Alpha-L-rhamnosidase, coding gene thereof and application of alpha-L-rhamnosidase and coding gene

Abstract

The invention belongs to the technical field of enzyme engineering and gene engineering, and discloses alpha-L-rhamnosidase and a coding gene and application thereof, and the alpha-L-rhamnosidase has an amino acid sequence as shown in SEQ ID NO.1; one or more amino acids are substituted, deleted or added on the basis of the amino acid sequence as shown in SEQ ID NO.1, and the enzyme activity of the amino acid sequence is unchanged and the amino acid sequence is derived from the SEQ ID NO.1. The alpha-L-rhamnosidase can catalytically hydrolyze alpha-L-rhamnoside in astilbin molecules so as to directionally synthesize taxifolin, meanwhile, the enzymolysis method has the advantages of being mild in condition, single in reaction product and environmentally friendly, astilbin can be efficiently catalyzed to be converted into taxifolin, and the method is suitable for industrial production. The preparation of taxifolin is more economical, practical and efficient, industrial production of high-purity taxifolin is facilitated, a foundation is laid for application of large-scale industrial biosynthesis of taxifolin in the future, and the method has very important significance for promoting wide application of taxifolin in industries such as medicine and food.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering and enzyme engineering, and in particular to an alpha-L-rhamnosidase and a coding gene thereof and applications thereof. Background Art

[0002] Taxifolin, also known as dihydroquercetin, is a dihydroflavonoid compound commonly found in pine plants and fruits and vegetables. Taxifolin exists in plants in two forms: aglycone and glycoside. Its molecular formula is C 15 H 12 O7, with a relative molecular mass of 304.25, is light yellow or colorless needle-shaped crystals, has no special smell, a melting point of 240°C, is easily soluble in solvents such as ethanol, acetic acid, and boiling water, and is slightly soluble in cold water. It has biological activities and pharmacological effects such as antioxidant, anti-inflammatory, anti-cancer, and antibacterial. It has certain therapeutic effects on chronic diseases such as atherosclerosis, dyslipidemia, and cardiovascular diseases, and is mainly used in the fields of food and medicine. Taxifolin is present in high levels in Larix olgensis, but its resources are precious. It exists in the fruit of Polygonum multiflorum and Pinus massoniana, which are rich in resources, but its content is low. The content of astilbin, the precursor of Taxifolin, is high in the leaves of Lycopodiella vulgaris. Therefore, we can extract, purify, and separate astilbin from Lycopodiella vulgaris leaves, and then perform enzyme-catalyzed reaction on astilbin to obtain Taxifolin.

[0003] At present, most of the hydrolysis of flavonoid glycoside bonds at home and abroad adopts chemical methods, but chemical hydrolysis reactions have many side reactions, resulting in complex components of hydrolysis products, which brings difficulties to subsequent purification. Enzymatic hydrolysis has the characteristics of high specificity and selectivity, and also has the advantages of no pollution, strong purpose, and high conversion rate. It is precisely these advantages that biological enzymatic hydrolysis has been favored by many researchers.

[0004] However, there is no report on the preparation of taxifolin by hydrolyzing astilbin with α-L-rhamnosidase. Summary of the invention

[0005] The invention aims to overcome the disadvantages of side reactions caused by traditional chemical hydrolysis of flavonoid glycoside bonds, high requirements on equipment, pollution to the environment, etc., and provide an alpha-L-rhamnosidase and a coding gene thereof and applications thereof. The alpha-L-rhamnosidase can catalyze the hydrolysis of alpha-L-rhamnoside in astilbin molecules to synthesize taxifolin in a directional manner. Meanwhile, the enzymolysis method has the advantages of mild conditions, relatively single reaction products and environmental friendliness, can efficiently catalyze the conversion of astilbin into taxifolin, makes the preparation of taxifolin more economical and efficient, is easy to industrially produce high-purity taxifolin, lays a foundation for the application of large-scale industrial biosynthesis of taxifolin in the future, and has very important significance for promoting its wide application in industries such as medicine and food.

[0006] In order to achieve the above-mentioned object, in a first aspect, the present invention provides an α-L-rhamnosidase, which has an amino acid sequence as shown in SEQ ID NO.1; or has an amino acid sequence derived from SEQ ID NO.1 by replacing, deleting or adding one or more amino acids on the basis of the amino acid sequence as shown in SEQ ID NO.1 and having unchanged enzyme activity.

[0007] In a second aspect, the present invention provides a gene capable of encoding the α-L-rhamnosidase described in the first aspect.

[0008] In a third aspect, the present invention provides a recombinant expression vector containing the gene described in the second aspect.

[0009] In a fourth aspect, the present invention provides a recombinant strain containing the recombinant expression vector described in the third aspect.

[0010] In a fifth aspect, the present invention provides a method for preparing α-L-rhamnosidase, the method comprising the following steps: (1) culturing the recombinant strain described in the fourth aspect to express the gene encoding α-L-rhamnosidase; and (2) isolating and purifying the expressed α-L-rhamnosidase.

[0011] In a sixth aspect, the present invention provides an enzyme preparation for catalyzing the hydrolysis of astilbin to direct the biosynthesis of taxifolin, wherein the enzyme preparation contains the α-L-rhamnosidase described in the first aspect as an active ingredient.

[0012] In the seventh aspect, the present invention provides the use of the α-L-rhamnosidase described in the first aspect, the gene described in the second aspect, the recombinant expression vector described in the third aspect, the recombinant strain described in the fourth aspect, and the enzyme preparation described in the sixth aspect that catalyzes the hydrolysis of astilbin to direct the biosynthesis of taxifolin in the preparation of taxifolin.

[0013] In the above technical scheme, this patent firstly uses α-L-rhamnosidase to catalyze the hydrolysis of α-L-rhamnoside in astilbin molecule, and directs the biosynthesis of taxifolin. The inventors analyzed the structures of astilbin and taxifolin and found that the difference between astilbin and taxifolin lies in one α-L-rhamnoside. α-L-rhamnosidase is used to hydrolyze the α-L-pyranose rhamnose on astilbin, and the bond breaking position is α-1,3 glycosidic bond, producing taxifolin with higher value.

[0014] At the same time, the enzymatic hydrolysis method has the advantages of mild conditions, relatively simple reaction products, and environmental friendliness. It is easy to industrially produce high-purity Taxifolin, laying the foundation for the future large-scale industrial biosynthesis of Taxifolin, and is of great significance for promoting its widespread application in the pharmaceutical, food and other industries.

[0015] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the following specific embodiments, they are used to explain the present invention but do not constitute a limitation of the present invention. In the accompanying drawings:

[0017] Figure 1 is the plasmid map of the recombinant plasmid pPIC9K-Rha of the present invention;

[0018] Figure 2 SDS-PAGE electrophoresis analysis of α-L-rhamnosidase of the present invention;

[0019] Figure 3 It is a schematic diagram of the reaction process of the α-L-rhamnosidase catalyzing the directed biosynthesis of taxifolin into astilbin of the present invention;

[0020] Figure 4 This is a high performance liquid chromatography standard product image of astilbin detected by HPLC;

[0021] Figure 5 This is a high performance liquid chromatography standard image of Taxifolin detected by HPLC;

[0022] Figure 6 The high performance liquid chromatogram of taxifolin, a product of the directed biosynthesis of astilbin catalyzed by α-L-rhamnosidase of the present invention. DETAILED DESCRIPTION

[0023] The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0024] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0025] In the present invention, unless otherwise specified, the term "enzyme activity" used refers to the amount of enzyme used, which is expressed in enzyme activity units, i.e., enzyme units (U). The definition of enzyme units in the present invention is: in 50 mmol / L citric acid-phosphate buffer (pH 6.0) at 60°C, the amount of enzyme required to release 1 μmol of p-nitrophenol (pNP) per minute is 1 enzyme activity unit.

[0026] In the first aspect, the present invention provides an α-L-rhamnosidase, which has an amino acid sequence as shown in SEQ ID NO.1; or an amino acid sequence derived from SEQ ID NO.1, in which one or more amino acids are substituted, deleted or added on the basis of the amino acid sequence as shown in SEQ ID NO.1 and the enzyme activity remains unchanged.

[0027] SEQ ID NO.1

[0028] MWSSWLLSALLATEALAVPYEEYILAPSSRDLAPASVRQVNGSVTNAAALTGAGGQATFNGVSSVTYDFGINVAGIVSVDVASASSESAFIGVTFTESSMWISNEACDATQDAGLDTPLWFAVGQGAGVYSVGKKYTRGAFRYMTVVSNTTATVSLNSVKINY TASPIQDLRAYTGYFHSSDELLNRIWYAGAYTLQLCSIDPTTGDALVGLGAITSSETITLPQTDKWWTNYTITNGSSTLTDGAKRDRLVWPGDMSIALESVAVSTEDLYSVRTALESLYALQKADGQLPYAGKPFYDTVSFTYHLHSLVGAASYYQYTGDRAWL TRYWGQYKKGVQWALSGVDSTGLANITASADWLRFGMGAHNIEANAILYYVLNDAISLAQSLNDNAPIRNWTATAARIKTVANELLWDDKNGLYTDNETTTLHPQDGNSWAVKANLTLSANQSAIISESLAARWGPYGAPAPEAGATVSPFIGGFELQAHYQAG QPDRALDLLRLQWGFMLDDPRMTNSTFIEGYSTDGSLVYAPYTNRPRVSHAHGWSTGPTSALTIYTAGLRVTGPAGATWLYKPQPGNLTQVEAGFSTRLGSFASSFSRSGGRYQELSFTTPNGTTGSVELGDVSGQLVSEGGVKVQLVGGKASGLQGGKWRLNV

[0029] Herein, the enzyme activity remains unchanged means that under the same assay conditions, the percentage (relative activity) between the enzyme activity of the α-L-rhamnosidase having the amino acid sequence derived from SEQ ID NO.1 and the enzyme activity of the α-L-rhamnosidase having the amino acid sequence shown in SEQ ID NO.1 is not less than 95% (or 96%, or 97%, or 98%, or 99%, or 100%).

[0030] The 20 amino acid residues that make up proteins can be divided into four categories according to the polarity of their side chains: 1. Non-polar amino acids: alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met), phenylalanine (Phe), tryptophan (Trp) and proline (Pro); 2. Polar uncharged amino acids: glycine (Gly), serine (Ser), threonine (Thr), cysteine ​​(Cys), aspartic acid (Asn), glutamine (Gln) and tyrosine (Tyr); 3. Positively charged amino acids: arginine (Arg), lysine (Lys) and histidine (His); 4. Negatively charged amino acids: aspartic acid (Asp) and glutamic acid (Glu) (see "Biochemistry" (Second Edition), Volume 1, Shen Tong, Wang Jingyan, pages 82-83, Higher Education Press, December 1990). If amino acid residues belonging to the same category are substituted in a protein, such as Arg replacing Lys or Leu replacing Ile, the role of the residue in the protein domain (such as providing positive charge or forming a hydrophobic pocket structure) does not change, so the three-dimensional structure of the protein is not affected, and the function of the protein can still be achieved. The amino acid residue substitution belonging to the same category can occur at any amino acid residue position of the above-mentioned α-L-rhamnosidase.

[0031] As mentioned above, the α-L-rhamnosidase provided by the present invention can also be modified or mutated to obtain a derived protein. The "derived protein" described in the present invention refers to an α-L-rhamnosidase having the above-mentioned amino acid sequence, which has a difference in amino acid sequence, and may also have a difference in the form of modification that does not affect the sequence, or both. These proteins include natural or induced genetic variants. The induced variants can be obtained by various techniques, such as random mutations produced by radiation or mutagens, or by techniques such as site-directed mutagenesis or other known molecular biology techniques. The "derived protein" also includes analogs with residues of natural L-type amino acids (such as D-type amino acids), and analogs with non-naturally occurring or synthetic amino acids (such as β-amino acids, γ-amino acids, etc.).

[0032] Modifications (usually without changing the primary structure, i.e., without changing the amino acid sequence) include: chemical derivatization of proteins in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those produced by glycosylation modification during protein synthesis and processing or in further processing steps. This modification can be accomplished by exposing the protein to a glycosylation enzyme (such as a mammalian glycosylase or deglycosylation enzyme). Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). Also included are proteins that have been modified to improve their resistance to proteolysis or optimize their solubility.

[0033] In order to facilitate purification, the amino acid sequence of α-L-rhamnosidase can also be modified by using common tags in the art. For example, it can be obtained by connecting the tags shown in Table 1 below (such as at least one of Poly-Arg, Poly-His, FLAG, Strep-tagⅡ and c-myc) to the amino terminus and / or carboxyl terminus of the amino acid sequence of α-L-rhamnosidase. The tag is, for example, at least one of Poly-Arg, Poly-His, FLAG, Strep-tagⅡ and c-myc. The tag will not affect the activity of the β-glucosidase mutant of the present invention. In actual application, it can be selected whether to add a tag according to needs.

[0034] Table 1

[0035]

[0036]

[0037] The above-mentioned α-L-rhamnosidase can be obtained by artificial synthesis, or its encoding gene can be synthesized first and then obtained by biological expression.

[0038] In a second aspect, the present invention provides a gene capable of encoding the α-L-rhamnosidase described in the first aspect.

[0039] It is well known in the art that among the 20 different amino acids constituting proteins, except for Met (ATG) or Trp (TGG) which are respectively encoded by a single codon, the other 18 amino acids are respectively encoded by 2-6 codons (Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, second edition, 1989, see Appendix D on page 950). That is, due to the degeneracy of the genetic code, there are mostly more than one codons that determine an amino acid, and the substitution of the third nucleotide in the triplet codon often does not change the composition of the amino acid, so the nucleotide sequence of the gene encoding the same protein can be different. According to the known codon table, those skilled in the art can completely deduce the nucleotide sequence of the gene that can encode them from the amino acid sequence disclosed in the present invention, and the amino acid sequence with unchanged α-L-rhamnosidase activity obtained from the amino acid sequence, and obtain the nucleotide sequence by biological methods (such as PCR method, mutation method) or chemical synthesis method, so this part of the nucleotide sequence should be included in the scope of the present invention. On the contrary, using the DNA sequence disclosed herein, it is also possible to obtain an amino acid sequence consistent with the α-L-rhamnosidase activity of the present invention by modifying the nucleic acid sequence provided by the present invention by methods known in the art, such as the method of Sambrook et al. (Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, Second Edition, 1989).

[0040] Preferably, the gene has the nucleotide sequence shown in SEQ ID NO.2.

[0041] SEQ ID NO.2

[0042]

[0043] As described above, correspondingly, the 5' end and / or 3' end of the nucleotide sequence may also be connected to a coding sequence of a tag as shown in Table 1 above.

[0044] The nucleotide sequence provided by the present invention can usually be obtained by polymerase chain reaction (PCR) amplification, recombination, or artificial synthesis. For example, those skilled in the art can easily obtain templates and primers based on the nucleotide sequence provided by the present invention, and amplify the relevant sequence using PCR.

[0045] Once the relevant nucleotide sequence is obtained, the relevant amino acid sequence can be obtained in large quantities by recombinant methods. The obtained nucleotide sequence is usually cloned into a vector, then transferred into genetically engineered bacteria, and then the relevant nucleotide sequence is separated from the proliferated host cells by conventional methods.

[0046] In addition, the relevant nucleotide sequences can also be synthesized by known artificial chemical synthesis methods.

[0047] In a third aspect, the recombinant expression vector provided by the present invention contains the gene provided by the present invention.

[0048] The "vector" used in the recombinant expression vector can be selected from various vectors known in the art, such as various commercially available plasmids, cosmids, phages and retroviruses, such as pPIC9K plasmid or pPICZα plasmid.

[0049] The recombinant expression vector can be constructed by using various nucleases that have cleavage sites in the vector multiple cloning site to obtain a linear plasmid, which is then connected to a gene fragment cleaved by the same nuclease to obtain a recombinant plasmid.

[0050] In a fourth aspect, the recombinant strain provided by the present invention contains the recombinant expression vector provided by the present invention.

[0051] The recombinant expression vector can be transformed, transduced or transfected into a host cell (strain) by conventional methods in the art, such as chemical transformation using the calcium chloride method, high-voltage electroporation transformation, and the like.

[0052] The host cell may be a prokaryotic cell or a eukaryotic cell, preferably a eukaryotic cell, and more preferably a yeast, such as Pichia pastoris GS115 or KM71, and the optimal expression host is GS115.

[0053] The present invention provides a method for preparing α-L-rhamnosidase, which comprises the following steps:

[0054] (1) culturing the recombinant strain described in the fourth aspect to express the gene encoding α-L-rhamnosidase;

[0055] (2) Isolate and purify the expressed α-L-rhamnosidase.

[0056] The culture conditions are conventional culture conditions, and the specific methods are conventional technical means of those skilled in the art, which will not be described in detail here.

[0057] In a sixth aspect, the present invention provides an enzyme preparation for catalyzing the hydrolysis of astilbin to direct the biosynthesis of taxifolin, wherein the enzyme preparation contains the α-L-rhamnosidase described in the first aspect as an active ingredient.

[0058] According to a preferred embodiment of the present invention, the content of the α-L-rhamnosidase can be 10-90% by weight based on the total weight of the enzyme preparation. The enzyme preparation can also contain solvents known to those skilled in the art (such as glycerol, sugars and protein protectants such as protease inhibitors).

[0059] In the seventh aspect, the present invention provides the use of the α-L-rhamnosidase described in the first aspect, the gene described in the second aspect, the recombinant expression vector described in the third aspect, the recombinant strain described in the fourth aspect, and the enzyme preparation that catalyzes the hydrolysis of astilbin to direct the biosynthesis of taxifolin described in the sixth aspect in the preparation of phloretin.

[0060] In the present invention, the method of using the α-L-rhamnosidase to catalyze the hydrolysis of astilbin to direct the biosynthesis of taxifolin may include: contacting an astilbin solution with the α-L-rhamnosidase and catalyzing according to conventional enzyme catalysis methods in the art.

[0061] Wherein, the concentration of the astilbin solution is 2 mg / mL;

[0062] The added amount of the α-L-rhamnosidase is 36U / mL;

[0063] The pH of the enzymatic hydrolysis system was 5.0, the reaction temperature was 40°C, and the reaction time was 60 h.

[0064] The present invention will be described in detail below through examples, but the protection scope of the present invention is not limited thereto.

[0065] (1) Methods and equipment used in the examples:

[0066] The experimental methods and equipment described in the following examples are conventional methods and equipment unless otherwise specified, and the drugs and pharmaceuticals are conventional commercial products.

[0067] (2) Culture medium, bacteria and enzymes used in the examples:

[0068] YPD medium: 1% yeast powder, 2% peptone, 2% glucose;

[0069] BMGY liquid medium: 1 g peptone, 0.5 g yeast powder, 2% glycerol, 10×YNB, biotin;

[0070] ·α-L-rhamnosidase was synthesized directly by Silicon Biotech.

[0071] (3) Solution used in the embodiment:

[0072] Wash Buffer: Tris-Hcl (1M Ph 8.0), Nacl (4M), imidazole (1M), DTT;

[0073] Elution Buffer: Tris-HCl (1M Ph 8.0), NaCl (4M), imidazole (1M), DTT.

[0074] (4) Sequence

[0075] ·The related genes of α-L-rhamnosidase were found from Aspergillus niger JMU-TS528 through gene mining.

[0076] The primer sequences used are shown in Table 2:

[0077] Table 2 Primer sequences

[0078]

[0079]

[0080] (5) Reaction system and reaction conditions

[0081] ·The PCR reaction system and reaction conditions are shown in Table 3 and Table 4 below:

[0082] The reaction system of Table 3 PCR amplification is 50 μL, and the proportions of each component are as follows:

[0083] Prime STAR Max 25μL Upstream primer F1 (20 μmol / L) 2.5μL Downstream primer R1 (20 μmol / L) 2.5μL Genome 2.5μL <![CDATA[ddH2O]]> 17.5μL Total volume 50μL

[0084] Table 4 PCR reaction conditions

[0085]

[0086] Example 1

[0087] This example is used to illustrate the acquisition of α-L-rhamnosidase:

[0088] The related genes of α-L-rhamnosidase were found from Aspergillus niger JMU-TS528 by gene mining, and then potential enzyme molecules were selected by using systematic bioinformatics and molecular simulation technology, and the related α-L-rhamnosidase encoding gene was designed and synthesized. The nucleotide sequence of the encoding gene is shown in SEQ ID NO.2.

[0089] Example 2

[0090] This example is used to illustrate the acquisition of recombinant α-L rhamnosidase:

[0091] Using the sequence obtained in Example 1 as a template, PrimeSTAR polymerase inverse PCR was used to amplify the vector portion without the α-factor signal peptide. The α-L-rhamnosidase encoding gene was amplified by polymerase chain reaction (PCR) using the primers in Table 2, wherein the primers contain 15-20 bp homologous sequences consistent with the pPIC9K vector sequence without the α-factor signal peptide at the 3' and 5' ends, respectively;

[0092] The α-L-rhamnosidase encoding gene obtained above was homologously recombined with the vector, and the ligation product was transformed into Escherichia coli JM109. The engineered bacteria were linearized and then electroporated into Pichia pastoris GS115. The high copy number was selected by G418 resistance. The screened single colony was inoculated on the MD screening plate for three generations to 4 mg / ml, inoculated in 5 ml YPD medium, and cultured at a temperature of 30°C and a rotation speed of 200 rpm for 24 hours. 1 ml of the bacterial solution in the YPD medium was inoculated in 50 ml BMGY liquid medium, and cultured at a temperature of 30°C and a rotation speed of 200 rpm to about OD600 = 1; the bacterial solution was transferred to a 50 ml centrifuge tube, centrifuged at 5000 rpm for 10 min to collect the bacteria, added BMMY liquid medium to resuspend the bacteria, and transferred to 50 ml In BMMY liquid culture medium, culture is carried out continuously for 5 days at a temperature of 30°C and a rotation speed of 200 rpm. Methanol is added every 24 hours. The amount of methanol added is 2% (v / v) of the total volume of the BMMY liquid culture medium. The supernatant obtained after centrifugation at 8000 rpm for 10 minutes is the crude enzyme solution of α-L-rhamnosidase Rha.

[0093] Purify α-L-rhamnosidase. First, combine the crude α-L-rhamnosidase solution obtained above with a nickel column at 100 rpm for 90 min, elute impurities with Wash Buffer, and elute the target protein with Elution Buffer to obtain pure α-L-rhamnosidase.

[0094] Example 3

[0095] This example is used to illustrate the method for determining the enzyme activity of α-L-rhamnosidase of the present invention:

[0096] The activity of α-L-rhamnosidase was determined using 50 mmol / L pNPR as substrate.

[0097] In 50mmol / L citric acid-phosphate buffer (pH 6.0), at 60℃, the amount of enzyme required to release 1μmol p NP per minute was defined as 1 unit of enzyme activity. After 40μL p NPR and 150μL buffer were incubated at 60℃ for 2min, 10μL enzyme solution was added, and the reaction was continued at 60℃ for 5min. 200μL of 1mol / L sodium carbonate solution was quickly added to terminate the reaction. The enzyme solution inactivated by boiling for 10min was used as a blank control and the absorbance was measured at 405nm.

[0098] Example 4

[0099] This example is used to illustrate the application of the α-L-rhamnosidase provided by the present invention in catalyzing the hydrolysis of astilbin to direct the biosynthesis of taxifolin

[0100] The pure α-L-rhamnosidase obtained in Example 2 was reacted with 2 g / L of astilbin at an enzyme dosage of 36 U. Due to the low solubility of astilbin, DMSO solvent was added to promote dissolution. The reaction temperature was 40°C and the reaction time was 50 h. Samples were taken every 10 h and the reaction was terminated with three times the volume of methanol. The reaction was then filtered through a 0.22 μm organic membrane and the conversion rate of astilbin was detected using a high performance liquid chromatography column method (HPLC).

[0101] The specific detection method is:

[0102] The contents of astilbin and taxifolin in the above catalytic reaction were simultaneously detected by high performance liquid chromatography column method, and the conversion rate of astilbin was calculated.

[0103] Chromatographic column conditions: C18 (5μm, 4.6mmx250mm),

[0104] Mobile phase: methanol and 0.1% formic acid aqueous solution, mixed in a volume ratio of 48:52, isocratic elution, flow rate 0.8 ml / min;

[0105] Detection wavelength: 290 nm, injection volume: 10 μL.

[0106] Depend on Figure 4 From the HPLC standard product graph of astilbin detected by HPLC, it can be seen that the peak time of astilbin standard product; Figure 5 From the HPLC standard product graph of Taxifolin detected by HPLC, it can be seen that the peak time of Taxifolin standard product; Figure 6The HPLC chromatogram of the directional biosynthesis product of astilbin catalyzed by α-L-rhamnosidase of the present invention is shown in FIG. Figure 4-5 As shown, it can be seen that the α-L-rhamnosidase of the present invention can catalyze the directed biosynthesis product of astilbin to taxifolin, and under the condition that the mass concentration of astilbin is 2 g / L, the reaction is carried out for 33 h, and the conversion rate of the astilbin substrate is as high as 90%, indicating that the α-L-rhamnosidase of the present invention can efficiently convert astilbin into taxifolin.

[0107] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0108] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0109] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. An α-L-rhamnosidase, characterized in that The α-L-rhamnosidase has the amino acid sequence shown in SEQ ID NO.1; or has an amino acid sequence derived from SEQ ID NO.1 with one or more amino acids substituted, deleted or added on the basis of the amino acid sequence shown in SEQ ID NO.1 and with unchanged enzyme activity.

2. A gene capable of encoding the α-L-rhamnosidase according to claim 1.

3. The gene according to claim 2, wherein The gene has a nucleotide sequence shown in SEQ ID NO.

2.

4. A recombinant expression vector, characterized in that: The recombinant expression vector contains the gene according to claim 2 or 3.

5. A recombinant strain, characterized in that: The recombinant strain contains the recombinant expression vector according to claim 4.

6. The recombinant strain according to claim 5, characterized in that The strain is a eukaryotic cell.

7. The recombinant strain according to claim 5, characterized in that The strain is Pichia pastoris GS115 or KM71.

8. A method for preparing α-L-rhamnosidase, characterized in that: The method comprises the following steps: (1) culturing the recombinant strain according to any one of claims 5 to 7 to express a gene encoding α-L-rhamnosidase; (2) Isolate and purify the expressed α-L-rhamnosidase.

9. An enzyme preparation for catalyzing the hydrolysis of astilbin to direct the biosynthesis of taxifolin, characterized in that: The enzyme preparation contains the α-L-rhamnosidase according to claim 1 as an active ingredient.

10. Use of the α-L-rhamnosidase according to claim 1, the gene according to claim 2 or 3, the recombinant expression vector according to claim 4, the recombinant strain according to any one of claims 5 to 7, and the enzyme preparation for catalyzing the hydrolysis of astilbin to direct biosynthesis of taxifolin according to claim 9 in the preparation of taxifolin.

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