An alpha-l-rhamnosidase from cryptococcus laurentii and its use in the preparation of icariin

Abstract

The application relates to the technical field of natural product biotransformation, and discloses a cryptosporidium curvatus alpha-L-rhamnosidase and application thereof in preparation of icariin. A cryptosporidium curvatus model strain DSM 28205 is cultured in an induction medium containing L-rhamnose 10 g / L, yeast extract powder 5 g / L, peptone 10 g / L, KH2PO4 2 g / L and MgSO4.7H2O 0.5 g / L at 28 DEG C and 180 rpm for 48 h; the bacterial cells are collected, ultrasonically broken and centrifuged at not more than 10 DEG C to obtain intracellular crude enzyme liquid. The crude enzyme liquid is directly used for enzymolysis of an epimedium water extract containing doriden C or a water extraction concentrate thereof, and doriden C is converted into icariin under the conditions of pH 6.0-7.0 and 40-55 DEG C for 3-15 h. Under preferred conditions, the molar yield of icariin is more than 83 %.

Description

Technical Field

[0001] This invention belongs to the field of microbial enzyme preparations and natural product biotransformation technology, specifically involving the acquisition of intracellular α-L-rhamnosidase using the model strain DSM 28205 of Cryptococcus lauriens, which can be obtained from public culture collection institutions, as an enzyme source under specific L-rhamnose-induced culture conditions, and the application of said α-L-rhamnosidase in catalyzing the conversion of icariin C into icariin. Background Technology

[0002] Epimedium is a commonly used traditional Chinese medicine. Its flavonoid components mainly include icariin, icariin A, icariin B, and icariin C. Icariin C is structurally similar to icariin, but it contains an additional L-rhamnosyl group in its sugar chain. By selectively cleaving the corresponding rhamnosyl group, the bioconversion of icariin C to icariin can be achieved. Compared with acid or alkaline hydrolysis, enzymatic conversion has the advantages of milder conditions and higher reaction selectivity, making it particularly suitable for epimedium extract systems containing multiple flavonoid glycosides.

[0003] Chinese patent application CN86105166A discloses a technique for converting saponin substrates into ginsenoside Rd using rhamnosidase and / or glucosidase, and explicitly lists *Cryptococcus lauriens* IFO 0609 as an applicable strain. In the specific embodiment of this document, IFO 0609 is cultured in a medium containing hesperidin, shaken at 28°C for 7 days, and the culture medium is filtered. The resulting filtrate is then used as the enzyme source for glycoside substrate conversion. This technique demonstrates that the model strain can generate rhamnosidase activity, but its enzyme source acquisition method, culture period, and target substrate differ from the conversion of ginsenoside Rd from icariin C.

[0004] Lou et al. reported in the Journal of Fungi, 2022, 8:644, on the *Cryptococcus laureus* ZJU-L07 strain obtained through soil screening and mutagenesis, and its intracellular α-L-rhamnosidase, which was then used for the conversion of epimedium C to icariin. The intracellular enzyme was prepared after culturing the strain at 28°C for 4 days in a medium containing 1% L-rhamnose. This report demonstrates that the intracellular α-L-rhamnosidase of a specific screened and mutagenic strain can be used for the conversion of epimedium C, but it does not provide a specific technical solution for obtaining the corresponding intracellular enzyme using the publicly available IFO 0609 / DSM 28205 model strain with a shorter induction period and directly processing epimedium water extract.

[0005] Therefore, given that the known *Cryptococcus lauriensis* model strain can produce common rhamnosidases and that the intracellular α-L-rhamnosidase of specific selected strains can convert icariin C, it is still necessary to determine the matching induction culture composition and culture cycle for the publicly available DSM 28205 model strain, and to establish a complete process for cell collection, intracellular enzyme release, and direct treatment of *Epimedium* water extract with crude enzyme solution. This would enable the model strain to achieve the conversion of icariin C to icariin C without relying on specific selected mutagenic strains, unlike the route of IFO 0609 which uses culture filtrate as the enzyme source after 7 days of culture. Summary of the Invention

[0006] In view of this, the present invention provides an α-L-rhamnosidase derived from Cryptococcus laurentii and its application in the preparation of icariin.

[0007] This invention uses the type strain of *Papiliotrema laurentii*, DSM 28205, as the enzyme-producing strain. DSM 28205 is publicly available from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH (DSMZ). The same type strain corresponding to DSM 28205 is listed in other microbial collections under the numbers ATCC 18803, CBS 139, JCM 9066, NBRC 0609, and NRRL Y-2536; the corresponding strain number can be used depending on the actual acquisition method.

[0008] In this invention, the seed culture of the model strain was inoculated into the induction medium at an inoculation rate of 5% (v / v). The induction medium contained 10 g of L-rhamnose, 5 g of yeast extract, 10 g of peptone, 2 g of KH2PO4, and 0.5 g of MgSO4·7H2O per liter, with an initial pH of 6.0. The cells were collected after culturing at 28°C and 180 rpm for 48 h.

[0009] The cultured bacterial cells were washed and resuspended in 0.10 mol / L, pH 7.4 PBS buffer. The suspension was then sonicated at a temperature not exceeding 10°C, and the lysate was centrifuged at 8000 rpm for 10 min. The supernatant was collected to obtain a crude enzyme solution containing intracellular α-L-rhamnosidase. This step releases the intracellular enzyme from the cultured bacterial cells, and the supernatant from the cell lysis is used as the enzyme source for subsequent enzymatic digestion.

[0010] The obtained crude enzyme solution was directly mixed with a substrate solution prepared from an aqueous extract of Epimedium containing icariin C or its concentrated aqueous extract. The concentration of the Epimedium aqueous extract or its concentrated aqueous extract in the reaction system was 2.5-7.5 g / L, and the concentration of icariin C in the reaction system was 500-1500 mg / L. The amount of α-L-rhamnosidase in the reaction system was 1.05-9.565 U / mL, and the reaction was carried out at pH 6.0-7.0 and 40-55℃ for 3-15 h to convert icariin C into icariin. This invention also provides the application of the aforementioned Cryptococcus laureus α-L-rhamnosidase in the preparation of icariin.

[0011] The α-L-rhamnosidase activity was determined using p-nitrophenyl-α-L-rhamnoside as a substrate; 1 U represents the amount of enzyme that catalyzes the release of 1 μmol of p-nitrophenol per minute at 55°C and pH 7.0.

[0012] In a preferred embodiment, the aqueous extract of Epimedium or its concentrated aqueous extract is prepared as a substrate solution using a 0.2 mol / L citrate-phosphate buffer solution with a pH of 6.0-7.0; more preferably, the concentration of Epimedium C in the reaction system is 500 mg / L, the amount of α-L-rhamnosidase is 9.565 U / mL, the pH is 7.0, the reaction temperature is 50°C, and the reaction time is 3 h.

[0013] Under the above-mentioned preferred conditions, the molar yield of icariin exceeded 83%, calculated based on the amount of icariin C before the reaction and the increase in the amount of icariin before and after the reaction. This molar yield was calculated based on the HPLC external standard quantitative results described in the examples and served as the main quantitative indicator for evaluating the degree of conversion of icariin C to icariin.

[0014] Compared with existing Cryptococcus lauriens rhamnosidase technology, the technical solution of this invention does not simply replace the screening strain with a publicly available model strain. Instead, it establishes a specific induction culture composition and 48-hour culture conditions for DSM 28205. After the culture is completed, the cells are collected and the intracellular enzyme is released. The resulting crude enzyme solution is then directly applied to the water extract of Epimedium or its water extract concentrate, thus forming a continuous process from obtaining the publicly available strain, 48-hour induction culture, preparation of intracellular enzyme to conversion of natural water extract.

[0015] The present invention has the following advantages over the prior art: When using the DSM 28205 model strain, a crude intracellular α-L-rhamnosidase enzyme solution suitable for the conversion of epimedium C can be obtained under the induction medium and culture conditions of 28℃, 180 rpm, and 48 h; the obtained crude enzyme solution directly treats the aqueous extract of epimedium or its aqueous extract concentrate, and achieves the conversion of epimedium C to icariin under near-neutral and mesophilic conditions. Compared with the route of using the culture filtrate as the enzyme source after 7 days of culture of IFO 0609, the present invention clearly adopts the intracellular enzyme route; compared with the existing epimedium C conversion schemes that rely on screening and mutagenesis strains, the present invention provides a reproducible process with publicly available model strains. Detailed Implementation

[0016] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are used to illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the present invention. Reagents, standards, culture medium components, chromatographic materials and instruments not specifically mentioned can all be commercially available products conventional in the art. The following "pNPR" refers to p-nitrophenyl-α-L-rhamnoside.

[0017] Example 1: Obtaining, reviving, and identifying the disclosed type strain The strain used in this embodiment is the type strain of *Papiliotrema laurentii*, DSM28205. DSM 28205 is publicly available from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH (DSMZ). The same type strain has been registered in other microbial collections under the numbers ATCC 18803, CBS 139, JCM 9066, NBRC 0609, and NRRL Y-2536; any of these numbered strains can be used to implement this invention.

[0018] The freeze-dried or activated cultures of the strains were revived according to the culture instructions provided by the supplier. For example, DSM 28205 could be revived and cultured using DSMZ Medium 186 at 22°C; this medium contains 10 g glucose, 5 g soybean peptone, 3 g malt extract, 3 g yeast extract, and 15 g agar per liter. After colony formation, single colonies were picked for expansion culture to obtain seed culture. Strain classification was confirmed based on identification data provided by the strain preservation institution and could also be verified by ITS sequencing.

[0019] Example 2: L-Rhamnose-induced enzyme production culture The seed culture obtained in Example 1 was transferred to the induction medium at an inoculation rate of 5% (v / v). The induction medium contained 10 g L-rhamnose, 5 g yeast extract, 10 g peptone, 2 g KH₂PO₄, and 0.5 g MgSO₄·7H₂O per liter, with the remainder being water. The initial pH was adjusted to 6.0. The culture was carried out at 28°C and 180 rpm for 48 h. After the culture was completed, the fermentation broth was centrifuged and the cells were collected for use in the preparation of crude enzyme solution.

[0020] Example 3 Preparation of crude enzyme solution The fermentation broth obtained in Example 2 was centrifuged at 3500 rpm for 10 min, and the cell pellet was collected and washed twice with ultrapure water. The washed cell pellet was resuspended in 0.10 mol / L, pH 7.4 PBS buffer to obtain a bacterial suspension. The bacterial suspension was ultrasonically disrupted in an ice bath, and the sample temperature was controlled not to exceed 10℃ during the disruption process. After disruption, the suspension was centrifuged at 8000 rpm for 10 min, and the supernatant was collected to obtain crude α-L-rhamnosidase from Cryptococcus larenus. The obtained cell disruption supernatant was used as the enzyme source for the subsequent step (3).

[0021] Example 4: Assay of α-L-rhamnosidase activity The α-L-rhamnoside activity of the crude enzyme solution was determined using p-nitrophenyl-α-L-rhamnoside (pNPR) as a substrate. 50 μL of a 10 mmol / L pNPR solution was mixed with 400 μL of a 200 mmol / L, pH 7.0 citrate-disodium hydrogen phosphate buffer solution and preheated at 55 °C for 5 min. Then, 50 μL of the enzyme solution was added, and the reaction was carried out at 55 °C for 10 min. After the reaction was complete, 750 μL of a 1.5 mol / L Na₂CO₃ solution was added to terminate the reaction, and the absorbance was measured at 405 nm. The release amount was calculated based on the p-nitrophenol standard curve. 1 U represents the amount of enzyme that catalyzes the release of 1 μmol of p-nitrophenol per minute at 55 °C and pH 7.0.

[0022] The enzyme activity of the crude enzyme solution obtained in Example 3 was determined according to the above method, and the volume of crude enzyme solution added in subsequent reactions was calculated based on the measured enzyme activity, so that the amount of α-L-rhamnosidase in the reaction system reached the U / mL set in each example.

[0023] Example 5 Isolation and purification of α-L-rhamnosidase Take the crude enzyme solution obtained in Example 3, add ammonium sulfate to 30% saturation, let stand at 4°C for 4 h, centrifuge at 10000 rpm for 15 min to remove the precipitate; continue to add ammonium sulfate to the obtained supernatant to 70% saturation, let stand at 4°C overnight, centrifuge at 10000 rpm for 15 min to collect the precipitate. Dissolve the precipitate with 0.2 mol / L, pH 7.0 citrate-phosphate buffer and dialyze, then concentrate using PEG6000. This example is used to illustrate that the intracellular enzyme can be further separated and purified, and is not a necessary step for the direct enzymatic hydrolysis of Epimedium water extract in Example 7.

[0024] The concentrated sample was purified by Sephacryl S-200 gel chromatography and DEAE Sepharose Fast Flow ion exchange chromatography. The fraction with α-L-rhamnosidase activity was collected to obtain the purified enzyme solution. In Example 7, the crude enzyme solution obtained in Example 3 was used directly for the conversion of Epimedium water extract.

[0025] Example 6: HPLC detection method and conversion verification of Asparagine C standard Icariin C and icariin were detected by HPLC. A C18 column (250 mm × 4.6 mm, 5 μm) was used. Mobile phase A was acetonitrile, and mobile phase B was water. The gradient elution program was as follows: 0–15 min, phase A increased from 22% to 28%; 15–30 min, phase A increased from 28% to 29%; 30–56 min, phase A increased from 29% to 100%. The flow rate was 1.0 mL / min, the column temperature was 40℃, the injection volume was 10 μL, and the detection wavelength was 270 nm. An external standard quantitative relationship was established using icariin C and icariin standards to quantify the two components before and after the reaction.

[0026] A substrate solution was prepared using ascorbic acid C standard to achieve a final concentration of 500 mg / L in the reaction system. The crude enzyme solution obtained in Example 3 was added, and the amount of crude enzyme solution added was adjusted according to the enzyme activity measured in Example 4. A reaction system was constructed using 0.2 mol / L, pH 7.0 citrate-phosphate buffer and reacted at 50°C. Samples were taken at 0 h, 3 h, 6 h, and 9 h, and the reaction was terminated by adding 3 volumes of methanol. The mixture was centrifuged at 8000 rpm for 5 min, and the supernatant was filtered through a 0.22 μm filter membrane.

[0027] A system without crude enzyme solution but with an equal volume of buffer solution was used as a blank control. HPLC results showed that after adding crude enzyme solution, the peak area of ​​icariin C decreased as the reaction proceeded, and a product peak with the same retention time as the icariin standard appeared, indicating that the crude enzyme solution obtained in Example 3 could catalyze the formation of icariin from icariin C.

[0028] Example 7 Direct enzymatic hydrolysis of Epimedium water extract An aqueous extract of Epimedium containing ascorbic acid C was prepared into a 5 g / L substrate solution using 0.2 mol / L, pH 7.0 citrate-phosphate buffer, resulting in an ascorbic acid C concentration of 500 mg / L in the reaction system. 1 mL of the substrate solution was mixed with the crude enzyme solution prepared in Example 3. The volume of the added solution was adjusted according to the enzyme activity measured in Example 4, so that the amount of α-L-rhamnosidase in the reaction system was 9.565 U / mL. The reaction was carried out at 50°C and pH 7.0 for 3 h.

[0029] After the reaction was completed, the mixture was placed in an ice bath, and 3 times its volume of methanol was added to terminate the reaction. The mixture was centrifuged at 8000 rpm for 5 min, and the supernatant was filtered through a 0.22 μm filter membrane and analyzed by HPLC according to the method described in Example 6. The molar yield of icariin was calculated as [(amount of icariin after enzymatic hydrolysis - amount of icariin before enzymatic hydrolysis) / amount of icariin C before enzymatic hydrolysis] × 100%; the amounts of icariin C and icariin were converted from the external standard quantification results described in Example 6. The results showed that under the above conditions, the molar yield of icariin exceeded 83%.

[0030] The results above show that the crude enzyme solution obtained using the DSM 28205 model strain can be directly used for the aqueous extract of Epimedium containing icariin C. Under the conditions described, icariin C can be converted into icariin, and a molar yield of icariin exceeding 83% can be obtained.

[0031] Table 1 Summary of key experimental results

[0032] Note: Example 8 uses a single-factor investigation. The results in the table are all detected by the HPLC method of Example 6 or quantified by external standard.

[0033] Example 8: Verification of Reaction Condition Range Based on the Epimedium aqueous extract system of Example 7, the concentrations of Asarum nitrate C in the reaction system were investigated at 500, 1000, and 1500 mg / L; the amount of α-L-rhamnosidase was investigated at 1.05, 1.975, 4.395, and 9.565 U / mL; the reaction temperatures were investigated at 40, 45, 50, and 55 °C; and the pH values ​​were investigated at 6.0, 6.5, and 7.0. Samples were taken from each group at 0, 3, 6, 9, 12, and 15 h, and the reactions were terminated, samples were processed, and HPLC analysis was performed using the same method as in Example 7.

[0034] The results showed that, within the pH range of 6.0-7.0 and 40-55℃, the decrease in icariin C and the product peak with the same retention time as the icariin standard could be detected by HPLC under all single-factor experimental conditions. The external standard method described in Example 6 could be used to quantify icariin C and icariin. Differences were found between the detection results of each enzyme-added group and the corresponding unenzyme-free blank control. Considering the icariin formation rate and endpoint yield, the optimal conditions were: icariin C concentration of 500 mg / L, α-L-rhamnosidase dosage of 9.565 U / mL, pH 7.0, 50℃, and reaction time of 3 h.

[0035] Comparative Example 1: Unenzyme-free blank control The reaction was carried out according to the method of Example 7, except that an equal volume of 0.2 mol / L, pH 7.0 citrate-phosphate buffer was used instead of the crude enzyme solution, while all other conditions remained the same. After the reaction, external standard quantification was performed using the HPLC method described in Example 6. The results showed that the blank control did not exhibit the changes in icariin C consumption and icariin production seen in the enzyme-added group, indicating that the transformation in Example 7 originated from the catalytic effect of the crude enzyme solution.

[0036] Comparative Example 2: Glucose Culture Control The culture was performed according to the method of Example 2, except that an equal mass of glucose was used instead of L-rhamnose, while the other culture conditions remained the same. Crude enzyme solutions were then prepared according to Examples 3 and 4, and α-L-rhamnosidase activity was measured. Finally, aspergillus C conversion was performed according to Example 6. The results showed that the α-L-rhamnosidase activity and aspergillus C conversion rate in the glucose culture group were both lower than those in the L-rhamnose-induced group, indicating that L-rhamnose-induced culture is beneficial for the model strain to form intracellular α-L-rhamnosidase activity for aspergillus C conversion.

Claims

1. A method for preparing icariin using α-L-rhamnosidase derived from Cryptococcus lauriens, characterized in that, Includes the following steps: (1) The seed culture of the revived Cryptococcus laurentis model strain DSM 28205 was amplified and the seed culture was inoculated into the induction medium at an inoculation rate of 5% v / v. The induction medium contained 10 g of L-rhamnose, 5 g of yeast extract, 10 g of peptone, 2 g of KH2PO4 and 0.5 g of MgSO4·7H2O per liter, and the initial pH was 6.

0. The cells were collected after culturing at 28℃ and 180 rpm for 48 h. (2) The bacterial cells were washed and resuspended in 0.10 mol / L, pH 7.4 PBS buffer. The suspension was sonicated at a temperature not higher than 10°C. The suspension was centrifuged at 8000 rpm for 10 min and the supernatant was collected to obtain a crude enzyme solution containing intracellular α-L-rhamnosidase. (3) The crude enzyme solution obtained in step (2) is directly mixed with the substrate solution prepared from the aqueous extract of Epimedium containing icariin C or its aqueous extract concentrate, wherein the concentration of the aqueous extract of Epimedium or its aqueous extract concentrate in the reaction system is 2.5-7.5 g / L, the concentration of icariin C in the reaction system is 500-1500 mg / L, the amount of α-L-rhamnosidase in the reaction system is 1.05-9.565 U / mL, and the reaction is carried out at pH 6.0-7.0 and 40-55℃ for 3-15 h, so that icariin C is converted into icariin.

2. The method according to claim 1, characterized in that, The α-L-rhamnosidase activity was determined using p-nitrophenyl-α-L-rhamnoside as a substrate. For the determination, 50 μL of a 10 mmol / L substrate solution was mixed with 400 μL of a 200 mmol / L citrate-disodium hydrogen phosphate buffer solution (pH 7.0), followed by the addition of 50 μL of enzyme solution and reaction at 55 °C for 10 min. The reaction was then terminated by adding 750 μL of a 1.5 mol / L Na₂CO₃ solution, and the absorbance was measured at 405 nm. 1 U represents the amount of enzyme that catalyzes the release of 1 μmol of p-nitrophenol per minute under the above conditions.

3. The method according to claim 1, characterized in that, The aqueous extract of Epimedium or its concentrated aqueous extract was prepared into a substrate solution using a 0.2 mol / L citrate-phosphate buffer solution at pH 6.0-7.

0.

4. The method according to claim 1, characterized in that, The reaction system contained 500 mg / L of astragaloside C, 9.565 U / mL of α-L-rhamnosidase, pH 7.0, a reaction temperature of 50℃, and a reaction time of 3 h.

5. The method according to claim 1 or 2, characterized in that, After the crude enzyme solution obtained in step (2) is tested for enzyme activity and the volume to be added is determined based on the measured enzyme activity, it is directly mixed with the substrate solution to carry out enzymatic hydrolysis in step (3).

6. The method according to claim 1, characterized in that, The substrate solution was prepared from an aqueous extract of Epimedium containing Aspergillus C, and the concentration of the aqueous extract of Epimedium in the reaction system was 5 g / L.

7. The method according to claim 1, characterized in that, The bacterial cells in step (2) are obtained by centrifuging the fermentation broth obtained in step (1) at 3500 rpm for 10 min. After collecting the bacterial cell precipitate, it is washed twice with ultrapure water and then resuspended with the PBS buffer.

8. The method according to claim 1, characterized in that, The resuscitation medium for the model strain described in step (1) contains 10 g of glucose, 5 g of soybean peptone, 3 g of malt extract, 3 g of yeast extract and 15 g of agar per liter, and the resuscitation culture temperature is 22℃. After the colony forms, a single colony is picked and expanded to obtain the seed liquid.

9. The method according to claim 6, characterized in that, The concentration of the Epimedium aqueous extract in the reaction system was 5 g / L, the concentration of Asarum nitrate C in the reaction system was 500 mg / L, and the substrate solution was prepared with 0.2 mol / L, pH 7.0 citrate-phosphate buffer; the amount of α-L-rhamnosidase in the reaction system was 9.565 U / mL, the reaction pH was 7.0, the reaction temperature was 50℃, and the reaction time was 3 h.

10. The application of a Cryptococcus lauriens-derived α-L-rhamnosidase in the preparation of icariin, characterized in that, The α-L-rhamnosidase was prepared from the model strain of Cryptococcus lauriens, DSM 28205. The seed culture of the model strain was inoculated into an induction medium at a rate of 5% v / v. The induction medium contained 10 g of L-rhamnose, 5 g of yeast extract, 10 g of peptone, 2 g of KH₂PO₄, and 0.5 g of MgSO₄·7H₂O per liter, with an initial pH of 6.

0. The culture was carried out at 28°C and 180 rpm for 48 hours, after which the cells were collected. The cells were washed, resuspended, and then sonicated and centrifuged at a temperature not exceeding 10°C. The supernatant was collected to obtain a crude enzyme solution containing intracellular α-L-rhamnosidase. The crude enzyme solution was mixed with an aqueous extract of Epimedium containing icariin C or its concentrated aqueous extract, causing the icariin C to be converted into icariin.

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