag aacaaagatg gcagccatat tgcgaaagcg 1380 gaaattaacc tgcaggatat gacctatcgc gtgaccgaaa acgatcagac cattagcttt 1440 gaataa 1446 <210> 3 <211> 1473 <212> DNA <213> Artificial Sequence <400> 3 atggagattc agaacaaagc gatgctgatt acctatgcgg atagcctggg caaaaatctg 60 aaagatgtgc atcaggtgct gaaagaagat 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 aggtttttgc 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 tgggaatac 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 tggcaccttt 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 ttttaaaaaag 300 gagcacctga acagcaagta taggatatatg tttatccgca tcaacgactt cttcccgccg 360 ggccgtccta atgaaaaaga tctggatctg atttataagc gcaaggaacaa agcgccgttt 420 480 cagattgatc tggatgtgac cgcggaagtg accaaagaat ttatcgcca gaccattaaa 540 aacatggcgg cgcatggctg cagcattctg cgcttagatg cgtttgcgta tgcgattaaa 600 aaactggata ccaacgattt cttcgtggag ccggaaattt gggatctgct ggatgaagtg 660 720 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 accctggatg cggtgtttcc gaatggcgcg accgaacgcg atctgctgac tgtttatcgc 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 gttttttctg 1140 ccgggcgtc cgcaggtgta ttgtgggc 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 cattacgcg ttaattttgc gagcctggat 1440 catgaactga gctgcagccg ccgcagacgc gatcgtgcgt ttgcatttag ctaa 1494

Claims

1. A method for preparing glycerol glucoside, characterized in that: This includes using an enzyme to catalyze the reaction of glycerol and sucrose to produce glycerol glucoside, wherein the enzyme is a sucrose phosphorylase; 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 occurs at pH 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 may be an immobilized enzyme or a non-immobilized enzyme.

21. 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: pure enzyme protein, microbial cells expressing the enzyme, fermentation broth of microbial cells expressing the enzyme, or lysate of microbial cells expressing the enzyme.

22. The preparation method according to any one of claims 1-6, characterized in that: Add buffer solution or water to the catalytic system.

23. 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.

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

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

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