Method for catalytically synthesizing ginsenoside Rd by immobilized enzyme

Abstract

The invention discloses a method for catalytically synthesizing ginsenoside Rd by an immobilized enzyme. The method comprises the following steps: adsorbing and fixing beta-glucosidase on a carrier UiO-66-OH to prepare the immobilized enzyme; the ginsenoside Rb1, the ginsenoside Rb2 and the ginsenoside Rb3 are catalyzed by the immobilized enzyme to be converted into the ginsenoside Rd, and the immobilized enzyme shows relatively good stability and can be recycled; the CB-gluu-coated UiO-66-OH obtained by the method disclosed by the invention can be used for efficiently converting ginsenoside Rb1, Rb2 and Rb3 into ginsenoside Rd, and the immobilized enzyme still keeps 100% of activity after being repeatedly used for 8 times.

Description

Technical Field

[0001] This invention relates to a method for synthesizing ginsenoside Rd using an immobilized enzyme catalysis, belonging to the field of immobilized enzyme technology. Background Technology

[0002] Ginsenoside Rd, a dammarane-type tetracyclic triterpenoid compound, is mainly derived from the genus Panax in the family Araliaceae. Panax Ginsenoside Rd, derived from ginseng, possesses a variety of pharmacological activities, including cardiovascular protection, antitumor activity, antidiabetic activity, neuroprotection, nephroprotection, and wound healing promotion. This makes ginsenoside Rd a very promising drug candidate. However, the content of ginsenoside Rd in its natural state is very low, and its direct extraction and isolation from ginseng or other medicinal plants is both difficult and expensive, limiting its application in the pharmaceutical and health industries.

[0003] Currently, protopanaxadiol (PPD) type ginsenosides Rb1, Rb2, Rb3, and Rc can be converted into ginsenoside Rd by removing the glycosyl group outside the C20 position. Methods for glycosyl removal include acid hydrolysis, alkaline hydrolysis, enzymatic hydrolysis, and microbial transformation. Among these, enzymatic hydrolysis offers advantages in efficiency and selectivity. However, this method cannot recover the enzyme, and the free enzyme mixed in the product necessitates unavoidable purification and separation steps, increasing production costs and resulting in high costs, making it unsuitable for industrial production.

[0004] Chemical enzyme engineering (i.e., enzyme immobilization technology) confines enzymes to a support, further stabilizing and recovering them; it is a chemical method for modifying enzymes. The selection and optimization of the support is a crucial step in enzyme immobilization. Metal-organic frameworks (MOFs) are porous materials formed by metal nodes and organic ligands through coordination bonds. They possess large surface areas and pore volumes, easily adjustable pore sizes, and modifiable metal nodes and organic ligands. Different MOF structures can be designed and synthesized as needed, making MOFs a promising enzyme immobilization material.

[0005] However, the compatibility between specific MOFs and specific enzymes still needs to be determined through experiments before further selection of materials and enzymes can be made. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a method for the immobilized enzyme-catalyzed synthesis of ginsenoside Rd. Specifically, β-glucosidase BC-glu is immobilized on MOF material UiO-66-OH to obtain the immobilized enzyme CB-glu@UiO-66-OH. The immobilized enzyme CB-glu@UiO-66-OH is then used to catalyze the conversion of ginsenosides Rb1, Rb2, and Rb3 into ginsenoside Rd. The immobilized enzyme exhibits good stability and reusability.

[0007] The objective of this invention is achieved through the following technical solution: (1) Synthesis of UiO-66-OH Zirconium chloride and 2-hydroxyterephthalic acid were dissolved in a mixture of N,N-dimethylformamide (DMF) and hydrochloric acid. After stirring and mixing, the mixture was transferred to a hydrothermal synthesis reactor and reacted at 115°C. The reaction product was separated into solid and liquid phases. The solid was washed and dried. The dried product was soaked in anhydrous ethanol for 6-8 hours, with the anhydrous ethanol being replaced every 2 hours. The solid was then dried to obtain UiO-66-OH. The N,N-dimethylformamide (DMF)-hydrochloric acid mixture is prepared by mixing N,N-dimethylformamide and ammonia hydrochloride in a volume ratio of 15:1. (2) Preparation of immobilized enzyme CB-glu@UiO-66-OH UiO-66-OH and β-glucosidase CB-glu were placed in pure water, stirred at room temperature for 1-3 hours, soaked for 1-2 hours, separated from solid and liquid, and the solid was washed to obtain immobilized enzyme CB-glu@UiO-66-OH. The mass ratio of UiO-66-OH to β-glucosidase CB-glu is 10-100:1.

[0008] (3) Ginsenosides Rb1, Rb2, and Rb3 were converted into ginsenoside Rd using CB-glu@UiO-66-OH, and their conversion efficiency and reusability were detected by high performance liquid chromatography (HPLC).

[0009] The amino acid sequence of the β-glucosidase is shown in SEQ ID NO:1, and it is derived from... Caldicellulosiruptor bescii , Gene accession number: ACM59590.

[0010] This invention immobilizes the enzyme β-glucosidase CB-glu on MOF material (UiO-66-OH), constructing an immobilized enzyme CB-glu@UiO-66-OH. Compared to the free enzyme, CB-glu@UiO-66-OH exhibits varying degrees of improvement in pH stability, thermal stability, and tolerance to metal ions and organic reagents. In particular, its stability at 85℃ is significantly enhanced, and its resistance to Zn is also improved.2 + Fe 3+ Al 3+ Mn 2+ The resistance to β-ME and Tween was significantly enhanced. More notably, the enzyme activity remained unchanged after immobilization, and the enzyme could be reused repeatedly. After being reused 8 times, it was still able to convert all ginsenosides Rb1, Rb2, and Rb3 into ginsenoside Rd, achieving efficient production of ginsenoside Rd and reducing production costs. Attached Figure Description

[0011] Figure 1 XRD patterns of UiO-66-OH and CB-glu@UiO-66-OH; Figure 2 SEM images of UiO-66-OH (Figure a) and CB-glu@UiO-66-OH (Figure b); Figure 3 The results show the effect of different amounts of material added on the activity of immobilized enzymes. Figure 4 The results show the effect of different immobilization times on the activity of immobilized enzymes; Figure 5 The results show the effect of different pH values ​​on the activities of free and immobilized enzymes. Figure 6 Results showing the stability of the isolated and immobilized enzymes at different pH values; Figure 7 The results show the effect of different temperatures on the activities of free and immobilized enzymes. Figure 8 Results for the temperature stability of the free enzyme; Figure 9 Results for the temperature stability of the immobilized enzyme; Figure 10 The results show the effect of ethanol on the activities of free and immobilized enzymes. Figure 11 The results show the effect of ethanol on the stability of free and immobilized enzymes. Figure 12 The results show the effects of reagents such as metal ions and β-mercaptoethanol on the activities of free and immobilized enzymes. Figure 13 The results show the effect of trypsin on the activities of free and immobilized enzymes. Figure 14 The results show the effect of glucose on the activities of free and immobilized enzymes. Figure 15 HPLC chromatogram of immobilized enzyme converting ginsenoside Rb1 to ginsenoside Rd; Figure 16HPLC chromatogram of immobilized enzyme converting ginsenoside Rb2 to ginsenoside Rd; Figure 17 The HPLC chromatogram shows the conversion of ginsenoside Rb3 to ginsenoside Rd by immobilized enzyme. Detailed Implementation

[0012] The present invention will be further described in detail below through embodiments, but the scope of protection of the present invention is not limited to the contents described. Unless otherwise specified, the methods in the embodiments are conventional methods, and the reagents used are conventional commercial reagents or reagents prepared according to conventional methods unless otherwise specified.

[0013] In the following examples, the 4-nitrophenol (pNP) method was used to determine the activity of CB-glu@UiO-66-OH or the free enzyme. The reaction system consisted of 450 μL of 2 mM 4-nitrophenol-β-D-glucoside (pNPG) and 50 μL of enzyme solution (0.1 mg / mL CB-glu@UiO-66-OH and 0.003 mg / mL free enzyme). The reaction was terminated with 2 mL of 1 M Na2CO3 after 10 min. Each reaction was performed in triplicate. Under specific conditions, enzyme activity was defined as the amount of enzyme required to degrade pNPG to generate 1 μmol of pNP per minute. Enzyme loading, immobilization yield, and recovery activity were calculated using the following formulas.

[0014] In the formula: c total The initial total enzyme concentration, c supernatant It refers to the enzyme concentration in the supernatant after immobilization; V This is the volume of the immobilized reaction system (2 mL). m matrace This represents the mass of UiO-66-OH; Activity supernatant and Activity composite These are the activities of the free enzyme and the immobilized enzyme in the supernatant, respectively. Activity total The initial total enzyme activity; Recovery activity refers to the enzyme activity after immobilization; Example 1: Synthesis of UiO-66-OH 186 mg of zirconium chloride and 290 mg of 2-hydroxyterephthalic acid were dissolved in 30 mL of N,N-dimethylformamide (DMF) and 2 mL of hydrochloric acid, and the mixture was magnetically stirred for 30 min. The mixture was then transferred to a 100 mL hydrothermal synthesis reactor and reacted in an oven at 115 °C for 24 h. After naturally cooling to room temperature, the solution was separated by centrifugation at 5000 rpm for 5 min, and the precipitate was retained. The precipitate was washed three times each with DMF and anhydrous ethanol, and centrifuged at 5000 rpm for 5 min each time. The solution was separated, and the precipitate was retained. The precipitate was dried in an oven at 60 °C for 12 h to obtain a white powder. The white powder was then soaked in anhydrous ethanol for 6 h, with the ethanol being replaced every 2 h. After soaking, the solution was separated by centrifugation at 5000 rpm for 5 min, and the precipitate was retained. The precipitate was dried in an oven at 60 °C for 12 h to obtain UiO-66-OH. The XRD pattern of UiO-66-OH is shown in [Figure number missing]. Figure 1 SEM image (see) Figure 2 -a, as can be seen from the figure, the material has been successfully synthesized.

[0015] Example 2: Construction of immobilized enzyme CB-glu@UiO-66-OH 1. Effect of different amounts of carrier added on the activity of immobilized enzymes Suspend 3, 5, 8, 10, 15, 20, 25, and 30 mg of UiO-66-OH in 1 mL of pure water, respectively, and then add 1 mL of β-glucosidase CB-glu (0.30 mg / mL). After stirring magnetically at room temperature for 2 h, let stand at 4°C for 1 h, and then centrifuge at 5000 rpm for 4 min to collect the precipitate. Wash the precipitate three times with pure water and centrifuge to obtain CB-glu@UiO-66-OH. See results Figure 3 As shown in the figure, when the amount of UiO-66-OH added is less than 10 mg, the enzyme loading rate is higher, but the recovery activity and immobilization rate decrease. When the amount of UiO-66-OH added is greater than 10 mg, the enzyme loading rate decreases, the immobilization rate remains basically unchanged, and the recovery activity decreases, but still remains above 80%. Among them, when the amount of carrier added is 10 mg, the recovery activity is 100%. The XRD pattern of the immobilized enzyme CB-glu@UiO-66-OH prepared with 10 mg of material is shown in the figure. Figure 1 SEM image (see) Figure 2 -b. As can be seen from the figure, the material retains its original structure before and after immobilization, indicating that UiO-66-OH can be used for the immobilization of CB-glu.

[0016] 2. Effects of different immobilization times on the activity of immobilized enzymes 10 mg UiO-66-OH was suspended in 1 mL of pure water, and then 1 mL of β-glucosidase CB-glu (0.30 mg / mL) was added. The mixture was then magnetically stirred at room temperature for 1, 2, 3, 4 and 5 h, respectively. After standing at 4 °C for 1 h, the precipitate was collected by centrifugation at 5000 rpm for 4 min. The precipitate was washed three times with pure water and centrifuged to obtain CB-glu@UiO-66-OH. See results Figure 4 As shown in the figure, the enzyme loading and recovery activity reached a high level within the immobilization time (1 h), indicating a strong interaction and adaptability between the enzyme and the material. Increasing the immobilization time did not affect the enzyme loading and recovery activity. Besides the strong interaction and adaptability between the enzyme and the material, the low enzyme-to-material mass ratio (0.3:10, providing ample adhesion surface for enzyme adsorption) shortened the immobilization time. The stability of CB-glu@UiO-66-OH ensured the enzyme's stability during long-term immobilization. Considering operational stability and reusability, a final immobilization time of 2 h was adopted for preparing CB-glu@UiO-66-OH.

[0017] Example 3: Determination of Enzymatic Properties In this embodiment, the reaction system consisted of 450 μL of 2 mM 4-nitrophenol-β-D-glucoside (pNPG) and 50 μL of enzyme solution (CB-glu@UiO-66-OH 0.1 mg / mL, free enzyme 0.003 mg / mL). The reaction was terminated with 2 mL of 1 M Na2CO3 after 10 min. 1. The optimal pH of immobilized enzyme CB-glu@UiO-66-OH or free enzyme (BC-glu) was determined at 75℃ and pH 4.5-7.0. Figure 5 The enzyme solution is prepared by mixing CB-glu@UiO-66-OH or free enzyme with pH 4.5-7.0 (citrate-disodium hydrogen phosphate buffer); After treating the reaction system at room temperature for 2.5 hours, the remaining enzyme activity in the solution was measured to determine the enzyme's pH stability. Figure 6 ).

[0018] See results Figure 5 , 6 As can be seen from the figure, the optimal pH for the immobilized enzyme CB-glu@UiO-66-OH is 5.5. The immobilized enzyme CB-glu@UiO-66-OH can still maintain the excellent performance of the free enzyme CB-glu. Compared with the free enzyme CB-glu, the immobilized enzyme shows higher stability at pH 4.5-5.5.

[0019] 2. The optimal temperature of CB-glu@UiO-66-OH or free enzyme (BC-glu) was determined under different temperatures (60 ℃-90 ℃) and optimal pH 5.5. The residual activity of the enzyme was measured after treatment at 75 ℃, 80 ℃, 85 ℃, and 90 ℃ for 30 min, 60 min, 90 min, and 120 min, respectively, to analyze the temperature stability of the enzyme. The reaction system was the same as above. See results Figure 7 , 8 Figure 9 shows that the optimal temperature range for CB-glu@UiO-66-OH is 75-85℃. The activity decreased slightly after incubation at 75℃ and 80℃ for 30 minutes, but remained essentially unchanged with prolonged incubation. This indicates that the structure of CB-glu@UiO-66-OH undergoes slight changes at 75℃ and 80℃. Notably, the stability of CB-glu@UiO-66-OH at 85℃ is significantly improved compared to the free enzyme CB-glu. While CB-glu essentially loses its activity after 30 minutes of incubation at 85℃, CB-glu@UiO-66-OH retains approximately 20% of its activity after 120 minutes of incubation at 85℃. Figure 8 , 9 ).

[0020] 3. Effects of ethanol on the activity of free and immobilized enzymes 3.1 The reaction system in this embodiment is the same as above, except that ethanol is added to a final concentration of 3%-30% (v / v), the ethanol resistance of CB-glu@UiO-66-OH or free enzyme CB-glu is determined, the catalytic reaction is carried out at the optimal pH 5.5 and 75℃, and the reaction system without ethanol is used as a control. 3.2 To investigate the enzyme’s tolerance to ethanol, the enzyme was first treated in a 3%-30% ethanol solution for 1 hour, and then placed in the above reaction system to carry out the catalytic reaction at the optimal pH (5.5) and optimal temperature (75℃). See results Figure 10 , 11 The experimental results showed that the immobilized enzyme CB-glu@UiO-66-OH and the free enzyme CB-glu exhibited similar performance in terms of ethanol resistance and ethanol stability, with no significant improvement or decrease in performance.

[0021] 4. Add metal ions to the reaction system at a final concentration of 5 mM, 5 mM β-mercaptoethanol (β-ME), Triton at a final concentration of 2% (v / v), and Tween at a final concentration of 2%, respectively, and determine their effects on the enzyme. See results Figure 12Among the effects of 15 metal ions and organic reagents (β-ME, Triton, Tween) on the enzyme, CB-glu@UiO-66-OH showed superior performance improvement compared to the free enzyme CB-glu. The free enzyme CB-glu exhibited better performance on Ag... 1+ Ni 2+ Ba 2 + Triton resistance is superior to CB-glu@UiO-66-OH. Furthermore, CB-glu@UiO-66-OH exhibits superior resistance to 14 other metal ions or organic reagents compared to the free enzyme CB-glu, especially to Zn. 2+ Fe 3+ Al 3+ Mn 2+ Resistance to β-ME and Tween was significantly enhanced. Interestingly, the free enzyme CB-glu showed improved resistance in 5 mM Al. 3+ The activity of the immobilized enzyme CB-glu@UiO-66-OH was very low (below 10%) under ionic conditions, while the activity of the immobilized enzyme CB-glu@UiO-66-OH was not only not lost under these conditions, but even slightly increased.

[0022] 5. Determine the effects of trypsin and glucose on this enzyme. CB-glu@UiO-66-OH or free enzyme CB-glu was added to a solution containing 0.1 mg / mL trypsin (pH 7.0), incubated at 37 °C for 1 h, and then the enzyme was placed in the above reaction system. The catalytic reaction was carried out at the optimal pH (5.5) and optimal temperature (75 °C), and the residual activity was detected. Prepare a reaction system containing 250-3000 mM glucose and determine the glucose resistance of CB-glu@UiO-66-OH or the free enzyme.

[0023] See results Figure 13 and Figure 14 The results showed that the immobilized enzyme CB-glu@UiO-66-OH and the free enzyme CB-glu exhibited similar performance in terms of glucose resistance and trypsin resistance, with no significant improvement or decrease in performance.

[0024] Example 4: CB-glu@UiO-66-OH catalytic conversion of ginsenosides 0.5 mL of ginsenosides Rb1, Rb2, and Rb3 (2 mg / mL) were added to 2 mL of citrate-disodium hydrogen phosphate buffer (pH 5.5), and then 10 mg of CB-glu@UiO-66-OH was added to the above solution. The mixture was magnetically stirred at 75 °C for 2 h, and the immobilized enzyme and solution were separated by centrifugation. The separated immobilized enzyme was reused. 1 mL of n-butanol was added to the separation solution to extract the reaction product. After drying the organic phase, the reaction product was obtained. The reaction product was dissolved in 1 mL of methanol, and the sample was prepared for HPLC. The control group was inactivated by high temperature with CB-glu@UiO-66-OH, and all other conditions were kept the same.

[0025] The HPLC detection conditions were as follows: The column was a Waters-XTerra-MS-C18 (5 μm, 250 mm × 4.6 mm, USA); the mobile phase was water and acetonitrile; gradient elution: 0–20 min, 100–25% acetonitrile; 20–40 min, 25–45% acetonitrile; 40–70 min, 45%–65% acetonitrile; 70–90 min, 65%–75% acetonitrile; 90–95 min, 75–100% acetonitrile. The flow rate was 1.0 mL / min, the column temperature was 30 ℃, and the detection wavelength was set to 203 nm. The HPLC results are shown in the figure. Figure 15-17 .

[0026] The results showed that there were no substrate residues in the ginsenosides Rb1, Rb2, and Rb3 during the reaction, and they were all completely converted into ginsenoside Rd, with the concentration of the product ginsenoside Rd reaching 1 mg / mL.

[0027] CB-glu@UiO-66-OH retained all its activity after being reused 8 times, and was still able to completely convert all substrates into ginsenoside Rd.

[0028] Comparative Example 1: Preparation of different MOF materials and preparation of immobilized enzymes 1. Preparation of MOF material UiO-66-NH2 746 mg of zirconium chloride and 573 mg of 2-aminoterephthalic acid were dissolved in 50 mL of N,N-dimethylformamide (DMF) and magnetically stirred for 30 min. The mixture was then transferred to a 100 mL hydrothermal synthesis reactor and reacted in an oven at 120 °C for 24 h. After naturally cooling to room temperature, the solution was separated by centrifugation at 5000 rpm for 5 min, and the precipitate was retained. The precipitate was washed three times with anhydrous ethanol, centrifuged at 5000 rpm for 5 min, and the solution was separated by centrifugation, and the precipitate was retained. The precipitate was dried in an oven at 60 °C for 12 h to obtain UiO-66-NH2.

[0029] 2. Construction of immobilized enzyme CB-glu@UiO-66-NH2 Same as step 1 in Example 2, using 10 mg UiO-66-NH2 as the carrier; 3. Enzyme activity assay of CB-glu@UiO-66-NH2 Enzyme activity was determined using the 4-nitrophenol (pNP) method described above. Experimental results showed that the material UiO-66-NH2 was poorly matched with the enzyme CB-glu. Its optimal enzyme loading, immobilization yield, and recovery acactivity were 13 mg / g, 63%, and 20%, respectively, far lower than the immobilization effect of UiO-66-OH. The recovery activity of CB-glu@UiO-66-NH2 was only 20%, indicating significant enzyme activity loss. It could not maintain the catalytic activity of the free enzyme CB-glu after immobilization. Therefore, UiO-66-NH2 is not suitable for immobilizing CB-glu.

[0030] This also indicates that the compatibility between specific MOFs and specific enzymes still needs to be determined through experiments. Among zirconium-based MOF materials, UiO-66-OH is more suitable for the immobilization of β-glucosidase CB-glu than UiO-66-NH2.

Claims

1. A method for synthesizing ginsenoside Rd using immobilized enzyme catalysis, characterized in that: Immobilized enzymes were prepared by adsorbing and immobilizing β-glucosidase on the carrier UiO-66-OH. The immobilized enzymes were used to catalyze the conversion of ginsenosides Rb1, Rb2, and Rb3 into ginsenoside Rd. The immobilized enzymes showed good stability and could be repeatedly recycled.

2. The method for synthesizing ginsenoside Rd by immobilized enzyme catalysis according to claim 1, characterized in that: The amino acid sequence of β-glucosidase is shown in SEQ ID NO:

1.

3. The method for synthesizing ginsenoside Rd by immobilized enzyme catalysis according to claim 1, characterized in that: The mass ratio of UiO-66-OH to β-glucosidase in the adsorption fixation was 10-100:1.

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