A rhamnose-based hydrolase mutant, its preparation method and application
By site-directed mutagenesis of TpeRha enzyme, a rhamnosyl hydrolase mutant was constructed, which solved the problem of insufficient α-L-rhamnosidase activity and achieved efficient preparation of hesperidin-7-O-glucoside, improving enzymatic hydrolysis efficiency and application range.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- FOSHAN GOLDEN HEALTH TECH CO LTD
- Filing Date
- 2022-11-29
- Publication Date
- 2026-05-08
AI Technical Summary
Existing α-L-rhamnosidases have insufficient activity when applied on a large scale, and the reaction environment is difficult to meet the optimal conditions for natural enzymes, resulting in low efficiency in the preparation of hesperidin-7-O-glucoside.
By site-directedly mutating lysine at position 513 of the TpeRha enzyme to a hydrophobic amino acid, such as alanine, tryptophan, or valine, a rhamnosyl hydrolase mutant was constructed, its catalytic activity pocket was optimized, and the hydrolysis efficiency of the enzyme was improved.
The mutants K513A and K513V have increased hydrolysis efficiency by 1.1 times and 1.2 times, respectively, shortening the enzymatic hydrolysis time. They can efficiently catalyze the conversion of hesperidin to hesperidin-7-O-glucoside and have a wide range of applications.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, and in particular to a rhamnosyl hydrolase mutant, its preparation method, and its application. Background Technology
[0002] Hesperidin-7-O-glucoside, with the molecular formula C 22 H 24 O 11 Hesperidin-7-O-glucoside is one of the components of citrus flavonoids. In recent years, research on hesperidin-7-O-glucoside has become more in-depth. The results show that hesperidin-7-O-glucoside has a shorter absorption time and higher bioavailability than hesperidin and neohesperidin. Moreover, after entering the animal body, hesperidin-7-O-glucoside also has prebiotic properties, which can promote the growth of beneficial intestinal bacteria, increase the content of short-chain fatty acids, a metabolite of the intestinal flora, and play a practical role in combating enteritis and solving intestinal problems.
[0003] Hesperidin-7-O-glucoside is typically formed from hesperidin or neohesperidin by removing one mole of rhamnose via α-L-rhamnosidase. α-L-rhamnosidase can cleave the glycosidic bond formed by the reaction of an alcohol hydroxyl group and a hemiacetal via exo- or endo-cleavage, thereby efficiently and directionally hydrolyzing natural active substances containing rhamnoside, such as hesperidin. However, significant technical barriers remain for the large-scale application of α-L-rhamnosidase. Natural α-L-rhamnosidase has insufficient activity, and its reaction environment often fails to meet the optimal conditions for natural enzymes. Summary of the Invention
[0004] To overcome the shortcomings of the prior art, the first objective of this invention is to provide a rhamnosyl hydrolase mutant that can solve the problem of insufficient rhamnosylase activity.
[0005] A rhamnosyl hydrolase mutant, wherein the rhamnosyl hydrolase mutant is formed by mutating the lysine at position 513 of the TpeRha enzyme, as shown in SEQ ID NO: 2, to an amino acid with a hydrophobic side chain.
[0006] Furthermore, the amino acid with the hydrophobic side chain is one of alanine, tryptophan, phenylalanine, or valine.
[0007] Further, the lysine at position 513 of the TpeRha enzyme is mutated to alanine to obtain mutant K513A, the amino acid sequence of which is shown in SEQ ID NO: 4; the lysine at position 513 of the TpeRha enzyme is mutated to valine to obtain mutant K513V, the amino acid sequence of which is shown in SEQ ID NO: 6.
[0008] Furthermore, the nucleotide sequence encoding the mutant K513A gene is shown in SEQ ID NO: 3; the nucleotide sequence encoding the mutant K513V gene is shown in SEQ ID NO: 5.
[0009] Furthermore, the TpeRha enzyme is derived from *Thermophyton floccosum* DSM 13995.
[0010] The second objective of this invention is to provide a method for preparing a rhamnosyl hydrolase mutant, which can solve the problem of preparing high-activity rhamnosylase.
[0011] A method for preparing a rhamnosyl hydrolase mutant includes the following steps:
[0012] Step 1: Ligate the gene encoding the TpeRha enzyme to be mutated into a plasmid to obtain a recombinant plasmid;
[0013] Step 2: Design and use site-directed mutagenesis primers to amplify the recombinant plasmid as a template, and obtain the mutant product after enzyme digestion; the site-directed mutagenesis primers mutate the lysine at position 513 of the TpeRha enzyme;
[0014] Step 3: Transform the mutant product into host cells, screen and induce host cells to express the rhamnosyl hydrolase mutant.
[0015] Further, the nucleotide sequence of the encoding gene of the TpeRha enzyme is shown in SEQ ID NO: 1; in step 2, the lysine at position 513 of the TpeRha enzyme is mutated to alanine using upstream primer K513A-F and downstream primer K513A-R, the nucleotide sequence of upstream primer K513A-F is shown in SEQ ID NO: 9, and the nucleotide sequence of downstream primer K513A-R is shown in SEQ ID NO: 10; the lysine at position 513 of the TpeRha enzyme is mutated to valine using upstream primer K513V-F and downstream primer K513V-R, the nucleotide sequence of upstream primer K513V-F is shown in SEQ ID NO: 17, and the nucleotide sequence of downstream primer K513V-R is shown in SEQ ID NO: 18.
[0016] Furthermore, the host cell is any one of Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris.
[0017] The third objective of this invention is to provide an application of a rhamnosyl hydrolase mutant that can solve the problem of preparing hesperidin-7-O-glucoside.
[0018] Application of a rhamnosyl hydrolase mutant, wherein the rhamnosyl hydrolase mutant is used to prepare a combination of one or both of hesperidin-7-O-glucoside or hesperidin.
[0019] Furthermore, the application involves the catalytic conversion of hesperidin to hesperidin-7-O-glucoside or a combination of one or both of hesperidin via a rhamnoside mutant.
[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0021] 1. The rhamnosyl hydrolase mutants K513A and K513V of the present invention can promote the hydrolysis of hesperidin, effectively improving the efficiency of hesperidin to hesperidin-7-O-glucoside. The mutants have shorter branched chains, which does not affect the binding of hydrophobic amino acids to the substrate. The hydrolysis efficiency is 1.1 times and 1.2 times that of the wild type, respectively, and the enzymatic hydrolysis time is shorter.
[0022] 2. The method for preparing the rhamnosyl hydrolase mutant of the present invention uses computer-aided protein engineering enzyme molecular design to analyze the active pocket region of α-L-rhamnosylase TpeRha derived from Thermoplasia petroleum, designs it molecularly, and then constructs a special enzyme through site-directed mutagenesis. The preparation method is precise and effective.
[0023] 3. The rhamnosyl hydrolase mutant of the present invention can catalyze the conversion of hesperidin to prepare compositions containing hesperidin-7-O-glucoside in different ranges, and has a wide range of applications. Attached Figure Description
[0024] Figure 1 This is a simulation diagram of the docking of TpeRha enzyme with hesperidin molecules;
[0025] Figure 2 Figure showing the hydrolysis efficiency of wild-type TpeRha and mutants in the conversion of hesperidin to hesperidin-7-O-glucoside.
[0026] Figure 3 This is an HPLC result of the reaction solution after whole-cell catalysis. Detailed Implementation
[0027] The technical solution of the present invention will now be clearly and completely described with reference to specific embodiments. Obviously, the described embodiments are merely some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.
[0028] Orange peel, a traditional medicinal resource, contains abundant flavonoids, which are the primary substances responsible for its antibacterial, antiviral, and antioxidant activities. Currently, over 60 flavonoids have been effectively isolated from orange peel, among which neohesperidin and hesperidin are relatively common. Neohesperidin and hesperidin have wide applications, including but not limited to inhibiting osteoporosis, lowering blood sugar and lipids and improving glucose tolerance, antioxidant effects, cardiovascular protection, promoting gastrointestinal digestion, antibacterial, antiviral, and antitumor effects. However, the absorption rate of neohesperidin and hesperidin in the human body is lower than that of hesperidin-7-O-glucoside, possibly because they require hydrolysis and metabolism by intestinal flora before they can be utilized by the body.
[0029] Hesperidin-7-O-glucoside, with the molecular formula C 22 H 24 O 11 Recent research results indicate that hesperidin-7-O-glucoside has a shorter absorption time and higher bioavailability than hesperidin and neohesperidin. Furthermore, after entering the animal body, hesperidin-7-O-glucoside also possesses prebiotic properties, promoting the growth of beneficial intestinal bacteria, increasing the content of short-chain fatty acids, a metabolic product of the gut microbiota, and playing a practical role in combating enteritis and addressing intestinal problems.
[0030] Hesperidin-7-O-glucoside is typically formed from hesperidin or neohesperidin by the removal of one rhamnose moiety from α-L-rhamnosidase. α-L-rhamnosidase can cleave glycosidic bonds formed by the reaction of an alcohol hydroxyl group and a hemiacetal via exo- or endo-cleavage, thereby efficiently and directionally hydrolyzing naturally occurring active substances containing rhamnoside, such as hesperidin. However, significant technical barriers remain for the large-scale application of α-L-rhamnosidase. Natural α-L-rhamnosidases have insufficient activity, and their reaction environments often fail to meet the optimal conditions for natural enzymes. With the increasing maturity of molecular biology, genetic engineering, and other related technologies, editing and modifying protein sequences can enhance the understanding of enzyme catalytic mechanisms and guide enzyme design. Therefore, this study aims to provide a highly active rhamnosyl hydrolase mutant to address the problem of insufficient rhamnosidase activity.
[0031] A rhamnosyl hydrolase mutant, wherein the rhamnosyl hydrolase mutant is formed by mutating the lysine at position 513 of the TpeRha enzyme, as shown in SEQ ID NO: 2, to an amino acid with a hydrophobic side chain.
[0032] Furthermore, the amino acid with the hydrophobic side chain is one of alanine, tryptophan, phenylalanine, or valine.
[0033] The mutation site of the rhamnosidase was first determined by simulating the three-dimensional structure of the wild-type rhamnosidase using computational biology, and then docking it with the substrate. Through docking simulation, the conformational relationship between the enzyme and substrate can be analyzed, sites related to the catalytic reaction can be quickly located, the mutant library can be reduced, and relevant targets can be efficiently obtained. Using this site-directed mutagenesis technique, based on the understanding of the structure-function relationship of proteins, proteins can be purposefully designed and modified to increase the hydrophobic attraction of the mutant enzyme's active pocket to the substrate hesperidin, thereby significantly improving its hydrolytic performance.
[0034] Furthermore, the lysine at position 513 of the TpeRha enzyme was mutated to alanine to obtain mutant K513A, the amino acid sequence of which is shown in SEQ ID NO: 4; the lysine at position 513 of the TpeRha enzyme was mutated to valine to obtain mutant K513V, the amino acid sequence of which is shown in SEQ ID NO: 6. After mutation at position 513 of the TpeRha enzyme, the hydrolysis efficiency was improved compared to the wild-type rhamnosidase.
[0035] Furthermore, the nucleotide sequence encoding the mutant K513A gene is shown in SEQ ID NO: 3; the nucleotide sequence encoding the mutant K513V gene is shown in SEQ ID NO: 5.
[0036] Furthermore, the TpeRha enzyme is derived from *Thermophyton floccosum* DSM 13995.
[0037] The second objective of this invention is to provide a method for preparing a rhamnosyl hydrolase mutant, which can solve the problem of preparing high-activity rhamnosylase.
[0038] A method for preparing a rhamnosyl hydrolase mutant includes the following steps:
[0039] Step 1: Ligate the gene encoding the TpeRha enzyme to be mutated into a plasmid to obtain a recombinant plasmid;
[0040] Step 2: Design and use site-directed mutagenesis primers to amplify the recombinant plasmid as a template, and obtain the mutant product after enzyme digestion; the site-directed mutagenesis primers mutate the lysine at position 513 of the TpeRha enzyme;
[0041] Step 3: Transform the mutant product into host cells, screen and induce host cells to express the rhamnosyl hydrolase mutant.
[0042] Further, the nucleotide sequence of the encoding gene for the TpeRha enzyme is shown in SEQ ID NO: 1; in step 2, the lysine at position 513 of the TpeRha enzyme is mutated to alanine using upstream primer K513A-F and downstream primer K513A-R, the nucleotide sequence of upstream primer K513A-F is shown in SEQ ID NO: 9, and the nucleotide sequence of downstream primer K513A-R is shown in SEQ ID NO: 10; the lysine at position 513 of the TpeRha enzyme is mutated to valine using upstream primer K513V-F and downstream primer K513V-R, the nucleotide sequence of upstream primer K513V-F is shown in SEQ ID NO: 17, and the nucleotide sequence of downstream primer K513V-R is shown in SEQ ID NO: 18. Further, the host cell is any one of *Escherichia coli*, *Saccharomyces cerevisiae*, and *Pichia pastoris*.
[0043] The third objective of this invention is to provide an application of a rhamnosyl hydrolase mutant that can solve the problem of preparing hesperidin-7-O-glucoside.
[0044] Application of a rhamnosyl hydrolase mutant, wherein the rhamnosyl hydrolase mutant is used to prepare a combination of one or both of hesperidin-7-O-glucoside or hesperidin.
[0045] Furthermore, the application involves the catalytic conversion of hesperidin to hesperidin-7-O-glucoside or a combination of one or both of hesperidin via a rhamnoside mutant.
[0046] The present invention will now be further described with reference to the accompanying drawings and specific embodiments. The following examples illustrate experimental methods with specific conditions, which are generally performed under standard experimental conditions or as recommended by the manufacturer. Unless otherwise specified, all reaction reagents involved in the examples can be purchased commercially. Molecular biology experimental methods not specifically described in these examples can be referred to *Molecular Cloning: A Laboratory Manual*.
[0047] Example 1: TpeRha enzyme tertiary structure model and molecular docking with hesperidin
[0048] The constructed three-dimensional structural model of the TpeRha enzyme was molecularly docked with the substrate hesperidin, and the results are as follows: Figure 1 As shown, the rhamnose of hesperidin is located in the active pocket of the TpeRha enzyme, while the rest of the hesperidin structure, excluding the rhamnose, is located on the enzyme surface. The remaining structure of hesperidin exhibits significant steric hindrance with three nearby amino acids: histidine at position 130, lysine at position 513, and arginine at position 514. Preferably, these three amino acid residues are mutated to alanine, and their importance is investigated by testing the activity of the mutant enzyme.
[0049] Example 2: TpeRha enzyme to be mutated and plasmid
[0050] The codon preference optimization of the α-L-rhamnosinase gene of *Thermoplasticum petroleum* DSM 13995 in *Escherichia coli* was performed as follows: The α-L-rhamnosinase gene with the nucleotide sequence shown in SEQ ID NO: 1 was ligated into the pET-28a plasmid to obtain the recombinant plasmid pET-28a-TpeRha; this plasmid was transformed into *Escherichia coli* BL21(DE3), and the recombinant strain was named BL21(DE3) / pET-28a-TpeRha. The amino acid sequence of the TpeRha enzyme expressed by this recombinant enzyme strain is shown in SEQ ID NO: 2.
[0051] Example 3: Obtaining the TpeRha mutant strain
[0052] 1. Construction of mutant enzyme vectors via whole plasmid PCR
[0053] Step 1: Extract a small amount of the recombinant plasmid pET-28a-TpeRha;
[0054] Step 2: Design mutation primers. The primers consist of a 15bp overlap region and a 15bp extension region, with the mutation sites designed in the overlap region. The mutation sites are His130, Lys513, and Arg514, respectively.
[0055] Step 3: Perform full plasmid PCR amplification using plasmid pET-28a-TpeRha as a template. The PCR system is shown in Table 1.
[0056] Table 1. PCR amplification system of the whole plasmid
[0057]
[0058] The primers Primer-F and Primer-R are upstream and downstream PCR primers designed according to different mutation sites. Specific primer information is shown in Table 2.
[0059] Table 2 Primers for whole plasmid PCR amplification
[0060]
[0061] Primer pairs H130A-F and H130A-R were used to obtain the mutant enzyme H130A; primer pairs R514A-F and R514A-R were used to obtain the mutant enzyme R514A.
[0062] Primer pairs K513A-F and K513A-R were used to obtain the mutant enzyme K513A, whose amino acid sequence is SEQ ID NO: 4.
[0063] PCR amplification program: pre-denaturation 98℃ for 3 min; cycling settings: denaturation 98℃ for 10 s, annealing 62℃ for 15 s, extension 72℃ for 3 min, 30 cycles; final extension 72℃ for 10 min; after the reaction, the PCR products were recovered using the kit.
[0064] Step 4: Remove template DNA by enzyme digestion. The recovered PCR product is then digested with enzymes. The enzyme digestion system is shown in Table 3.
[0065] Table 3 Enzyme digestion system for whole plasmid PCR products
[0066] QuickCut DpnⅠ 1μL PCR products ≤1μg 10×QuickCut Buffer 3μL ddH2O Add to 30μL
[0067] The above enzyme digestion system was placed in a metal bath at 37°C for 1 hour for digestion. After the reaction, the enzyme digestion products were recovered using a kit.
[0068] 2. Sequencing to verify whether the mutant enzyme strain was successfully constructed.
[0069] The enzyme digestion products were transformed into Escherichia coli BL21(DE3) competent cells and cultured overnight at 37°C with the cells inverted. Positive transformants were selected for sequencing verification, and mutant enzyme expression strains BL21(DE3) / pET-28a-H130A, BL21(DE3) / pET-28a-K513A and BL21(DE3) / pET-28a-R514A were successfully obtained.
[0070] Example 4 Whole-cell catalytic reaction of TpeRha mutant enzyme
[0071] 1. Induction of TpeRha recombinant strain and preparation of whole-cell enzyme solution by mutant enzyme recombinant strain
[0072] Step 1: Take BL21(DE3) / pET-28a-TpeRha and BL21(DE3).
[0073] Strains / pET-28a-H130A, BL21(DE3) / pET-28a-K513A, and BL21(DE3) / pET-28a-R514A were activated by streaking on LB agar plates containing Kan (100 μg / mL). After overnight incubation at 37°C with inverted incubation, single colonies were picked and inoculated into 5 mL of LB liquid medium containing Kan, and cultured at 37°C with shaking at 200 rpm for 12–16 h. The overnight seed culture was then inoculated into 20 mL of fresh TB liquid medium containing Kan at a 1% inoculation rate, and cultured at 37°C with shaking at 200 rpm for 2–3 h until the OD600 reached 0.6–0.8. IPTG was then added to a final concentration of 0.5 mM, and the culture was incubated at 37°C with shaking at 200 rpm for 16 h to induce protein expression.
[0074] Step 2: After protein induction, collect the bacterial cells by centrifugation at 8000 rpm for 8 min at 4℃. Wash the bacterial cells once with citrate-phosphate buffer (pH 4.6), and then resuspend the bacterial cells in an appropriate amount of citrate-phosphate buffer (pH 4.6) according to the wet weight of the bacterial cells to obtain a cell suspension with a total cell concentration of 200 mg / mL. All operations are performed on ice or at 4℃.
[0075] 2. Whole-cell reaction
[0076] The whole-cell suspensions of wild-type TpeRha enzyme and mutant H130A, K513A, and R514A enzymes obtained in the above experiments were used as enzyme solutions, and the reaction systems shown in Table 4 were prepared:
[0077] Table 4 Whole-cell reaction solution system
[0078] Whole cell enzyme solution 50mg / mL Hesperidin solution (using DMSO as solvent) 1mg / mL pH 4.6 citrate-phosphate buffer Add to 500μL
[0079] Step 2: The whole-cell reaction system was reacted at 55℃ for 1 hour. After the reaction was completed, 2 volumes of DMSO were added to terminate the reaction. After thorough vortexing, the cells were removed by centrifugation at 12000 rpm for 1 minute. The reaction product was filtered through a 0.45 μm organic filter and then analyzed by HPLC.
[0080] 3. Detection method for hesperidin-7-O-glucoside
[0081] Quantitative analysis of hesperidin-7-O-glucoside was performed using HPLC, and the chromatographic conditions are as follows:
[0082] High Performance Liquid Chromatography (HPLC): Agilent 1100 Series
[0083] Chromatographic column: C18 (250mm×4.6mm×5μm)
[0084] Detector: VWD detector, detection wavelength 282nm
[0085] Mobile phase ratio and elution conditions: flow rate 1 mL / min; column temperature 30℃; injection volume 10 μL; gradient elution system as shown in Table 5:
[0086] Table 5 HPLC gradient elution system
[0087]
[0088]
[0089] 4. Results Analysis
[0090] The results of the whole-cell catalytic reaction are as follows Figure 2 and Figure 3 As shown, under reaction conditions of 55℃ and pH 4.6, after 1 h of reaction, the hydrolysis efficiency of hesperidin-7-O-glucoside produced using the wild-type TpeRha enzyme was 8.29%. However, the hydrolysis efficiency of hesperidin-7-O-glucoside produced using the mutant H130A and R514A enzymes was lower than that of the wild-type TpeRha enzyme, decreasing to 4.98% and 6.62%, respectively. Compared to the histidine site at position 130 and the arginine site at position 514, the hydrolysis efficiency of hesperidin-7-O-glucoside produced using the mutant K513A enzyme increased to 19.85%, which is 2.39 times higher than that of the wild-type enzyme.
[0091] Example 5 Construction of hydrophobic re-evolution mutant and its whole-cell catalysis
[0092] 1. Construction of hydrophobic re-evolution mutants
[0093] Based on the results of Example 4, the hydrolysis efficiency of substrate amino acids was improved after the lysine position at position 513 of the TpeRha enzyme was mutated to alanine with a hydrophobic side chain. Therefore, the lysine position at position 513 was mutated to other amino acids with similar hydrophobic side chains, such as tryptophan, phenylalanine, and valine, to increase the hydrophobic attraction of the active pocket to the substrate hesperidin. The steps for constructing the mutant and the whole-cell catalytic steps in Examples 3 and 4, and the primers used are shown in Table 6 below.
[0094] Table 6. Primers used to obtain the hydrophobic TpeRha evolutionary mutant enzyme
[0095]
[0096]
[0097] Primer pairs K513W-F and K513W-R were used to obtain the mutant enzyme K513W.
[0098] Primer pairs K513F-F and K513F-R were used to obtain the mutant enzyme K513F.
[0099] Primer pairs K513V-F and K513V-R were used to obtain the mutant enzyme K513V, whose amino acid sequence is SEQ ID NO: 6.
[0100] 2. Results of whole-cell hydrophobic re-evolution
[0101] Under reaction conditions of 55℃ and pH 4.6, with a reaction time of 1 hour, the results of the whole-cell catalytic reaction are as follows: Figure 2As shown. Using mutant K513W, K513F, and K513V enzymes, the hydrolysis efficiencies of the product hesperidin-7-O-glucoside were 13.46%, 14.62%, and 51.96%, respectively, which were 1.62, 1.76, and 6.27 times higher than those of the wild type.
[0102] Example 6: Small-scale reaction of hesperidin monoglucoside
[0103] Based on the hydrophobic TpeRha enzyme mutation results in Example 5, the mutant enzyme strain K513V was selected as the fermentation strain. Referring to the strain induction and whole-cell preparation methods in Example 4, the reaction system ratio was adjusted to a 1:20 concentration ratio of hesperidin solution (using DMSO as solvent) to whole-cell enzyme solution, and the reaction time was extended to 20 h. Its hydrolysis efficiency and the reaction catalyzed by whole-cell catalysis were also tested by HPLC. The small-scale results are as follows: Figure 2 and Figure 3 As shown in the figure, the hydrolysis efficiency of mutant K513V reached as high as 98.52% after increasing the enzyme solution and extending the reaction time.
[0104] The above description is merely a preferred embodiment of the present invention. Those skilled in the art can make various improvements and modifications based on the technical solutions and concepts described above, without departing from the principles of the present invention. These improvements and modifications should also be considered within the scope of protection of the present invention, and all such changes should fall within the scope of protection of the claims of the present invention.
Claims
1. A rhamnosyl hydrolase mutant, characterized in that, The rhamnosyl hydrolase mutant is obtained by mutating lysine at position 513 of the TpeRha enzyme, as shown in SEQ ID NO: 2, to any one of tryptophan, phenylalanine, or valine.
2. The rhamnosyl hydrolase mutant as described in claim 1, characterized in that, The TpeRha enzyme has a 513 lysine residue mutated to valine (K513V), and its amino acid sequence is shown in SEQ ID NO:
6.
3. The rhamnosyl hydrolase mutant as described in claim 2, characterized in that, The nucleotide sequence of the gene encoding the K513V is shown in SEQ ID NO:
5.
4. The rhamnosyl hydrolase mutant according to any one of claims 1-3, characterized in that, The TpeRha enzyme is derived from *Thermophyton fume* (…). Thermotoga petrophila DSM 13995.
5. A method for preparing a rhamnosyl hydrolase mutant as described in any one of claims 1-4, characterized in that, Includes the following steps: (1) The TpeRha enzyme encoding gene to be mutated was ligated into a plasmid vector to obtain a recombinant plasmid; (2) Design site-directed mutagenesis primers, use the recombinant plasmid as a template for amplification, and obtain a mutant product containing the mutation site by enzyme digestion; wherein, the site-directed mutagenesis primers are used to mutate the 513th lysine of TpeRha enzyme; (3) The mutant product is transformed into the host cell, screened and induced to express, to obtain the rhamnosyl hydrolase mutant.
6. The preparation method according to claim 5, characterized in that, The nucleotide sequence of the TpeRha enzyme encoding gene is shown in SEQ ID NO: 1; in step (2), the lysine at position 513 of the TpeRha enzyme is mutated to valine using upstream primer K513V-F and downstream primer K513V-R, wherein the nucleotide sequence of upstream primer K513V-F is shown in SEQ ID NO: 17 and the nucleotide sequence of downstream primer K513V-R is shown in SEQ ID NO:
18.
7. The preparation method according to claim 5 or 6, characterized in that, The host cell is any one of Escherichia coli, Saccharomyces cerevisiae, or Pichia pastoris.
8. The application of a rhamnosyl hydrolase mutant as described in any one of claims 1-4, characterized in that, The rhamnosyl hydrolase mutant is used to prepare compositions containing hesperidin-7-O-glucoside and / or hesperidin.
9. The application as described in claim 8, characterized in that, The application involves using the rhamnosyl hydrolase mutant to catalyze the conversion of hesperidin to generate hesperidin-7-O-glucoside and / or hesperidin.