Application and method of a glycoside hydrolase with dual enzyme activity and its recombinant strain in the preparation of rare ginsenosides

Abstract

This invention relates to the field of catalysis. It provides an application and method for a glycoside hydrolase with dual enzyme activity and its recombinant strain in the preparation of rare ginsenosides. The amino acid sequence of the glycoside hydrolase is shown in SEQ ID NO.1. Using the glycoside hydrolase of this invention or the recombinant strain expressing this enzyme, ginsenoside R1 can be catalyzed to produce rare ginsenoside Rh1, and ginsenoside Rb1 can be catalyzed to produce rare ginsenoside C-K. The glycoside hydrolase of this invention possesses both xylosidase and glucosidase activities. Therefore, using this enzyme or the recombinant strain, the xylosidic bond and glucosidic bond at the C-6 position of the aglycone of ginsenoside R1 can be simultaneously cleaved to generate rare ginsenoside Rh1. Compared with methods for preparing rare ginsenosides using glycoside hydrolases with single enzyme activity, this method has the advantages of simple process and economic efficiency, making it very suitable for large-scale industrial production.

Description

Technical Field

[0001] This invention relates to the field of catalysis technology, and in particular to the application and method of a glycoside hydrolase with dual enzyme activity and its recombinant strain in the preparation of rare ginsenosides. Background Technology

[0002] Ginsenosides, the core active ingredients of ginseng (Panax ginseng CAMey.), a traditional Chinese medicine, exhibit a wide range of biological activities, including but not limited to their significant anti-cancer, anti-inflammatory, and anti-aging properties. In-depth scientific research reveals that rare ginsenosides derived from proto-ginsenosides through biotransformation processes, due to their structural differences, not only enhance intestinal absorption but also improve bioavailability. This allows rare ginsenosides such as ginsenoside F1, Rh1, and CK to surpass their proto-molecules in exhibiting biological activity.

[0003] The superior pharmacological properties of these rare ginsenosides have laid a solid foundation for their widespread application in the pharmaceutical and functional food industries. However, the technical challenges in preparing rare ginsenosides constitute a significant bottleneck, limiting their large-scale production and application. In light of this, in recent years, researchers have dedicated themselves to developing and optimizing a series of preparation strategies aimed at improving the yield and purity of rare ginsenosides, thereby promoting their widespread use in clinical and healthcare fields.

[0004] The industrial production of rare ginsenosides mainly relies on a precise hydrolysis strategy targeting specific glycosyl sites of the original ginsenosides. Compared to the limitations of traditional chemical synthesis methods, such as low yield, numerous byproducts, and complex wastewater treatment, enzymatic hydrolysis demonstrates significant advantages. These advantages include mild reaction conditions, environmental friendliness, and high efficiency, making it the preferred method for the industrial preparation of rare ginsenosides. In this process, glycoside hydrolases (GHs) play a crucial role, effectively catalyzing the cleavage of glycosyl groups on ginsenoside aglycones to generate the target rare ginsenosides.

[0005] However, actual industrial raw materials typically contain diverse mixtures of proto-ginsenosides, such as ginsenosides Rb1, Rg1, and Rd. The glycosyl structures of these saponins primarily involve glucose units, while ginsenosides R1 and R2, for example, carry an extra-xylose group at the 6-carbon position of their aglycone. Given this complexity, a single type of glycoside hydrolase is insufficient to efficiently hydrolyze all target glycosyl groups. This necessitates the integration of multiple specific glycoside hydrolases into the production process for synergistic operation. This enzyme combination strategy not only increases the complexity of the process but also poses challenges to cost control and economic efficiency, highlighting the urgent need to optimize enzyme selection and reaction conditions to simplify operations and improve overall economics. Summary of the Invention

[0006] The purpose of this invention is to provide a glycoside hydrolase with dual enzyme activity and its application and method in the preparation of rare ginsenosides and recombinant bacteria. Ginsenoside R1 can be obtained by using the glycoside hydrolase or by using recombinant whole cells to catalyze ginsenoside R1, and ginsenoside CK can be obtained by catalyzing ginsenoside Rb1.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a glycoside hydrolase with dual enzyme activity, the amino acid sequence of which is shown in SEQ ID NO.1.

[0009] Preferably, the nucleotide sequence of the glycoside hydrolase is shown in SEQ ID NO.2.

[0010] The present invention also provides the application of the glycoside hydrolase in the preparation of rare ginsenosides.

[0011] The present invention also provides a method for preparing rare ginsenosides using the glycoside hydrolase, comprising the following steps: adding the glycoside hydrolase to a ginsenoside solution, catalyzing and then adding methanol to terminate the reaction, thereby obtaining rare ginsenosides;

[0012] The solvent for the ginsenoside solution is Tris buffer, the concentration of ginsenoside in the ginsenoside solution is 0.2–2 mg / mL, the final concentration of the glycoside hydrolase is 0.1–1 mg / mL, the catalytic conditions are 30–50 °C and pH 5.5–8, the catalytic time is 12–36 h, and the volume ratio of methanol to ginsenoside solution is 1:0.8–1.2.

[0013] When the ginsenoside in the ginsenoside solution used is ginsenoside R1, the rare ginsenoside obtained is ginsenoside Rh1; when the ginsenoside in the ginsenoside solution used is ginsenoside Rb1, the rare ginsenoside obtained is ginsenoside CK.

[0014] The present invention also provides a method for constructing a recombinant bacterium containing the aforementioned glycoside hydrolase gene, comprising the following steps:

[0015] (1) After PCR amplification of the nucleotide sequence shown in SEQ ID NO.2, it was introduced into a plasmid by homologous recombination to obtain a recombinant plasmid;

[0016] (2) The recombinant plasmid was introduced into Escherichia coli BL21(DE3) by chemical transformation to obtain recombinant bacteria;

[0017] The plasmid is either pETduet1 or pET28a.

[0018] The present invention also provides recombinant bacteria prepared by the method.

[0019] The present invention also provides the application of the recombinant bacteria in the preparation of rare ginsenosides.

[0020] The present invention also provides a method for preparing rare ginsenosides using the recombinant bacteria, comprising the following steps: adding the recombinant bacteria to a ginsenoside solution, catalyzing and then adding methanol to terminate the reaction, thereby obtaining rare ginsenosides;

[0021] The solvent for the ginsenoside solution is Tris buffer solution, the concentration of ginsenoside in the ginsenoside solution is 0.2-2 mg / mL, the final concentration of the recombinant bacteria is OD600 = 1-4, the catalytic conditions are 30-50℃, pH 5.5-8, the catalytic time is 12-36 h, and the volume ratio of methanol to ginsenoside solution is 1:0.8-1.2.

[0022] When the ginsenoside in the ginsenoside solution used is ginsenoside R1, the rare ginsenoside obtained is ginsenoside Rh1; when the ginsenoside in the ginsenoside solution used is ginsenoside Rb1, the rare ginsenoside obtained is ginsenoside CK.

[0023] The present invention also provides the application of the recombinant bacteria in the preparation of the glycoside hydrolase.

[0024] The present invention also provides a method for preparing glycoside hydrolase using the recombinant bacteria, comprising the following steps: inoculating the recombinant bacteria into LB medium, culturing at 30-40°C, adding IPTG when the bacterial concentration reaches OD600 = 0.4-0.8, inducing at 15-25°C, centrifuging and resuspending with buffer to obtain whole-cell bacterial solution, homogenizing under high pressure to obtain crude enzyme solution, and purifying by Ni-NTA column to obtain pure enzyme;

[0025] The final concentration of IPTG in the bacterial culture is 0.1–0.4 mM. The induction conditions are 180–220 rpm and 14–18 °C, with an induction time of 18–22 h. The centrifugation conditions are 5500–6500 rpm. The buffer solution is PBS buffer. The bacterial concentration in the whole-cell culture is OD600 = 1.9–2.1. The high-pressure homogenization pressure is 750–850 MPa, and the time is 4–6 min.

[0026] This invention provides a glycoside hydrolase with dual enzyme activity and its application and method in the preparation of rare ginsenosides and recombinant bacteria. The amino acid sequence of the glycoside hydrolase is shown in SEQ ID NO.1. Using the glycoside hydrolase of this invention or recombinant bacteria expressing this enzyme, ginsenoside R1 can be catalyzed to produce rare ginsenoside Rh1, and ginsenoside Rb1 can be catalyzed to produce rare ginsenoside CK. The glycoside hydrolase of this invention possesses both xylosidase and glucosidase activities. Therefore, using this enzyme or recombinant cells, the xylosidic bond and glucosidic bond at the C-6 position of the ginsenoside R1 aglycone can be simultaneously cleaved to generate rare ginsenoside Rh1. Compared with methods for preparing rare ginsenosides using glycoside hydrolases with single enzyme activity, this method has the advantages of simple process and economic efficiency, making it very suitable for large-scale industrial production.

[0027] The beneficial effects of this invention include the following:

[0028] (1) The glycoside hydrolase B6 in this invention has two activities: hydrolyzing xylose and glucose. It can catalyze the hydrolysis of ginsenoside R1 to Rh1. The yield can reach 0.3122 mg / mL in 24 hours, with a yield of 67.4%.

[0029] (2) The glycoside hydrolase B6 in this invention has two activities: hydrolyzing xylose and glucose. It can hydrolyze ginsenoside R1 to Rh1 through whole-cell catalysis. The yield can reach 0.2191 mg / mL in 24 hours, with a yield of 49.3%.

[0030] (3) The glycoside hydrolase B6 in this invention can catalyze the hydrolysis of ginsenoside Rb1 to CK, with a yield of 0.7655 mg / mL in 24 hours and a yield of 68.2%.

[0031] (4) The glycoside hydrolase B6 in this invention can utilize various prototype saponins in the industrial production of rare ginsenoside raw materials, which can simplify the operation process and improve the overall economy. Attached Figure Description

[0032] Figure 1 This is a schematic diagram of a recombinant gene vector;

[0033] Figure 2Schematic diagram of electrophoresis of denatured proteins of glycoside hydrolase B6;

[0034] Figure 3 Schematic diagram of pNPG, a universal substrate for glucose hydrolysis by glycoside hydrolase B6, and pNPX, a universal substrate for xylose;

[0035] Figure 4 Schematic diagram of the conversion pathway for preparing ginsenoside R1 from glycoside hydrolases / recombinant cells;

[0036] Figure 5 Schematic diagram of the transformation pathway for preparing ginsenoside Rb1 from glycoside hydrolases / recombinant cells;

[0037] Figure 6 The image shows the products of ginsenosides R1, Rg1, F1, and Rh1 as determined by HPLC.

[0038] Figure 7 The image shows the product of ginsenosides Rb1, GYP17, GYP75, and CK as determined by HPLC. Detailed Implementation

[0039] This invention provides a glycoside hydrolase with dual enzyme activity, the amino acid sequence of which is shown in SEQ ID NO.1.

[0040] SEQ ID NO.1:

[0041] MTPTYLTAPDGTRFRDLNGNGVMDPYEDPRLSADERAADLVARMSLEEKCGLMFQTVIEVGEEGELLEAPGRISKSPTTTVVRGKHLSHFNVHAIRSARQAAVWNNNLQALAAQTPHGVPVTVSTDPRHAFVENTGVGFAAGPFSQWPEGLGLAAIDDVETVRRFADVARQEYRAVGIRAALHPQIDLATEPRWG RQAQTLGQDAGRVAEFTAAYLQGFQGDALGPDSVACTTKHFPGGGPQKDGEDAHFPYGREQVYPGGMFEYHLEPFREAIRRGTAAMMPYYGMPIGLERNGVPIEEVGFGYNRQIVTDLLRGELGFDGVVVTDWELVNDNHVGDQVLPARAWGVEELSPSERMLKILDAGADQFGGEECVDLLIALVRAGRVDEARIDASALRLLRVKFQLGLFDDPFVDPDEAERIVGNAQFRAEGERAQARSLTVVQNRPTPGADHPVLPLSGAGRRVYVEGFRPEDVAELGEIVADPADADLALVRLGAPFEPRDDLFLEAWFHQGSLEFPPGRVYRMRSIAAHCPLVLVVNLDRPGILTPFAAFASAIVVDFGSSSRAVVDVLTGRIAPEGRLPIELPRSMDAVRSSREDVPSDTGDPLFPVHFGLELPMRVGGRADA。

[0042] In the present invention, the nucleotide sequence of the glycoside hydrolase is as shown in SEQ ID NO.2;

[0043] SEQ ID NO.2:

[0044]

[0045] The present invention also provides the application of the glycoside hydrolase in the preparation of rare ginsenosides.

[0046] The present invention also provides a method for preparing rare ginsenosides using the glycoside hydrolase, comprising the following steps: adding the glycoside hydrolase to a ginsenoside solution, catalyzing and then adding methanol to terminate the reaction, thereby obtaining rare ginsenosides;

[0047] The solvent for the ginsenoside solution is preferably Tris buffer solution; the concentration of ginsenosides in the ginsenoside solution is preferably 0.2–2 mg / mL, more preferably 1 mg / mL; the final concentration of the glycoside hydrolase is preferably 0.1–1 mg / mL, more preferably 0.5 mg / mL; the catalytic conditions are preferably 30–50 °C and pH 5.5–8, more preferably 30–33 °C and pH 7.0–7.5; the catalytic time is preferably 12–36 h, more preferably 24 h; and the volume ratio of methanol to ginsenoside solution is preferably 1:0.8–1.2, more preferably 1:1.

[0048] When the ginsenoside in the ginsenoside solution used is ginsenoside R1, the rare ginsenoside obtained is ginsenoside Rh1; when the ginsenoside in the ginsenoside solution used is ginsenoside Rb1, the rare ginsenoside obtained is ginsenoside CK.

[0049] The present invention also provides a method for constructing a recombinant bacterium containing the aforementioned glycoside hydrolase gene, comprising the following steps:

[0050] (1) After PCR amplification of the nucleotide sequence shown in SEQ ID NO.2, it was introduced into a plasmid by homologous recombination to obtain a recombinant plasmid;

[0051] (2) The recombinant plasmid was introduced into Escherichia coli BL21(DE3) by chemical transformation to obtain recombinant bacteria;

[0052] The plasmid is either pETduet1 or pET28a.

[0053] The present invention also provides recombinant bacteria prepared by the method.

[0054] The present invention also provides the application of the recombinant bacteria in the preparation of rare ginsenosides.

[0055] The present invention also provides a method for preparing rare ginsenosides using the recombinant bacteria, comprising the following steps: adding the recombinant bacteria to a ginsenoside solution, catalyzing and then adding methanol to terminate the reaction, thereby obtaining rare ginsenosides;

[0056] The solvent for the ginsenoside solution is preferably Tris buffer solution; the concentration of ginsenosides in the ginsenoside solution is preferably 0.2–2 mg / mL, more preferably 1 mg / mL; the final concentration of the recombinant bacteria is preferably OD600 = 1–4, more preferably OD600 = 2–3; the catalytic conditions are preferably 30–50℃ and pH 5.5–8, more preferably 30–33℃ and pH 7.0–7.5; the catalytic time is preferably 12–36 h, more preferably 24 h; and the volume ratio of methanol to ginsenoside solution is preferably 1:0.8–1.2, more preferably 1:1.

[0057] When the ginsenoside in the ginsenoside solution used is ginsenoside R1, the rare ginsenoside obtained is ginsenoside Rh1; when the ginsenoside in the ginsenoside solution used is ginsenoside Rb1, the rare ginsenoside obtained is ginsenoside CK.

[0058] The present invention also provides the application of the recombinant bacteria in the preparation of the glycoside hydrolase.

[0059] The present invention also provides a method for preparing glycoside hydrolase using the recombinant bacteria, comprising the following steps: inoculating the recombinant bacteria into LB medium, culturing at 30-40°C, adding IPTG when the bacterial concentration reaches OD600 = 0.4-0.8, inducing at 15-25°C, centrifuging and resuspending with buffer to obtain whole-cell bacterial solution, homogenizing under high pressure to obtain crude enzyme solution, and purifying by Ni-NTA column to obtain pure enzyme;

[0060] In this invention, the culture temperature is preferably 35°C, the IPTG is preferably added when the OD600 is 0.6, and the induction temperature is preferably 20°C.

[0061] In this invention, the final concentration of IPTG in the bacterial culture is preferably 0.1–0.4 mM, more preferably 0.2–0.3 mM; the induction conditions are preferably 180–220 rpm and 14–18°C, more preferably 200 rpm and 16°C; the induction time is preferably 18–22 h, more preferably 20 h; the centrifugation conditions are preferably 5500–6500 rpm, more preferably 6000 rpm; the buffer solution is preferably Tris buffer; the concentration of bacteria in the whole-cell bacterial culture is preferably OD600 = 1.9–2.1, more preferably OD600 = 2.0; and the high-pressure homogenization pressure is 750–850 MPa, and the time is 4–6 min.

[0062] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0063] Example 1

[0064] This embodiment utilizes glycoside hydrolase B6 to catalyze the preparation of rare ginsenoside Rh1 from ginsenoside R1. Specifically, 0.65 mg of ginsenoside R1 was weighed and dissolved in Tris buffer (pH 7.5) containing 0.3 mg of glycoside hydrolase B6. The reaction was carried out at 30°C for 24 h, and then 1 mL of methanol was added to stop the reaction. The content of ginsenoside Rh1 in the system was detected by HPLC (acetonitrile:water = 1:3, UV detector 203 nm, C18 reversed-phase column, flow rate 1 mL / min, column temperature 35°C), and the final substrate conversion rate was calculated to be 100%, the yield to be 67.4%, and the production rate to be 0.3122 mg / mL.

[0065] Example 2

[0066] This embodiment utilizes recombinant cells expressing glycoside hydrolase B6 to catalyze the preparation of rare ginsenoside Rh1 from ginsenoside R1. Specifically, 0.65 mg of ginsenoside R1 was weighed and dissolved in Tris buffer (pH 7.5) containing recombinant cells, achieving a final bacterial concentration of OD600 = 1. The reaction was carried out at 30°C for 24 h, and then 1 mL of methanol was added to stop the reaction. The content of ginsenoside Rh1 in the system was detected by HPLC (acetonitrile:water = 1:3, UV detector 203 nm, C18 reversed-phase column, flow rate 1 mL / min, column temperature 35°C), and the final substrate conversion rate was calculated to be 100%, the yield to be 49.3%, and the production rate to be 0.2191 mg / mL.

[0067] Example 3

[0068] This embodiment utilizes glycoside hydrolase B6 to catalyze the preparation of rare ginsenoside CK from ginsenoside Rb1. Specifically, 2 mg of ginsenoside Rb1 was weighed and dissolved in Tris buffer (pH 7.5) containing 1 mg of glycoside hydrolase B6. The reaction was carried out at 30°C for 24 h, and then 1 mL of methanol was added to stop the reaction. The contents of ginsenoside Rb1 and CK in the system were detected by HPLC (acetonitrile:water = 1:3, UV detector 203 nm, C18 reversed-phase column, flow rate 1 mL / min, column temperature 35°C), and the final substrate conversion rate was calculated to be 100%, the yield to be 68.2%, and the production rate to be 0.7655 mg / mL.

[0069] Example 4

[0070] This embodiment utilizes recombinant cells expressing glycoside hydrolase B6 to catalyze the preparation of rare ginsenoside CK from ginsenoside Rb1. Specifically, 1 mg of ginsenoside Rb1 was weighed and dissolved in Tris buffer (pH 7.5) containing recombinant cells, achieving a final bacterial concentration of OD600 = 1. The reaction was carried out at 30°C for 24 h, and then 1 mL of methanol was added to stop the reaction. The content of ginsenoside CK in the system was detected by HPLC (acetonitrile:water = 1:3, UV detector 203 nm, C18 reversed-phase column, flow rate 1 mL / min, column temperature 35°C), and the final substrate conversion rate was calculated to be 100%, the yield to be 31.9%, and the production rate to be 0.3582 mg / mL.

[0071] Note: The detection method used in the embodiments of this invention is as follows:

[0072] Sample preparation: Add an equal volume of methanol (chromatographic grade) to the reaction system and filter through a 0.22 μm filter membrane;

[0073] Chromatographic column: C18 column, 250×4.6mm; Column temperature: 35℃;

[0074] Mobile phase: Phase A acetonitrile, Phase B ultrapure water;

[0075] Detector: Ultraviolet detector; Detection wavelength: 203 nm; Injection volume: 20 μL;

[0076] Elution conditions: The hydrolysis of R1 was carried out using the gradient elution method in Table 1, and the hydrolysis of Rb1 was carried out using the gradient elution method in Table 3; the peak times are detailed in Tables 2 and 4.

[0077] Table 1. Mobile phase operating gradient conditions 1

[0078] Time / min Mobile phase A% Mobile phase B% 0.01 25 75 15.00 50 50 15.01 25 75 25.00 25 75

[0079] Table 2 Mobile Phase Operating Gradient Conditions 1 - Peak Position

[0080] Standard products Peak time R1 6.454 Rg1 7.479 F1 12.423 Rh1 15.980

[0081] Table 3. Mobile phase operating gradient conditions 2

[0082]

[0083]

[0084] Table 4 Mobile Phase Operating Gradient Conditions 2 - Peak Position

[0085] Standard products Peak time Rb1 13.932 Rd 16.990 Gyp17 18.188 F2 22.423 Gyp75 25.85 CK 32.684

[0086] As can be seen from the above embodiments, the present invention provides an application and method of a glycoside hydrolase with dual enzyme activity and its recombinant strain in the preparation of rare ginsenosides. The amino acid sequence of the glycoside hydrolase is shown in SEQ ID NO.1. Using the glycoside hydrolase of the present invention or the recombinant strain expressing this enzyme, ginsenoside R1 can be catalyzed to produce rare ginsenoside Rh1, and ginsenoside Rb1 can be catalyzed to produce rare ginsenoside CK. The glycoside hydrolase of the present invention has both xylosidase and glucosidase activities. Therefore, using this enzyme or the recombinant strain, the xylosidic bond and glucosidic bond at the C-6 position of the aglycone of ginsenoside R1 can be simultaneously broken to generate rare ginsenoside Rh1. Compared with the method of preparing rare ginsenosides using a single-enzyme glycoside hydrolase, this method has the advantages of simple process and economic efficiency, and is very suitable for large-scale industrial production.

[0087] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing rare ginsenosides using recombinant bacteria, characterized in that, The method comprises the following steps: adding the recombinant bacteria into a ginsenoside solution, adding methanol to terminate the reaction after catalysis, and obtaining rare ginsenoside; The solvent of the ginsenoside solution is Tris buffer, the concentration of ginsenoside in the ginsenoside solution is 0.2-2 mg / mL, the final concentration of the recombinant bacteria is OD600=1-4, the catalysis condition is 30-50 DEG C, pH 5.5-8, the catalysis time is 12-36 h, and the volume ratio of methanol to ginsenoside solution is 1:0.8-1.2; The ginsenoside in the ginsenoside solution is ginsenoside R1, and the obtained rare ginsenoside is ginsenoside Rh1; The recombinant bacteria are constructed by the following steps: (1) the nucleotide sequence shown in SEQ ID NO. 2 is subjected to PCR amplification, and then introduced into a plasmid by homologous recombination to obtain a recombinant plasmid; (2) the recombinant plasmid is introduced into E. coli BL21(DE3) by chemical transformation method to obtain the recombinant bacteria; The plasmid is pETduet1 or pET28a.

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