Rhamnosidase and application thereof in production of icariin

Abstract

The invention relates to rhamnosidase and application of rhamnosidase in production of icariin. The amino acid sequence of the rhamnosidase provided by the invention is shown as SEQ ID NO: 1, when the rhamnosidase is used for catalyzing the conversion of epimedin C into icariin, 93% or more of epimedin C can be consumed within 2 hours, the yield of icariin is always maintained to be 91% or more, and the production efficiency of icariin is very high.

Description

Technical Field

[0001] The present application relates to the technical field of enzyme preparations, and in particular to a rhamnosidase and its application in the production of icariin. Background Art

[0002] Rhamnosidase plays an important role in the food and pharmaceutical industries. It is involved in the production of food additives, biochemical agents, the preparation of a variety of drugs and drug precursors, etc. In addition, studies have found that rhamnose is related to the aging of cells and skin tissues, and has great application potential in the pharmaceutical industry and the cosmetics field. α-L-rhamnosidase can also efficiently hydrolyze the rhamnosyl groups at the ends of many natural glycosidase compounds, such as hydrolyzing natural flavonoid glycoside compounds such as hesperidin, naringin, rutin, and epimedin C. However, the research on rhamnosidase in China started late, and there is a large gap with foreign countries in all aspects. At present, the fermentation level of rhamnosidase is low. Although commercial production has been carried out at home and abroad, the price is expensive, which hinders the large-scale application of rhamnosidase. At present, rhamnosidase preparations from commercial sources are generally derived from Aspergillus niger and Penicillium. Rhamnosidases from these sources are difficult to purify and contain other hydrolases. It is easy to produce by-products in production that affect the purity of the product. Its enzyme activity is only about 80% of the selected Pichia pastoris in this application. Therefore, it is imperative to take the directed evolution principle of enzymes as the core, combine genetic engineering methods, and use directed induced evolution technology and ultraviolet mutagenesis technology to increase enzyme production and industrialize it. This is also the current missing pain point.

[0003] On the whole, although the existing rhamnosidase production scheme can meet the commercialization means, it still has the following shortcomings. First, the existing production scheme is still limited by low fermentation activity, low yield and high production cost, and the market price is expensive and difficult to be widely used. Secondly, the production method of the existing production scheme is complicated, and a large number of repeated operations such as screening and purification are required, and the production cycle is also long. Finally, the purification operation of the rhamnosidase obtained by the current production scheme is difficult, or the presence of other hydrolases contained in the cultivation of Aspergillus niger and Penicillium is easy to produce by-products in production, affecting the purity of the product, and further increasing the production cost. Therefore, it is necessary to design and invent a new production scheme of rhamnosidase that can integrate multiple functions, achieve high enzyme activity, high yield and low cost, and put into formal commercialization as soon as possible. Summary of the invention

[0004] The present application provides a rhamnosidase and its application in the production of icariin, so as to solve the problem of low efficiency of rhamnosidase in the production of icariin in the related art. The rhamnosidase provided in the present application can consume more than 93% of icariin C in 2 hours when catalyzing the conversion of epimedin C into icariin, and the yield of icariin is maintained at more than 91%, and the production efficiency of icariin is relatively high.

[0005] Based on this, this application provides the following technical solutions:

[0006] In a first aspect, the present application provides a rhamnosidase having an amino acid sequence as shown in SEQ ID NO:1.

[0007] In a second aspect, the present application provides a rhamnosidase mutant, which has a sequence homology of more than 80% and less than 100% with the amino acid sequence of SEQ ID NO: 1.

[0008] The third aspect of the present application provides a nucleic acid molecule comprising a nucleic acid sequence capable of encoding the above-mentioned rhamnosidase or the above-mentioned rhamnosidase mutant.

[0009] The fourth aspect of the present application provides a vector, which contains the above-mentioned nucleic acid molecule.

[0010] The fifth aspect of the present application provides a microorganism, which contains one or more of the above-mentioned rhamnosidase, the above-mentioned rhamnosidase mutant, the above-mentioned nucleic acid molecule, and the above-mentioned vector.

[0011] The sixth aspect of the present application provides the use of the above-mentioned rhamnosidase mutant in the production of icariin using rhamnosidase.

[0012] In a seventh aspect, the present application provides a method for producing icariin using rhamnosidase: the rhamnosidase is contacted with epimedin C in a reaction system with a pH of 4.0 to 7.0 and a temperature of 40 to 60° C., and icariin is obtained after the reaction is completed.

[0013] According to the seventh aspect of the present application, in some embodiments, the concentration of epimedin C in the reaction system is 8 to 20 g / L.

[0014] According to the seventh aspect of the present application, in some embodiments, the reaction system uses phosphate buffer as the reaction liquid.

[0015] According to the seventh aspect of the present application, in some embodiments, the reaction time is 2 to 3 hours.

[0016] Compared with the prior art, this application has at least the following beneficial effects:

[0017] The rhamnosidase of the present application shows high catalytic activity in the production of icariin using rhamnosidase. The reaction system can react 93% of icariin C in 2 hours, and the yield of icariin is maintained at more than 91%. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0019] Figure 1 :Overall flow chart of this application.

[0020] Figure 2 : Schematic diagram of the principle of UV mutagenesis.

[0021] Figure 3 : The final standard curve obtained.

[0022] Figure 4 : Optimal pH for RhaE-F8.

[0023] Figure 5 : Optimum temperature of RhaE-F8.

[0024] Figure 6 :RhaE1 and RhaE-F8 hydrolyze epimedin C.

[0025] Figure 7 : RhaE1 and RhaE-F8 catalyze the hydrolysis of rutin. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solution and advantages of the present application clearer, the technical solution of the present application will be clearly and completely described below in combination with the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present application.

[0027] In one embodiment of the present application, a rhamnosidase is provided, the amino acid sequence of which is shown in SEQ ID NO: 1. In the process of catalyzing the conversion of epimedin C to icariin, the rhamnosidase can consume more than 93% of epimedin C in 2 hours, and the yield of icariin is maintained at more than 91%, and the production efficiency of icariin is relatively high. In addition, the rhamnosidase can also be used to catalyze the hydrolysis of rutin to remove rhamnose to generate the product quercetin. Compared with the existing rhamnosidase, the conversion rate of rutin to quercetin is increased by 68%.

[0028] The present application defines rhamnosidase as a protein having an amino acid sequence as shown in SEQ ID NO: 1. There may be meaningless sequences or naturally occurring mutations before and after the amino acid sequence shown in SEQ ID NO: 1. If such a protein has the same or corresponding activity as a protein composed of a peptide chain of the amino acid sequence shown in SEQ ID NO: 1, it should fall within the scope of rhamnosidase of the present application. This is obvious to those skilled in the art.

[0029] As a specific example, the rhamnosidase of the present application has an amino acid sequence as shown in SEQ ID NO: 1 or 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, or may be a protein containing an amino acid sequence having homology or identity. In addition, as long as the amino acid sequence has such homology or identity and exhibits the efficacy corresponding to the above-mentioned protein, a protein having an amino acid sequence in which part of the sequence is deleted, modified, substituted or added may also be considered. Obviously, the present application is included in the scope of the protein to be mutated in the present application.

[0030] Even if the present application describes "a protein or polypeptide having an amino acid sequence described by a specific sequence number" or "a protein or polypeptide comprising an amino acid sequence described by a specific sequence number", it is obvious that a protein corresponding to an amino acid sequence formed by deleting, modifying, replacing or adding a part of the sequence can also be used in the present application, as long as it has the same or corresponding activity as the polypeptide or protein consisting of the amino acid sequence shown in SEQ ID NO: 1. For example, "a polypeptide comprising the amino acid sequence of SEQ ID NO: 1" belongs to "a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1" if it has the same or corresponding activity.

[0031] In the present application, the term "mutant" refers to an amino acid sequence that is different from the amino acid sequence shown in SEQ ID NO: 1 in one or more conservative substitutions and / or modifications, and refers to a protein in which the function or characteristics of the above-mentioned protein are retained. The difference between the mutant and the amino acid sequence shown in SEQ ID NO: 1 is that there are multiple amino acid substitutions, deletions or additions. Such mutants can usually be identified by modifying one or more amino acids in the amino acid sequence of the protein and evaluating the properties of the modified protein. That is, the ability of the mutant can be increased compared to the rhamnosidase of the present application. In addition, some mutants may include mutants in which one or more parts (such as an N-terminal leader sequence or a transmembrane domain) have been removed. Other mutants may include mutants in which parts are removed from the N and / or C termini of the mature protein.

[0032] Another aspect of the present application may provide a nucleic acid molecule comprising a nucleic acid sequence capable of encoding the above-mentioned rhamnosidase or the above-mentioned rhamnosidase mutant.

[0033] In the present application, the term "nucleic acid molecule" refers to a DNA or RNA chain of a certain length or longer, which is a nucleotide polymer in which nucleotide units (monomers) are linked by covalent bonds to form a long chain, and is a polynucleotide fragment encoding the above-mentioned rhamnosidase or the above-mentioned rhamnosidase mutant.

[0034] The nucleic acid molecule can be used to construct a vector, which is introduced into a microorganism, and the microorganism is used to mass-produce rhamnosidase with an amino acid sequence as shown in SEQ ID NO: 1, so that the rhamnosidase can catalyze the conversion of epimedin C into icariin for application in industrial production.

[0035] The nucleic acid molecule can also be used to prepare a nucleic acid probe, which hybridizes with all or part of the complementary sequence of the above nucleic acid molecule under stringent conditions to detect whether the above nucleic acid molecule is effectively amplified in a microorganism.

[0036] Another aspect of the present application can provide a vector containing the above-mentioned nucleic acid molecule. The vector is loaded with the above-mentioned nucleic acid molecule and can be used to construct an engineered cell. The vector used in the present application is not particularly limited, and any vector known in the art can be used.

[0037] Another aspect of the present application may provide a microorganism that produces rhamnosidase, which contains one or more of the above-mentioned rhamnosidase, the above-mentioned rhamnosidase mutant, the above-mentioned nucleic acid molecule, and the above-mentioned vector.

[0038] The microorganism of the present application may be a microorganism expressing rhamnosidase or a mutant thereof whose amino acid sequence is shown in SEQ ID NO: 1, or a microorganism into which the above nucleic acid molecule or the above vector is introduced.

[0039] In the present application, the term "rhamnosidase-producing microorganism" refers to a microorganism that has the ability to produce rhamnosidase added to a parent strain that does not have the ability to produce rhamnosidase. Specifically, the microorganism is a microorganism that expresses rhamnosidase with an amino acid sequence as shown in SEQ ID NO: 1. For example, the microorganism is not limited to the RhaE1 strain screened in the present application.

[0040] Another aspect of the present application can provide the use of rhamnosidase in the production of icariin using rhamnosidase. Specifically, the present application provides a method for producing icariin using rhamnosidase: the above-mentioned rhamnosidase is contacted with epimedin C in a reaction system with a pH of 4.0 to 7.0 and a temperature of 40 to 60°C, and icariin is obtained after the reaction is completed.

[0041] In some embodiments of the present application, the concentration of epimedin C in the reaction system is 8 to 20 g / L, preferably 10 to 15 g / L, and more preferably 12 g / L.

[0042] In some embodiments of the present application, the concentration of rhamnosidase in the reaction system is 50 mL / L.

[0043] In some embodiments of the present application, the reaction system consists of the above-mentioned rhamnosidase, epimedin C and phosphate buffer.

[0044] In some embodiments of the present application, the reaction time is 2 to 3 hours.

[0045] Below, the present application will be explained in more detail by examples. However, these examples are for illustrative explanation of the present application, and the scope of the present application is not limited to these examples.

[0046] In the following examples, the activity of rhamnosidase (hereinafter referred to as enzyme activity) was detected by the following method: 0.0713 g of 4-nitrophenyl α-L-arabinofuranoside was accurately weighed to 0.0001 g, and the corresponding buffer solution was slowly added to nearly 50 mL, and magnetic stirring was performed for about 10 min. The corresponding pH value was adjusted with 2 mol / L citric acid or sodium hydroxide, and the volume was finally fixed to 50 mL, and the enzyme solution was diluted to an appropriate multiple with a sodium citrate buffer solution of pH 4.8, and the absorbance value was controlled in the range of 0.2-0.4.

[0047] Specifically, it includes drawing of standard curve, enzyme activity determination and calculation.

[0048] Accurately dilute the 5mmol / L p-nitrophenol solution 10 times, and then dilute it 2, 4, 6, 8, 10, 12, and 16 times respectively.

[0049] Take 0.5 mL of the above-mentioned p-nitrophenol dilution (take buffer solution for blank control), add 2 mL of sodium carbonate solution, add 0.5 mL of substrate solution, mix well, adjust to zero with blank control, and measure the absorbance value at 410 nm.

[0050] With the content of p-nitrophenol in the system as the horizontal axis (X) and the absorbance value as the vertical axis (y), a standard curve y=kx+b is drawn, and r2 is calculated to determine whether the curve fit is good.

[0051] Take an appropriate amount of substrate and preheat at 50℃ for 5min;

[0052] Take four 15*150 test tubes (one blank tube and three sample tubes), and accurately add 0.5mL of diluted enzyme solution to each of the four tubes; place the four test tubes in a 50±0.1℃ water bath at the same time and preheat for 2min; accurately add 0.5mL of substrate solution to the sample test tube and accurately time for 15min; quickly and accurately add 2.0mL of sodium carbonate solution to each tube, and accurately add 0.50mL of substrate solution to the blank tube and shake well.

[0053] Use a blank tube to zero and measure the absorbance of the sample solution in the three sample tubes at a wavelength of 410nm using a 10mm colorimetric cup. Take the average value. Calculate the content of p-nitrophenol by checking the standard curve or using the linear regression equation.

[0054] p-Nitrophenol is the product of rhamnosidase hydrolysis of 4-nitrophenyl α-L-arabinofuranoside. Its content is proportional to the enzyme activity. The enzyme activity is calculated using the following formula A=X×1 / 0.5×n / 15:

[0055] Where:

[0056] A——rhamnosidase activity / u·g -1 (or u·mL -1 );

[0057] X——the content of p-nitrophenol found (or calculated) by absorbance on the standard curve / μmol;

[0058] 1 / 0.5——the volume of enzyme solution added;

[0059] n——dilution multiple of enzyme sample;

[0060] 15——Time conversion factor.

[0061] Embodiment 1:

[0062] Step 1: Cultivation of starter Pichia pastoris RhaE1, induced expression of enzyme and enzyme activity determination

[0063] The Pichia RhaE1 purchased from Shaanxi Bokesiming Biotechnology Co., Ltd. was inoculated on a yeast extract powder peptone glucose agar (YPD) plate and cultured at 30°C for 2-3 days; a single colony was picked and inoculated in a test tube containing 1.5mL BMGY liquid culture medium (for Pichia), and after shaking culture at 30°C and 250rpm for 1 day, centrifuged, the upper culture medium was removed, and 200μL BMM culture medium (for Pichia) was added, and shaking culture was carried out at 30°C and 250rpm for 2 days, and methanol was added at 0.5% of the volume of the reaction system every day; then centrifuged, the lower layer was the bacteria, and the upper layer was the enzyme-containing supernatant, and the enzyme activity of rhamnosidase in the supernatant was measured.

[0064] Step 2: UV mutagenesis of bacteria

[0065] The bacteria were spread on a YPD plate and cultured at 30°C for 48 h. The bacteria spread on the YPD plate were then washed with sterile water to prepare a suspension. The suspension was diluted to a bacterial concentration of 1×10 6 / mL, irradiate with UV lamp (40W) for 2-10min, at a distance of about 22cm, the lethality rate reaches over 90%. Figure 2 shown.

[0066] Step 3: Cultivation of mutant bacteria

[0067] After multiple rounds of UV mutagenesis, each single colony was inoculated into a 96-deep-well plate containing 200 μL BMGY liquid culture medium (for Pichia pastoris), cultured at 30°C and 250 rpm for 1 day, centrifuged, the upper culture medium was removed, and 200 μL BMM culture medium (for Pichia pastoris) was added, and cultured at 30°C and 250 rpm for 2 days. Methanol was added at 0.5% of the volume of the reaction system every day to induce expression.

[0068] Step 4: Screening of mutant strains

[0069] After 2 days of induction expression, centrifugation is performed, the lower layer is the bacterial body, and the upper layer is the enzyme-containing supernatant. The enzyme activity of rhamnosidase in the supernatant is measured and recorded as the initial measurement result of the mutant strain. Using the starting strain Pichia pastoris RhaE1 as a control, the mutant strain with relatively improved rhamnosidase activity is screened out. These mutant strains are cultured according to the conditions of step 1 to increase their number, and induce them to express rhamnosidase, and the enzyme activity of the obtained rhamnosidase is measured respectively, and compared with the initial measurement result of the mutant strain, and the mutant strain with an error within a reasonable range is retained and named Pichia pastoris RhaE-F8.

[0070] Step 5: Comparison of rhamnosidase activity

[0071] The starting strain Pichia pastoris RhaE1 and the mutant strain Pichia pastoris RhaE-F8 were cultured according to the conditions of step 1, and the supernatants were collected and the rhamnosidase activity in the supernatants was determined. The results showed that the activity of rhamnosidase expressed by the mutant strain Pichia pastoris RhaE-F8 was 24% higher than that of rhamnosidase expressed by the starting strain Pichia pastoris RhaE1.

[0072] Step 6: Optimal pH of rhamnosidase obtained from Pichia pastoris RhaE-F8

[0073] The enzyme activity is different at different pH values. The present invention cultured the mutant strain Pichia pastoris RhaE-F8 according to the conditions of step one, and finally collected the supernatant. The supernatant and the crude extract of the reaction substrate epimedin C (0.1 g) were diluted and prepared with buffer solutions of pH 3, 3.5, 4, 5, 6, 6.5, 7, 8, and 8.5, respectively, and the rhamnosidase activity was determined at 50°C to calculate the enzyme activity. Control group: The inactivated rhamnosidase and the crude extract of the reaction substrate epimedin C (0.1 g) were diluted and prepared with buffer solutions of pH 3, 3.5, 4, 5, 6, 6.5, 7, 8, and 8.5, respectively, and the rhamnosidase activity was determined at 50°C as a control for enzyme activity calculation. The results are as follows: Figure 4 As shown, the optimum pH of the rhamnosidase recombinantly expressed by Pichia pastoris RhaE-F8 provided in the present application is 4.0-7.0.

[0074] Step 7: Optimal Action Temperature of Rhamnosidase from Pichia pastoris RhaE-F8

[0075] The present invention cultivates the mutant strain Pichia pastoris RhaE-F8 according to the conditions of step 1, collects the supernatant, dilutes the supernatant and the reaction substrate epimedin C crude extract (0.1 g) with a buffer solution of pH 5.0, and sets a series of temperature gradients: 20, 30, 40, 50, 60, 70° C., so as to measure the rhamnosidase activity at different temperatures.

[0076] Control group: The inactivated rhamnosidase and the reaction substrate, the crude extract of ophiopogonadine C (0.1 g), were diluted and prepared with a buffer solution of pH 5.0, and a series of temperature gradients were set: 20, 30, 40, 50, 60, 70°C to measure the rhamnosidase activity at different temperatures. Figure 5 As shown, the optimal temperature of rhamnosidase expressed by the mutant strain Pichia pastoris RhaE-F8 is 40°C.

[0077] Step 6: Sequencing the target gene of the mutant bacteria Pichia pastoris RhaE-F8, the following results were obtained: the amino acid sequence of rhamnosidase is shown in SEQ ID NO: 1, and the nucleotide sequence encoding rhamnosidase is shown in SEQ ID NO: 2.

[0078] Example 2: Application of the enzymes obtained from the starting strain Pichia pastoris RhaE1 and the mutant strain Pichia pastoris RhaE-F8 to hydrolyze epimedin C

[0079] (1) The starting strain Pichia pastoris RhaE1 and the mutant strain Pichia pastoris RhaE-F8 were inoculated on yeast extract peptone glucose agar (YPD) plates, respectively, and cultured at 30°C for 2-3 days; a single colony was picked and inoculated into a test tube containing 1.5 mL of BMGY liquid culture medium (for Pichia pastoris), and cultured at 30°C and 250 rpm for 1 day, followed by centrifugation, the upper culture medium was removed, and 200 μL of BMM culture medium (for Pichia pastoris) was added, and cultured at 30°C and 250 rpm for 2 days, with methanol added every day at 0.5% of the volume of the reaction system; and then centrifuged, with the lower layer being the bacterial cells and the upper layer being the enzyme-containing supernatant.

[0080] (2) The enzyme-containing supernatant of the starting strain Pichia pastoris RhaE1 and the enzyme-containing supernatant of the mutant strain Pichia pastoris RhaE-F8 obtained in step (1) were respectively prepared according to the following reaction system (5 mL):

[0081] Phosphate buffer pH 6.0 4.75mL

[0082] Epimedin C crude extract (0.1g) 0.1g

[0083] 0.25 mL of supernatant containing enzyme

[0084] RhaE1 enzyme reaction system and RhaE-F8 enzyme reaction system were obtained respectively, wherein the epimedin C crude extract (epimedin C content is 60wt%) was purchased from Nanjing Boyan Biotechnology Co., Ltd. with the item number 110642-44-9.

[0085] (3) The above reaction system was reacted at 40°C, and samples were taken from the two enzyme reaction systems every 0.5 h. After the reaction, the samples were diluted 10 times with methanol, centrifuged, and the supernatant was taken for HPLC detection. The results were as follows: Figure 6 As shown in the figure, epimedin C can be converted into icariin in both the RhaE1 enzyme reaction system and the RhaE-F8 enzyme reaction system. Figure 6 As shown in the figure, with the extension of reaction time, the conversion rate of epimedin C continued to increase. When the reaction time was 2h, the conversion rate of epimedin C in the RhaE1 enzyme reaction system reached 89%, and the conversion rate of epimedin C in the RhaE-F8 enzyme reaction system reached 93%, and the conversion rate of epimedin C was significantly improved. As shown in Table 1, at 0.5h, the conversion rate of epimedin C in the RhaE-F8 enzyme reaction system was as high as 93.9%, and at 3h of reaction, the yield of icariin could reach 95.6%.

[0086] Table 1 Concentrations of epimedin C and icariin at different time points in the RhaE-F8 enzyme reaction system

[0087]

[0088]

[0089] Among them, m1 represents the initial mass of epimedin C; m2 represents the residual mass of epimedin C; m3 represents the mass of generated icariin; M1 represents the relative molecular mass of epimedin C; M2 represents the relative molecular mass of icariin.

[0090] Example 3: Application of enzymes obtained from the starting strain Pichia pastoris RhaE1 and the mutant strain Pichia pastoris RhaE-F8 to hydrolyze rutin

[0091] (1) The starting strain Pichia pastoris RhaE1 and the mutant strain Pichia pastoris RhaE-F8 were inoculated on yeast extract peptone glucose agar (YPD) plates, respectively, and cultured at 30°C for 2-3 days; a single colony was picked and inoculated into a test tube containing 1.5 mL of BMGY liquid culture medium (for Pichia pastoris), and cultured at 30°C and 250 rpm for 1 day, followed by centrifugation, the upper culture medium was removed, and 200 μL of BMM culture medium (for Pichia pastoris) was added, and cultured at 30°C and 250 rpm for 2 days, with methanol added every day at 0.5% of the volume of the reaction system; and then centrifuged, with the lower layer being the bacterial cells and the upper layer being the enzyme-containing supernatant.

[0092] (2) The enzyme-containing supernatant of the starting strain Pichia pastoris RhaE1 and the enzyme-containing supernatant of the mutant strain Pichia pastoris RhaE-F8 obtained in step (1) were respectively prepared according to the following reaction system (5 mL):

[0093] Phosphate buffer pH 6.0 4.5mL

[0094] Rutin crude extract 1mL

[0095] 500 μL of enzyme-containing supernatant

[0096] RhaE1 enzyme reaction system and RhaE-F8 enzyme reaction system were obtained respectively, wherein the crude rutin extract was purchased from Nanjing Boyan Biotechnology Co., Ltd. with the product number 153-18-4, and the concentration of rutin was 8 g / L.

[0097] (3) The above reaction system was reacted in a water bath at 40°C. Samples were taken from the two enzyme reaction systems every 0.5 h. After the reaction, the enzyme reaction system was diluted 10 times with methanol, centrifuged, and the supernatant was taken for HPLC detection. The results were as follows: Figure 7As shown in the figure, in both the RhaE1 enzyme reaction system and the RhaE-F8 enzyme reaction system, rutin can be hydrolyzed to remove rhamnose to generate the product quercetin. When the reaction time is 3h, the conversion rate of rutin in the RhaE-F8 enzyme reaction system is increased by 68% compared with the RhaE1 enzyme reaction system, greatly improving the conversion efficiency of rutin to quercetin.

[0098] Table 2 Conversion rate of rutin at different time points in two enzyme reaction systems

[0099] Time (min) 0.5 1 1.5 2 2.5 3 3.5 RhaE-F8(%) 19.8 35.8 45 60.5 85.4 90.6 93.4 RhaE1(%) 7.3 9.8 15.1 17.5 20.4 22.7 25.3

[0100] The above description is only a specific implementation of the present application, so that those skilled in the art can understand or implement the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown herein, but will conform to the widest range consistent with the principles and novel features applied for herein.

Claims

1. A rhamnosidase, characterized in that: Its amino acid sequence is shown in SEQ ID NO:

1.

2. A rhamnosidase mutant, characterized in that: The rhamnosidase mutant has a sequence homology of more than 80% and less than 100% with the amino acid sequence of SEQ ID NO:

1.

3. A nucleic acid molecule, characterized in that It comprises a nucleic acid sequence capable of encoding the rhamnosidase according to claim 1 or the rhamnosidase mutant according to claim 2.

4. A carrier, characterized in that: It contains the nucleic acid molecule according to claim 3.

5. A microorganism producing rhamnosidase, characterized in that: The microorganism contains one or more of the rhamnosidase according to claim 1, the rhamnosidase mutant according to claim 2, the nucleic acid molecule according to claim 3, and the vector according to claim 4.

6. Use of the rhamnosidase according to claim 1 or the rhamnosidase mutant according to claim 2 in producing icariin using rhamnosidase.

7. A method for producing icariin using rhamnosidase, characterized in that: The rhamnosidase according to claim 1 is contacted with epimedin C in a reaction system with a pH of 4.0 to 7.0 and a temperature of 40 to 60° C. After the reaction is completed, the icariin is purified to obtain icariin.

8. The method for producing icariin using rhamnosidase according to claim 7, characterized in that: The concentration of epimedin C in the reaction system is 8-20 g / L.

9. The method for producing icariin using rhamnosidase according to claim 7, characterized in that: The reaction time is 2 to 3 hours.

10. Use of the rhamnosidase according to claim 1 or the rhamnosidase mutant according to claim 2 in producing quercetin using rutin.

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