An α-rhamnosidase mutant and its application

Abstract

This invention discloses an α-rhamnosylase mutant and its applications, belonging to the field of microbial engineering technology. The α-rhamnosylase mutant disclosed in this invention has the amino acid sequence shown in SEQ ID NO.2, which consists of a total of 268 amino acid residue point mutations and two frameshift mutations adding 14 amino acid residues based on the parental amino acid sequence of α-rhamnosylase SEQ ID NO.1. Results show that the parental α-rhamnosylase and the α-rhamnosylase IgRha M24-5 mutant enzyme have conversion rates of 72.84% and 99.76% for astragalus root C, respectively; and conversion rates of 90.33% and 99.81% for rutin, respectively. This indicates that the α-rhamnosylase mutant has good application prospects in the selective biotransformation of flavonoid glycosides to prepare derhamnosylflavones under high-temperature conditions.

Description

Technical Field

[0001] This invention belongs to the field of microbial engineering technology, and more specifically, relates to an α-rhamnosidase mutant and its application. Background Technology

[0002] Flavonoids are a class of polyphenolic secondary metabolites widely found in plants. The variety of flavonoids is extremely rich, and they can be classified into flavones, isoflavones, flavanols, flavonols, chalcones, anthocyanins, and others. Furthermore, those with a glycosyl group are called flavonoid glycosides, while those without are called flavonoid aglycones. Due to their unique chemical structure, flavonoids exhibit a wide range of pharmacological activities, including antioxidant, antitumor, anticancer, antibacterial, antiviral, and immunomodulatory activities, showing great potential for application in the pharmaceutical and food industries. However, the unique molecular structure of flavonoids leads to poor solubility, significantly limiting their bioavailability. In particular, the type and number of glycosides significantly affect their biological activity. Taking rutin as an example, its selective de-terminal rhamnose product, quercetin-3-glucoside (isoquercetin), has been shown to exhibit anti-proliferative activity against various cancer cells, including colon cancer, breast cancer, hepatocellular carcinoma, and lung cancer. Tests on the anti-proliferative activity of rutin and isoquercetin revealed that isoquercetin possesses significantly greater anti-proliferative activity than rutin. Therefore, structural modification to alter its solubility and improve its bioavailability, enabling its widespread application in food, medicine, and other fields, has become a research hotspot.

[0003] α-Rhamnosidases selectively hydrolyze non-reducing α-rhamnoside bonds, releasing L-rhamnose. Currently, over four thousand rhamnosidases from different sources have been isolated, identified, and cloned; however, only two strains, derived from *Aspergillus niger* and *Penicillium* respectively, are widely used commercially. The current problems and limitations of α-rhamnosidase applications are analyzed as follows: 1) Fungal rhamnosidases are easy to purify, but most are crude enzyme solutions after fermentation. These solutions contain not only α-rhamnosidases but also large amounts of β-glucosidases, β-galactosidases, endomannosidases, and other glycoside hydrolases with different functions. If β-glucosidases are not removed, byproducts are unavoidable, leading to a decrease in the yield of the target product. 2) Most reported rhamnosidases have low activity, making them unsuitable for large-scale production. Furthermore, the fungal fermentation process has many influencing factors, hindering production control. (3) At present, most rhamnosidases from different sources are mesophilic enzymes with low reaction temperatures. In particular, the solubility of flavonoids in hot water is significantly higher than that in cold water. Therefore, when the temperature of the catalytic system is increased to convert some natural products with low solubility, most enzymes cannot maintain high thermal stability under high temperature conditions, which affects production efficiency.

[0004] In conclusion, discovering heat-resistant α-rhamnosidases or modifying their structures to improve enzyme activity and stability, and achieving selective and efficient conversion of flavonoid glycosides such as rutin under high-temperature conditions, is of great significance for realizing the widespread application of flavonoids in multiple fields. Summary of the Invention

[0005] To address the aforementioned problems in the existing technology, the technical problem to be solved by the present invention is to provide an α-rhamnosidase mutant. Another technical problem to be solved by the present invention is to provide applications of the α-rhamnosidase mutant for regulating the selective conversion efficiency of flavonoids.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0007] An α-rhamnosylase mutant, the amino acid sequence of which is shown in SEQ ID NO.2.

[0008] The amino acid sequence of the α-rhamnosylase mutant is based on the parental amino acid sequence SEQ ID NO.1 of α-rhamnosylase, with a total of 268 amino acid residue point mutations and 2 frameshift mutations that add 14 amino acid residues.

[0009] The recombinant strain E. coli-IgRha M24-5 containing the α-rhamnosidase mutant.

[0010] Application of α-rhamnosidase mutants in regulating the selective conversion efficiency of flavonoids.

[0011] Application of α-rhamnosidase mutants in regulating the selective conversion efficiency of flavonoid glycosides.

[0012] Application of α-rhamnosidase mutant in the selective conversion of icariin C to produce icariin.

[0013] Application of α-rhamnosidase mutant in the selective conversion of rutin to prepare isoquercitrin.

[0014] Application of recombinant strain E. coli-IgRha M24-5 containing α-rhamnosidase mutant in the selective transformation of icariin C to prepare icariin.

[0015] Application of recombinant strain E. coli-IgRha M24-5 containing an α-rhamnosidase mutant in the selective transformation of rutin to prepare isoquercetin.

[0016] The application of the recombinant strain E. coli-IgRha M24-5 containing the α-rhamnosidase mutant includes the following steps:

[0017] 1) Prepare recombinant plasmids containing α-rhamnosidase mutants;

[0018] 2) The recombinant plasmid was transformed into Escherichia coli to obtain an α-rhamnosidase mutant strain;

[0019] 3) Cultivate and screen recombinant strain E. coli-IgRha M24-5 that improves the selective transformation efficiency of icariin C or rutin for the preparation of icariin or isoquercitrin.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] The α-rhamnosylase mutant disclosed in this invention has the amino acid sequence shown in SEQ ID NO.2. It consists of a total of 268 amino acid residue point mutations and two frameshift mutations that add 14 amino acid residues based on the parental α-rhamnosylase amino acid sequence SEQ ID NO.1. Results show that the α-rhamnosyl glycoside parent enzyme and the α-rhamnosyl glycoside IgRhaM24-5 mutant enzyme have conversion rates of 72.84% and 99.76% for astragalus root extract, respectively; and conversion rates of 90.33% and 99.81% for rutin, respectively. At the same addition amount, the mutant enzyme IgRhaM24-5 can almost completely convert a high concentration of flavonoid glycosides (10 g / L) to the derhamnosylated product at 95 °C. Compared with the parent enzyme, the mutant enzyme exhibits excellent thermostability and catalytic activity at 95 °C. The α-rhamnosidase mutant can better catalyze the conversion of rutin and asparagine C into derhamnosylflavonoid isoquercitrin and icariin, respectively, and shows a higher conversion rate at 100℃. This indicates that the α-rhamnosidase mutant has good application prospects in the selective biotransformation of flavonoid glycosides to prepare derhamnosylflavonoids under high temperature conditions. Attached Figure Description

[0022] Figure 1 A comparison of the optimal reaction temperatures of the rhamnosinase mutant IgRha M24-5 and the parent enzyme;

[0023] Figure 2 A comparison of the temperature stability of the rhamnosinase mutant IgRha M24-5 and the parent enzyme;

[0024] Figure 3 The biotransformation pathways and enzymatic reaction routes of rutin and astragaloside C are shown in the diagrams (a. the products and reaction pathways obtained by biotransformation of astragaloside C by rhamnosidase, glucosidase or a combination of the two enzymes; b. the products and reaction pathways obtained by biotransformation of rutin by rhamnosidase, glucosidase or a combination of the two enzymes).

[0025] Figure 4The HPLC chromatograms show the conversion of α-rhamnosidase mutant IgaRhaM24-5 and parental enzyme into icariin C products (STD is the standard substrate icariin C, icariin and icariin I; reaction time is 60 min).

[0026] Figure 5 The HPLC chromatograms show the products obtained by converting rutin with the α-rhamnosidase mutant IgaRhaM24-5 and the parent enzyme, respectively (STD is the standard substrate rutin and isoquercitrin; reaction time is 60 min). Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described below with reference to specific embodiments. Unless otherwise described in detail, the technical means used in the following embodiments are all conventional means well known to those skilled in the art.

[0028] Example 1

[0029] 1. Recombinant expression of parental IgRha

[0030] The α-rhamnosaccharidase IgRha (NCBI accession number MEM1526583.1) from the thermophilic archaea (Ignisphaera sp.) was used as the parent, and its amino acid sequence is shown in SEQ ID NO.1. The gene sequence of the artificially synthesized α-rhamnosaccharidase IgRha was inserted into the multiple cloning site (NcoI / XhoI) of the pET-28a vector to obtain pET-28a-IgRha; pET-28a-IgRha was transformed into Escherichia coli BL21(DE3) competent cells and placed in a shaker at 37℃ and 180 rpm for 60 min for rejuvenation; after rejuvenation, the bacterial culture was plated on LB agar plates containing 50 μg / mL kanamycin and incubated at 37℃. Invert the plate and incubate overnight; pick a single colony from the plate and inoculate it into 30 mL of LB medium (250 mL Erlenmeyer flask) containing 50 μg / mL kanamycin antibiotic. Incubate at 37°C and 220 rpm for 12 hours without adding an inducer; collect the bacterial cells at 12000 rpm, 4°C, and 10 min. Wash the collected bacterial cells twice with 1×PBS buffer solution. Use the washed bacterial cells (wet cells) directly for screening or store them in a -80°C freezer for later use.

[0031] 2. Construction of α-rhamnosidase mutant

[0032] Using pET-28a-IgRha plasmid as a template, error-prone PCR primers were designed. The primer sequences are shown below:

[0033] F1: 5'-AATTTTGTTTAACTTTAAGAAGGAGATATACCATG-3',

[0034] F2: 5'-AGAGGATGGGCTGGCTAGGAGATGCATGGCTGAG-3';

[0035] F3: 5'-ACTCTTTGGGAAAGGTGGGAGAAAACTCACCGGC-3',

[0036] R1: 5'-ACTCAGCCATGCATCTCCTAGCCAGCCCAT CCTCTC-3';

[0037] R2: 5'-ACTGCCGGTGAGTTTCTCCCACCTTTCCCAAAGAGT-3',

[0038] R3: 5'-CCGGATCTCAGTGGTGGTGGTGGTGGTGCTCGAGT-3'.

[0039] The PCR reaction system consisted of: 1 ng template, 1.5 μL each of upstream and downstream segmented PCR primers (10 μM), 5 μL of 2×PCR Buffer, 7 mmol / L MgCl2, 0.5 mmol / L MnCl2, and finally, sterile distilled water was added to bring the volume to 50 μL.

[0040] The PCR reaction program was as follows: pre-denaturation at 94℃ for 2 min; denaturation at 94℃ for 20 s; annealing at 60 s according to the mutant primers; extension at 60℃ for 3 min; 28 cycles; extension at 60℃ for 10 min; and storage of the PCR product at 12℃.

[0041] After purification and recovery of the PCR product by 0.8% agarose gel electrophoresis, it was digested with NcoI and XhoI, and then mixed with the pET-28a plasmid fragment purified by 0.8% agarose gel electrophoresis. T4 ligase was added, and the mixture was reacted at 16°C for 4-16 hours. The resulting inoculum was then transformed into *E. coli* TOP10F' competent cells. The recovered bacterial culture was plated on LB agar plates containing 50 μg / mL kanamycin and incubated overnight at 37°C. Transformation efficiency was verified by selecting 1-50 transformants based on the number of transformants.

[0042] Based on the results of the previous round of transformant screening, positive mutant transformants with significantly improved transformation performance (using sterile toothpicks to pick up E. coli colonies and adding them to the error-prone PCR reaction system) were selected as templates. The same method was used to perform error-prone PCR on the basis of one or more transformants from the previous round, so as to obtain mutants with more mutation points.

[0043] 3. Screening of positive transformants of α-rhamnosidase mutants

[0044] Using rutin as a screening substrate, the efficiency of α-rhamnosidase mutants in selectively converting rutin to isoquercetin at different temperatures was determined. The specific method is as follows:

[0045] Fifty single colonies of transformants were randomly selected from the plate and added to a centrifuge tube (10 mL) containing 5 mL of LB liquid medium. The culture was carried out at 37°C and 220 rpm without the addition of an inducer for 12 hours. Then, 50 μL of rutin substrate was added to a final concentration of 1 g / L, and the mixture was incubated at 85-100°C for 1 hour. 500 μL of the transformation product was mixed with 500 μL of methanol and centrifuged at 12000 rpm for 5 minutes at 4°C. The mixture was then filtered through a 0.22 μm organic filter. The content was determined by HPLC. The substrate conversion rate (%) was calculated as: (molar concentration of isoquercitrin in the product / initial molar concentration of rutin in the substrate) × 100%.

[0046] The specific screening process is as follows:

[0047] Taking level A screening as an example, using the parental gene IgRha as a template, after obtaining mutant transformants through the previous error-prone PCR step, rutin is used as the screening substrate. The mutant transformants are reacted at 85℃ for 1 hour, and the transformation products are analyzed by HPLC. Screening is performed with a rutin conversion rate ≥50%, yielding first-round PCR positive transformants 1-27 and 1-39 (where, for example, 1-27 refers to the 27th transformant in the first round of PCR). If the number of positive transformants obtained from the first round of error-prone PCR is less than 5, the process continues using the parental gene... Using IgRha as a template, a second round of error-prone PCR is performed and screened to obtain second-round positive transformants 2-17 (where, for example, 2-17 refers to the 27th transformant in the second round of error-prone PCR; the same applies below); no positive transformants meeting the screening criteria are found in the third round, and then positive transformants 4-9 are obtained in the fourth round; positive transformants 5-41 and 5-28 are obtained in the fifth round; when the total number of positive transformants obtained from the first-level error-prone PCR screening is ≥5, the next level of screening is performed, and the positive transformants obtained in this level are used as the template for the next round of error-prone PCR.

[0048] Taking level b screening as an example, using the six positive transformants (1-27, 1-39; 2-17; 4-9; 5-41, 5-28) obtained from level a screening as templates, a sixth round of error-prone PCR was performed. Rutin was used as the screening substrate. After reacting the mutant transformants at 85℃ for 1 hour, the transformation products were analyzed by HPLC. Screening was conducted under the condition that the rutin conversion rate was ≥80%, and the sixth round of positive transformants 6-20, 6-22, and 6-44 were obtained. A seventh round of error-prone PCR was then performed, and positive transformants 7-14 and 7-33 were obtained. When the total number of positive transformants obtained from the level b screening error-prone PCR is greater than or equal to 5, the next level of screening is performed, and the positive transformants obtained in this level are used as templates for the next round of error-prone PCR.

[0049] The results are shown in Table 1. Similarly, following the screening conditions in Table 1, after passing 8 levels of screening conditions and 24 rounds of error-prone PCR screening, the transformants obtained in the 24th round of error-prone PCR were screened at 95℃ for 1 hour, and then screened for rutin conversion rate ≥80%, to obtain the mutant recombinant strain E.coli-IgRha M24-5 that can efficiently transform rutin at 95℃.

[0050] Table 1. Results of activity assays for α-rhamnosidase mutants.

[0051]

[0052] 4. Purification of the target protein

[0053] 20 μL of the parental α-rhamnosidase IgRha and the α-rhamnosidase mutant recombinant strain E. coli-IgRha M24-5 were added to 25 mL of LB medium containing 50 μg / mL kanamycin and cultured overnight at 37 °C and 180 rpm. 2.5 mL of the bacterial culture was added to 350 mL of 200 mL LB medium containing 50 μg / mL kanamycin and cultured for 14 h at 37 °C and 180 rpm. The culture was collected by centrifugation at 6000 rpm and 4 °C for 10 min, washed twice with 1×PBS buffer, and the resulting wet cells were stored at -80 °C for later use. After induction of expression, the cells were cultured at a ratio of 10 g (wet cells): 100 mL of equilibration buffer (500 mM NaCl, 20 mM imidazole, 20 mM... Resuspend the cells in Tris-HCl buffer (pH=7.2), sonicate on ice for 1 second, turn off for 2 seconds, for a total of 30 min; centrifuge the cell lysate at 12000 rpm at 4℃ for 15 min, and filter the supernatant through a 0.22 μm filter membrane for subsequent purification.

[0054] Nickel column affinity chromatography purification: The purification column is a 5 mL pre-packed column. First, the entire purification system is equilibrated with equilibration buffer (500 mM NaCl, 20 mM imidazole, 20 mM Tris-HCl buffer, pH = 7.2). Then, the sample is loaded at a flow rate of 1 mL / min. The uncoated contaminating proteins are further removed with equilibration buffer (500 mM NaCl, 20 mM imidazole, 20 mM Tris-HCl buffer, pH = 7.2). Then, gradient elution is performed with elution buffer (400 mM NaCl, 500 mM imidazole, 20 mM Tris-HCl buffer, pH = 7.2). The target protein is eluted at an imidazole concentration of approximately 250 mM-300 mM. The target protein is collected, concentrated and desalted using an ultrafiltration tube (50 kDa cutoff) to a final volume of 1 mL. Finally, the protein solution is aliquoted and stored at -80°C for later use.

[0055] The amino acid sequence of the α-rhamnosaccharidase IgRha M24-5 mutant is shown in SEQ ID NO.2. It consists of a total of 268 amino acid point mutations and two frameshift mutations that add 14 amino acid residues based on the parental amino acid sequence of α-rhamnosaccharidase, SEQ ID NO.1. The nucleotide sequence of its encoding gene is shown in SEQ ID NO.3.

[0056] Example 2

[0057] The reaction system was as follows: 85 μL of 100 mmol / L citrate-disodium hydrogen phosphate buffer (pH 7.0) was added to 5 μL of 20 mmol / L p-nitrophenyl-α-L-rhamnoside (pNPR). The mixture was incubated at 100 °C for 3 min, followed by the addition of 2-10 μg of enzyme and a 10 min reaction. The reaction was then terminated by adding 600 μL of 1 mol / L sodium carbonate solution. After color development, the absorbance was measured at 405 nm. Specific enzyme activity (U) is defined as the amount of enzyme required to hydrolyze the substrate to produce 1 μmol of p-nitrophenol per minute under optimal enzyme reaction conditions.

[0058] 1. Optimal reaction temperature

[0059] Enzyme activity was measured at 5°C intervals within the range of 70–100°C. The buffer was 50 mmol / L citrate-disodium hydrogen phosphate buffer, pH 7.0.

[0060] The results are as follows Figure 1 As shown, the optimal reaction temperatures for the α-rhamnosylase mutant IgRha M24-5 and the parent enzyme are 100℃ and 80℃, respectively. The α-rhamnosylase mutant IgRha M24-5 is currently the only reported rhamnosylase with an optimal reaction temperature of 100℃.

[0061] 2. Temperature stability

[0062] Under pH 7 conditions, the enzyme was incubated at 80℃, 90℃, 95℃, and 100℃ for 60 min at each temperature, and then the enzyme activity was measured. The enzyme activity without incubation (stored at 4℃) was taken as 100%.

[0063] The results are as follows Figure 2 As shown, the α-rhamnosidase mutant IgRha M24-5 exhibits a residual enzyme activity of over 85% after incubation at 95℃ for 1 hour and shows almost no inactivation after incubation at 90℃ for 1 hour. However, the parent enzyme shows significant loss of enzyme activity after incubation at temperatures above 80℃ for more than 1 hour.

[0064] 3. The activity of α-rhamnosidase in selectively converting flavonoid glycosides

[0065] The standards for flavonoid glycosides such as rutin, isoquercitrin, ascorbic acid C, and icariin were all purchased from Chengdu Manster Biotechnology Co., Ltd.

[0066] 1) The conversion pathway for the selective conversion of α-rhamnosidase into icariin from citric acid is as follows: Figure 3 As shown in a.

[0067] The final concentration of Asarum nitrate C in the 50 μL reaction system was 10 g / L. 5 μL of buffer (500 mmol / L citrate-disodium hydrogen phosphate buffer, pH 7) was added to the reaction system to a final concentration of 50 mmol / L. The amount of both the parental enzyme IgRha and the mutant enzyme IgRhaM24-5 added was 47 μg. After reacting at 80℃ and 95℃ for 60 min, samples were taken, and the reaction was terminated by adding one volume of methanol. Changes in product content were quantitatively analyzed by HPLC.

[0068] The results are as follows Figure 4 As shown, the conversion rates of the parent enzyme and the mutant enzyme for astragalus C were 72.84% and 99.76%, respectively.

[0069] 2) The hydrolytic pathway for the selective conversion of rutin to isoquercetin using α-rhamnosidase is shown in the diagram below. Figure 3 As shown in b.

[0070] The final concentration of rutin in the 50 μL reaction system was 10 g / L. 5 μL of buffer (500 mmol / L citrate-disodium hydrogen phosphate buffer, pH 7) was added to the reaction system to a final concentration of 50 mmol / L. Both the parental enzyme IgRha and the mutant enzyme IgRhaM24-5 were added at 56 μg. After reacting at 80℃ and 95℃ for 60 min, samples were taken, and the reaction was terminated by adding one volume of methanol. Changes in product content were quantitatively analyzed by HPLC.

[0071] The results are as follows Figure 5 As shown, the conversion rates of rutin by the parent enzyme and the mutant enzyme were 90.33% and 99.81%, respectively.

[0072] In summary, at the same addition level, the mutant enzyme IgRha M24-5 can almost completely convert high-concentration substrate flavonoid glycosides (10 g / L) into derhamnosyl products at 95 °C. Compared with the parent enzyme, the mutant enzyme exhibits superior thermostability and catalytic activity at 95 °C. The α-rhamnosylase mutant can better catalyze the conversion of rutin and ascorbic acid C into derhamnosyl flavonoid isoquercitrin and icariin, respectively, showing higher conversion rates under high-temperature conditions. This indicates that the α-rhamnosylase mutant has good application prospects in the selective biotransformation of flavonoid glycosides to prepare derhamnosyl flavonoids under high-temperature conditions.

[0073] The technical solutions provided by the embodiments of this application have been described in detail above. Specific examples have been used in this article to illustrate the principles and implementation methods of the embodiments of this application. The description of the above embodiments is only applicable to help understand the principles of the embodiments of this application. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the embodiments of this application.

Claims

1. An α-rhamnosidase mutant, the amino acid sequence of which is shown in SEQ ID NO.

2.

2. A recombinant strain E. coli containing the α-rhamnosidase mutant of claim 1.

3. The use of the α-rhamnosidase mutant of claim 1 in the preparation of icariin.

4. The use of the α-rhamnosidase mutant of claim 1 in the preparation of isoquercitrin.

5. The use of recombinant strain E. coli containing the α-rhamnosidase mutant of claim 1 in the preparation of icariin.

6. The use of recombinant strain E. coli containing the α-rhamnosidase mutant of claim 1 in the preparation of isoquercitrin.

7. The application according to any one of claims 5 or 6, characterized in that step include: 1) Prepare a recombinant plasmid containing the α-rhamnosidase mutant of claim 1; 2) The recombinant plasmid was transformed into Escherichia coli to obtain an α-rhamnosidase mutant strain; 3) Recombinant strains of E. coli for the preparation of icariin or isoquercitrin were cultivated and screened.

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