Application of marine sucrose phosphorylase Suc75290 in synthesis of alpha-salicin and alpha-isosalicin

Abstract

The invention relates to application of marine sucrose phosphorylase Suc75290 in synthesis of alpha-salicin and alpha-isosalicin, and belongs to the field of enzymology, the application method is as follows: o-hydroxybenzyl alcohol (salicin) and sucrose are taken as substrates for simultaneous catalytic synthesis of the alpha-salicin and the alpha-isosalicin, the reaction conditions are that the sucrose concentration is (192-288) g / L, the salicin concentration is (6-18) g / L, and the pH value is (7.0-8.0); the enzyme addition amount is (150-250) U / ml, and the reaction time is (16-24) h at the temperature of (40-50) DEG C. Under the optimal condition, the yield of the alpha-salicin reaches 8.57 g / L, the yield of the alpha-isosalicin is 0.52 g / L, and the conversion rate of the salicyl alcohol reaches up to 79.17 + / -0.64%.

Description

Technical Field

[0001] The present invention belongs to the field of enzymology, and particularly relates to the application of a marine sucrose phosphorylase Suc75290 in the synthesis of α - salicin and α - isosalicin. Background Art

[0002] Sucrose phosphorylase (EC2.4.1.7, Sucrose phosphorylase, abbreviated as SPase) is a member of the glycoside hydrolase 13 family, with hydrolytic activity and transglycosylation activity. It can catalyze the reversible conversion of sucrose and phosphate into D - fructose and α - D - glucose 1 - phosphate. It can also act as a glycosyl donor to transfer a glucose group of the sucrose molecule to different receptors, and the receptors have broad specificity. Glucose, fatty alcohols, aromatic alcohols and sugar alcohols, ascorbic acid and kojic acid, and fructose can all serve as its receptors. Eventually, it can catalyze the synthesis of oligosaccharides with one more glucose group. Oligosaccharides can be applied in food additives, health products, dairy products, feed additives, etc. After glycosylation modification of hydroquinone and L - ascorbic acid, α - arbutin and L - ascorbic acid - 2 - glucoside (AA - 2G) with good skin care effects and high stability are obtained. Therefore, this enzyme has extensive applications in the fields of food, medicine, cosmetics, etc.

[0003] As a multifunctional enzyme, SPase has important application value in the synthesis of glycoside substances. Through the catalytic action of SPase, various bioactive compounds can be glycosylated modified, thereby improving their water solubility, stability and bioavailability, and at the same time reducing or eliminating their potential toxicity.

[0004] The recombinant Escherichia coli BL21(DE3) / pET24a - Suc75290 was constructed and preserved by our laboratory. The amino acid sequence number of the expressed protein Suc75290 is PP496818. The previous experimentalists comprehensively analyzed its enzymatic properties, including enzyme activity, stability, optimal reaction conditions, etc. It efficiently catalyzes the reaction of sucrose and glycerol to generate 2 - αGG with important commercial value. Through fermentation optimization, an economical and feasible medium scheme has been developed under IPTG induction, laying a foundation for subsequent industrial production. For the marine - derived sucrose phosphorylase Suc75290, the research on its glycosylation ability and potential applications is still in its infancy. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide the application of a marine sucrose phosphorylase Suc75290 in the synthesis of α - salicin and α - isosalicin.

[0006] The present invention is realized by the following technical solutions:

[0007] Application of a marine sucrose phosphorylase Suc75290 in the synthesis of α - salicin and α - isosalicin, and the application method is to simultaneously catalyze the synthesis of α - salicin and α - isosalicin using salicyl alcohol and sucrose as substrates.

[0008] Preferably, the optimal transglycosylation reaction conditions are as follows: sucrose concentration (192 - 288) g / L, salicyl alcohol concentration (6 - 18) g / L, pH value (7.0 - 8.0), enzyme dosage (150 - 250) U / ml, and reaction at (40 - 50) °C for (16 - 24) h.

[0009] Most preferably, the optimal transglycosylation reaction conditions are as follows: sucrose concentration 240 g / L, salicyl alcohol concentration 12 g / L, pH value 7.5, enzyme dosage 200 U / ml, and reaction at 45 °C for 20 h.

[0010] Advantages of the present invention compared with the prior art: The present invention provides a new use of a marine sucrose phosphorylase Suc75290 for catalytic simultaneous synthesis of α - salicin and α - isosalicin. Under the optimal conditions, the yield of α - salicin reaches 8.57 g / L, the yield of α - isosalicin is 0.52 g / L, and the conversion rate of salicyl alcohol is as high as 79.17 ± 0.64%. Description of the Drawings

[0011] Figure 1 It is the liquid chromatography analysis chart before and after the reaction; black curve: the detection result before the reaction, red short dotted line: the detection result after the reaction; (a) tyrosol; (b) salicyl alcohol; (c) vanillin; (d) ethyl vanillin; (e) p - hydroxybenzyl alcohol;

[0012] Figure 2 It is the optimal reaction temperature chart of the enzyme - catalyzed salicyl alcohol;

[0013] Figure 3 It is the optimal pH chart of the enzyme - catalyzed salicyl alcohol;

[0014] Figure 4 It is the optimal substrate concentration chart of the enzyme - catalyzed salicyl alcohol;

[0015] Figure 5 It is the optimal donor - acceptor ratio chart of the enzyme - catalyzed salicyl alcohol;

[0016] Figure 6 It is the optimal enzyme activity chart of the enzyme - catalyzed salicyl alcohol;

[0017] Figure 7 It is the optimal enzyme activity chart of the enzyme - catalyzed salicyl alcohol;

[0018] Figure 8 It is the high - performance liquid analysis chart after sample separation and purification;

[0019] Figure 9 TLC analysis chart after sample separation and purification;

[0020] Figure 10 13C and 1H nuclear magnetic resonance charts of Product I; a is the 13C analysis chart and b is the 1H analysis chart;

[0021] Figure 11 13C and 1H nuclear magnetic resonance charts of Product II; a is the 13C analysis chart and b is the 1H analysis chart. Detailed implementation manners

[0022] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and implementation schemes of the present invention.

[0023] Example 1. Screening of substrates

[0024] 1. Materials: Recombinant Escherichia coli BL21 / pET24a-Suc75290 was constructed and preserved by our laboratory. The amino acid serial number of the expressed protein marine sucrose phosphorylase Suc75290 is PP496818; the screened substrates are tyrosol, salicyl alcohol, kojic acid, menthol, p-hydroxybenzyl alcohol, vanillin, ethyl vanillin, salicylic acid, sucrose: Yuanye Biochemical Reagent Co., Ltd.; Morpholinoethanesulfonic Acid (MES): Shanghai Macklin Biochemical Co., Ltd.

[0025] 2. Instruments: Constant temperature water bath shaker: Shanghai Centrifuge Machinery Research Institute; SW-CJ ultra-clean workbench: Suzhou Antai Air Technology Co., Ltd.; HANPING electronic balance: Shanghai Jingke Co., Ltd.; GI54DS high-pressure steam sterilizer: Xiamen Zhwei Instruments Co., Ltd.; AKTA explorer liquid chromatography protein purification system: General Electric Company of the United States; IKA rotary evaporator: IKA (Guangzhou) Instrument Equipment Co., Ltd.; High performance liquid chromatography: Shimadzu Corporation of Japan. GL Science C18 (4.6mm×250mm): GL Science (Shanghai) Trading Co., Ltd.

[0026] 3. Transglycosylation reaction of Suc75290 with different substrates

[0027] To screen for substrates suitable for glycosylation reactions, using the glucosyltransferase ability of sucrose phosphorylase Suc75290, it was reacted with a variety of receptor substrates containing different functional groups (tyrosol, salicyl alcohol, kojic acid, menthol, p-hydroxybenzyl alcohol, vanillin, ethyl vanillin, salicylic acid). The reaction system was set up as follows: In 10 mL of 20 mmol / L MES-NaOH buffer at pH 7.0, 120 g / L of sucrose, 6 g / L of substrate, and 150 U / mL of sucrose phosphorylase Suc75290 were added, and the reaction system was placed in an environment at 40 °C for 24 h. A control group was set up, and the inactivated sucrose phosphorylase Suc75290 was added, with other conditions the same as the experimental group. After the reaction was completed, the reaction system was placed in a boiling water bath and heated for 5 min to inactivate the enzyme and terminate the reaction. After centrifugation, the reaction solution was filtered using a 0.22 μm filter membrane for subsequent analysis. High-performance liquid chromatography (HPLC) was used to analyze the filtered reaction solution to quantitatively detect the formation of glycosylation products. Through this experiment, the efficiency and effect of recombinant Suc75290 in catalyzing the synthesis of glycosylation products from different substrates can be evaluated.

[0028] 4. Results: A preliminary experiment was carried out with sucrose and receptor compounds as substrates under the conditions of pH 7.0 and temperature 40 °C. The results of the reaction solution are as Figure 1 , shown in Table 1. Inactivated Suc75290 was used as a control. New products were detected in the high-performance liquid chromatography diagrams of the experimental groups of tyrosol, salicyl alcohol, p-hydroxybenzyl alcohol, vanillin, and ethyl vanillin, while no new substances were detected in the HPLC diagrams of the control group. No new substances were formed in the reaction solutions of kojic acid and menthol. Among them, two products were detected in the reaction solutions of tyrosol, p-hydroxybenzyl alcohol, and salicyl alcohol, and one product was detected in the reaction solutions of vanillin and ethyl vanillin.

[0029] Table 1. Product intensity analysis

[0030]

[0031]

[0032] Example 2 Optimization of the conditions for the synthesis of α-salicyl glycoside and α-isosalicyl glycoside by marine sucrose phosphorylase Suc75290 using salicyl alcohol and sucrose as substrates

[0033] 1. Effect of temperature on the catalysis of α-salicyl glycoside and α-isosalicyl glycoside

[0034] Temperature optimization: 120g / L sucrose, 6g / L salicyl alcohol, 150U / mL sucrose phosphorylase were added to 20mmol / L pH 7.0 MES buffer, with a total reaction volume of 10mL, and reacted at 30℃, 35℃, 40℃, 45℃, and 50℃ for 24h, with a rotation speed of 200rpm / min to ensure that the substrate and enzyme in the reaction system were fully mixed. A control group was set up, in which inactivated Suc75290 was added, and other conditions were the same as those of the experimental group. After the reaction was completed, the reaction system was placed in a boiling water bath and heated for 10min to inactivate the enzyme and terminate the reaction. The reaction solution after centrifugation was filtered with a 0.22μm filter membrane and the product was analyzed by HPLC.

[0035] like Figure 2 As shown in the figure, the experimental results show that when the reaction temperature is between 40℃ and 45℃, the conversion rate of salicyl alcohol reaches the highest value, and the sum of the peak areas of the generated α-salicin and α-isosalicin products also presents the maximum value, which means that the reaction efficiency and product generation are at a relatively optimal level in this temperature range. However, when the reaction temperature rises to 50℃, the substrate conversion rate drops sharply. This is most likely due to the fact that the excessively high temperature has a significant inhibitory effect on the activity of the Suc75290 enzyme, causing its catalytic ability to be greatly reduced, and thus it is unable to effectively catalyze the conversion of substrates into products. Taking all factors into consideration, it was finally determined that the optimal temperature for the catalytic reaction was 45℃, which can provide stable and efficient basic conditions for subsequent reaction optimization.

[0036] 2. Effect of pH on the catalysis of α-salicylosidin and α-isosalicylosidin

[0037] 120g / L sucrose, 6g / L salicyl alcohol, 150U / mL sucrose phosphorylase were reacted in 20mmol / L MES buffer (5.5-8.0) with different pH values ​​at 45℃ for 24h. After the reaction was completed, the reaction system was placed in a boiling water bath and heated for 10min to inactivate the enzyme and terminate the reaction. The reaction solution after centrifugation was filtered with a 0.22μm filter membrane and the product was analyzed by HPLC according to the detection conditions of 2.2.3.

[0038] like Figure 3 As shown in the figure, experimental data show that when the pH value of the reaction system is in the range of 6.5 to 7.5, the yield of α-salicin and α-isosalicin is relatively high, indicating that the activity of the enzyme can be well maintained in this pH range, which is conducive to the catalytic reaction and thus promotes the formation of products. Further research found that when the pH value is 7.5, the yield of α-salicin and α-isosalicin generated by the reaction reaches a peak, which indicates that this pH value is the optimal pH value for Suc75290 to catalyze this reaction, providing a key parameter basis for the optimization of the subsequent reaction system.

[0039] 3. Effect of Substrate Concentration on the Catalysis of α-Salicylglucoside and α-Isosalicylglucoside

[0040] 120 g / L sucrose was respectively reacted with salicyl alcohol at different concentrations (3 - 30 g / L) and 150 U / mL sucrose phosphorylase in 2 mL (pH 7.5) of 20 mmol / L MES buffer at 45 °C for 24 h, then boiled in a water bath for 10 min. The reaction solution after centrifugation for 10 min was filtered through a 0.22 μm filter membrane and analyzed by HPLC for the products.

[0041] As Figure 4 shown in the figure, the experimental results show that with the gradual increase of the salicyl alcohol concentration, the yields of α-salicylglucoside and α-isosalicylglucoside show a gradually increasing trend, indicating that the increase in substrate concentration is beneficial to the formation of products to a certain extent. However, when the salicyl alcohol concentration increases to 12 g / L, although the product yield is still increasing, the conversion rate of the substrate remains at about 49%; when the salicyl alcohol concentration continues to increase, its conversion rate drops rapidly. This may be due to the imbalance of the ratio of substrate to enzyme in the reaction system caused by too high substrate concentration, or the phenomenon of substrate inhibition, etc., which affects the catalytic efficiency of the enzyme and the conversion effect of the substrate. In order to ensure that both the yield of glycosylated substances and the conversion rate of salicyl alcohol can be maintained at a relatively high level, considering various factors comprehensively, the concentration of salicyl alcohol in the reaction system was finally selected as 12 g / L as the optimized condition.

[0042] 4. Effect of Donor-Acceptor Ratio on the Catalysis of α-Salicylglucoside and α-Isosalicylglucoside

[0043] Salicyl alcohol (12 g / L) was reacted with sucrose at different concentrations (molar ratio of sucrose / salicyl alcohol was 4:1, 8:1, 12:1, 16:1, 20:1, 24:1, 28:1) and 150 U / mL sucrose phosphorylase in 2 mL (pH 7.5) of 20 mmol / L MES buffer at 45 °C for 24 h. After the reaction, it was boiled in a water bath for 10 min. The reaction solution after centrifugation was filtered through a 0.22 μm filter membrane and analyzed by HPLC for the products, and the analysis was carried out according to the detection conditions in 2.2.3.

[0044] As Figure 5As shown, the experimental data indicate that when the donor-acceptor ratio is less than 5:1, with the gradual increase of the donor-acceptor ratio, the yields of α-salicin and α-isosalicin show an obvious upward trend, which means that increasing the relative content of sucrose within a certain range helps to increase the production of the product. However, when the donor-acceptor ratio is greater than 20:1, continuing to increase the sucrose concentration results in a significant decrease in the yields of α-salicin and α-isosalicin. This may be because the high sucrose concentration causes a substantial increase in the viscosity of the reaction solution, hindering the contact and interaction between the substrate and the enzyme, thus being unfavorable for the smooth progress of the reaction. Considering factors such as yield and reaction efficiency comprehensively, the ratio of sucrose to salicyl alcohol of 20:1 is finally determined as the optimized donor-acceptor ratio condition to ensure the efficient and stable progress of the reaction.

[0045] 4. Effect of Enzyme Dosage on the Catalysis of α-Salicin and α-Isosalicin

[0046] Salicyl alcohol (12 g / L) and sucrose were mixed at a molar ratio of 1:20. Different experimental groups were set to add 50, 100, 150, 200, 250, and 300 U / mL of sucrose phosphorylase respectively. After reacting at 45 °C for 24 h, a 10-min boiling water bath was carried out, and the reaction solution after centrifugation for 10 min was filtered through a 0.22-μm filter membrane and analyzed by HPLC for the product.

[0047] In the catalytic synthesis reaction, adding an appropriate amount of enzyme dosage is crucial for the smooth progress of the reaction and the efficient production of the product. Insufficient enzyme dosage will lead to a slow reaction rate, unable to fully utilize the substrate for product synthesis, thus reducing the yield; while excessive enzyme dosage will cause waste of the enzyme, increase production costs, and may also trigger some unnecessary side reactions, bringing adverse effects to the purity of the product and the subsequent separation and purification work. To determine the optimal enzyme dosage, the effects of different enzyme dosages on the yields of α-salicin and α-isosalicin were systematically studied, and the results are as Figure 6 shown. Through detailed analysis and comparison of the experimental data, it was found that when the enzyme dosage was 200 U / mL, the yields of α-salicin and α-isosalicin reached the highest value, and the stability of the reaction system and the quality of the product were both good at this enzyme dosage. Therefore, 200 U / mL was determined as the optimal enzyme dosage for this catalytic synthesis reaction, providing an important reference basis for subsequent industrial production or reaction optimization at the laboratory scale.

[0048] 6. Effect of Reaction Time on the Catalysis of α-Salicin and α-Isosalicin

[0049] Salicyl alcohol (12 g / L) and sucrose (the molar ratio of sucrose to salicyl alcohol is 20:1), 200 U / mL sucrose phosphorylase were reacted at 45°C. Samples were taken every 4 h and then placed in a boiling water bath for 10 min. The reaction solution after centrifugation for 10 min was filtered through a 0.22 μm filter membrane and analyzed by HPLC for the products.

[0050] The results are as Figure 7 shown. The experimental data show that within the first 4 hours of the reaction, the yields of α-salicyl glucoside and α-isosalicyl glucoside showed a rapid increasing trend, indicating that the reaction proceeded relatively rapidly in the initial stage and the substrate could be quickly converted into products. As the reaction time continued to extend, the increasing rate of the yield gradually slowed down. This may be due to the fact that as the reaction proceeded, the substrate concentration gradually decreased and the product concentration gradually increased, resulting in a gradual decline in the reaction rate. At the same time, the influence of some side reactions or reversible reactions may have gradually emerged. When the reaction reached 20 hours, the yield reached the maximum value. At this time, the conversion rate of salicyl alcohol was close to 79%, and thereafter the yield basically remained stable without obvious increase. This indicates that within 20 hours of the reaction time, the reaction had basically reached an equilibrium state, and both the conversion of the substrate and the formation of the product had tended to be stable. Considering factors such as yield and production efficiency, 20 hours was determined as the optimal reaction time for this catalytic synthesis reaction, providing a scientific basis for the control of the reaction time in the actual production process.

[0051] In summary, the reaction conditions for the purified recombinant Suc75290 to catalyze tyrosol, salicyl alcohol, p-hydroxybenzyl alcohol, and vanillin were optimized.

[0052] Through a series of experiments, the following optimal conditions were determined: using salicyl alcohol as the substrate, the optimal pH was 7.5, the optimal temperature was 45°C, the optimal substrate concentration was 12 g / l, the optimal donor-acceptor ratio of sucrose to salicyl alcohol was 20:1, the optimal enzyme addition amount was 200 U / ml, the optimal reaction time was 20 h, and the substrate conversion rate was 79.15%.

[0053] Example 3

[0054] 1. Salicyl alcohol (12 g / L) and sucrose were mixed at a molar ratio of 1:20 according to the optimized conditions in Example 2 above, 200 U / mL sucrose phosphorylase Suc75290, and the reaction was carried out at 45°C. Samples were taken every 4 h and then placed in a boiling water bath for 10 min. The reaction solution after centrifugation for 10 min was filtered through a 0.22 μm filter membrane and analyzed by HPLC for the products.

[0055] 2. The sample was separated by a semi-preparative column and purified. After collecting the sample, it was loaded onto the column by rotary evaporation. The sample was detected by high-performance liquid chromatography (HPLC) to check its purity and analyzed by TLC to determine the purity of the purified sample. HPLC detection conditions: Shimadzu PDA detector; semi-preparative C18 column; mobile phase: water: methanol = 70:30; flow rate: 3 mL / min; column temperature: 30 °C; detection wavelength: 280 nm. The separation results are shown in Figure 8 and Figure 9 .

[0056] 3. The salicyl alcohol glycosylation product was collected by freeze-drying

[0057] The collected effluent was rotary evaporated for 30 min at a water bath temperature of 40 °C, a pressure of 50 mbar, and a rotation speed of 100 r / min. It was concentrated by rotary evaporation to about 2 mL and then freeze-dried.

[0058] 4. NMR identification and analysis of the salicyl alcohol glycosylation product

[0059] The structures of salicin analogues and α-salicin were determined by 13C and 1H nuclear magnetic resonance (Tables 2 and 3, Figure 10 、 Figure 11 ).

[0060] By 13C NMR and 1H NMR analysis, Product 1 showed significant differences from β-salicin, and its characteristics indicated an α-configuration glycoside. The chemical shift of C1 on the glucose ring decreased from 76.02 ppm to 73.06 ppm, C3 shifted from 75.51 ppm to 72.60 ppm, C4 changed from 72.88 ppm to 71.27 ppm, and C5 decreased from 100.37 ppm to 96.62 ppm. These significant shifts reflected the influence of the glycosidic bond configuration change on the chemical environment. The chemical shifts of C8, C9, C10, C11, C12, and C13 on the benzene ring were also adjusted, indicating the influence of the overall structural change on the electronic effect. In the 1H NMR spectrum, the chemical shift range of the 1-proton on the glucose ring changed to 3.72 - 3.64 ppm, shifting downfield, the 3-proton was also in this range, the 4-proton shifted to 3.94 ppm, and the 5-proton shifted significantly to 5.62 ppm. These changes were significant, and the benzene ring proton signals were different from those of β-salicin. The coupling constants (J) of the anomeric protons were 3.70 Hz and 3.65 Hz, confirming that the glycosidic bond was in the α-configuration. Through comprehensive analysis, Product 1 was determined to be α-salicin.

[0061] The 13C NMR spectrum of product II shows a signal pattern that is significantly different from that of β-salicylin and α-salicylin. C1 on the glucose ring is 73.11ppm, C3 shifts to 71.78ppm, and C5 is 97.77ppm. The obvious changes in these carbon signals suggest the presence of special substituents, molecular conformations, or chemical bond connections in the molecular structure. The chemical shifts of C8, C9, C10, C11, C12, and C13 on the benzene ring are significantly different from those of the other two, further reflecting the particularity of its structure. In the 1H NMR spectrum, proton 1 of the glucose ring is at 3.56-3.52ppm, proton 5 is at 5.03ppm, and the benzene ring proton signal is also significantly different from the other two. The coupling constant of the terminal proton also confirms that its glycosidic bond is in α-configuration. Comprehensive analysis determined that product II is α-isosalicylin.

[0062] The chemical shift of α-salicin C1 δ = 73.06 ppm (singlet) is significantly different from that of β-type (δ = 76.02 ppm, doublet), confirming the α-glycosidic bond characteristics. Glycosylation of the primary hydroxyl group causes C7 to shift to δ = 65.54 ppm, which is consistent with the anomeric effect reported in the literature (Kemmei et al., 2024).

[0063] Table 2.1H NMR analysis

[0064]

[0065] Table 3. 13 C NMR analysis

[0066]

[0067] In summary, the best reaction conditions for salicyl alcohol are pH 7.5, temperature 45°C, substrate concentration 12g / l, donor / acceptor ratio sucrose / salicylic alcohol 20:1, enzyme dosage 200U / ml, reaction time 20h, and substrate conversion rate 79.15%. High performance liquid chromatography was used to separate and analyze the glycoside products, and nuclear magnetic resonance spectroscopy identified the two main products as α-salicylic acid and α-isosalicylic acid.

Claims

1. Use of marine sucrose phosphorylase Suc75290 in the synthesis of α-salicylin and α-isosalicylin, characterized in that: The application method is to use o-hydroxybenzyl alcohol and sucrose as substrates to simultaneously catalyze the synthesis of α-salicin and α-isosalicin.

2. The use according to claim 1, characterized in that: The reaction conditions are as follows: sucrose concentration of 192-288 g / L, o-hydroxybenzyl alcohol concentration of 6-18 g / L, pH value of 7.0-8.0, enzyme addition amount of 150-250 U / ml, and reaction at 40-50° C. for 16-24 hours.

3. The use according to claim 1, characterized in that: The reaction conditions were as follows: sucrose concentration 240 g / L, o-hydroxybenzyl alcohol concentration 12 g / L, pH 7.5, enzyme addition amount 200 U / ml, and reaction at 45°C for 20 h.

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