Alpha-rhamnosidase mutant and application thereof

Abstract

The invention discloses an alpha-rhamnosidase mutant and application thereof, and relates to the technical field of microbial engineering. The amino acid sequence of the alpha-rhamnosidase mutant disclosed by the invention is as shown in SEQ ID NO.2, and the alpha-rhamnosidase mutant has 268 amino acid residue point mutation in total on the basis of the amino acid sequence SEQ ID NO.1 of an alpha-rhamnosidase parent, and 14 amino acid residues are increased by 2-segment frameshift mutation. The results show that the conversion rates of the alpha-rhamnoside parent enzyme and the alpha-rhamnoside IgRha M24-5 mutant enzyme to the epimedin C are respectively 72.84% and 99.76%; and the conversion rates of rutin are respectively 90.33% and 99.81%. Therefore, the alpha-rhamnosidase mutant has a good application prospect in the aspect of preparing rhamnosylated flavonoids by selectively biologically converting flavone glycoside compounds under a high-temperature condition.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial engineering, and more specifically relates to an alpha-rhamnosidase mutant and application thereof. Background Art

[0002] Flavonoids are a class of polyphenolic secondary metabolites widely found in plants. Flavonoids are extremely rich in variety, and the flavonoid nucleus can be divided into flavonoids, isoflavones, flavanols, flavonols, chalcones, anthocyanidins, etc. In addition, those with sugar groups are flavonoid glycosides, and those without sugar groups are flavonoid aglycones. Due to their special chemical structure, flavonoids show a wide range of pharmacological activities, including antioxidant, anti-tumor, anti-cancer, antibacterial, antiviral, immunomodulatory and other biological activities, and have great application potential in the fields of medicine and food. However, the special molecular structure of flavonoids leads to their unsatisfactory solubility, and their bioavailability is greatly limited, especially the type and amount of glycosides have a significant impact on their biological activity. Taking rutin as an example, its selective removal of terminal rhamnose product quercetin-3-glucoside (isoquercetin) has an anti-proliferative effect on a variety of cancer cells, including colon cancer, breast cancer, hepatocellular carcinoma and lung cancer. The results showed that the anti-proliferative effect of isoquercetin is much greater than that of rutin. Therefore, it has become a research hotspot to modify its structure to change its solubility and improve its bioavailability so that it can be widely used in food, medicine and other fields.

[0003] α-Rhamnosidase can selectively hydrolyze the α-rhamnosidic bond at the non-reducing end and release L-rhamnose. At present, more than 4,000 rhamnosidases from different sources have been isolated, identified, cloned and expressed, but only two strains from Aspergillus niger and Penicillium have been widely used in commercial applications. The problems and limitations of α-rhamnosidase in the current application process are analyzed as follows: 1) Fungal rhamnosidase is easy to purify, but most of them are crude enzyme liquid after fermentation, which contains not only α-rhamnosidase but also a large amount of β-glucosidase, β-galactosidase, endomannosidase and other different functional glycoside hydrolases. If β-glucosidase is not removed, by-products will inevitably be produced, resulting in a decrease in the yield of the target product. 2) Most of the rhamnosidases reported so far have low activity and are not suitable for large-scale production. At the same time, there are many influencing factors in the process of fungal fermentation enzyme production, which is not conducive to production regulation. (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 relatively low solubility, most enzymes cannot maintain high thermal stability under high temperature conditions, affecting production efficiency.

[0004] In summary, discovering thermostable α-rhamnosidase or modifying its structure, improving enzyme activity and stability, and realizing the selective and efficient conversion of flavonoid glycosides such as rutin under high temperature conditions are of great significance for the wide application of flavonoid compounds in multiple fields. Summary of the invention

[0005] In view of the above problems existing in the prior art, 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 an application of the α-rhamnosidase mutant for regulating the selective conversion efficiency of flavonoid compounds.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0007] An α-rhamnosidase mutant, whose amino acid sequence is shown in SEQ ID NO.2.

[0008] The amino acid sequence of the α-rhamnosidase mutant is based on the α-rhamnosidase parent amino acid sequence SEQID NO.1, with a total of 268 amino acid residue point mutations and 2 frameshift mutations adding 14 amino acid residues.

[0009] Recombinant strain E. coli-IgRha M24-5 containing 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 mutants in regulating the selective conversion of epimedin C to prepare icariin.

[0013] Application of α-rhamnosidase mutants in regulating the selective conversion of rutin to produce isoquercetin.

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

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

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

[0017] 1) preparing a recombinant plasmid encoding an α-rhamnosidase mutant;

[0018] 2) transforming the recombinant plasmid into Escherichia coli to obtain an α-rhamnosidase mutant strain;

[0019] 3) Cultivating and screening a recombinant strain E. coli-IgRha M24-5 that has improved selective conversion efficiency for epimedin C or rutin and is used to prepare icariin or isoquercetin.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The α-rhamnosidase mutant disclosed by the present invention has an amino acid sequence as shown in SEQ ID NO.2, which is a mutant of α-rhamnosidase parent amino acid sequence SEQ ID NO.1, with a total of 268 amino acid residues point mutations and 2 frameshift mutations adding 14 amino acid residues. The results show that the conversion rates of α-rhamnosidase parent enzyme and α-rhamnosidase IgRhaM24-5 mutant enzyme to epimedin C are 72.84% and 99.76% respectively; and the conversion rates to rutin are 90.33% and 99.81% respectively. Under the same addition amount, the mutant enzyme IgRha M24-5 can almost completely convert high-concentration substrate concentration flavonoid glycoside (10g / L) into de-rhamnosyl products at 95°C, and compared with the parent enzyme, the mutant enzyme shows excellent thermal stability and catalytic activity at 95°C. The α-rhamnosidase mutant can better catalyze rutin and epimedin C to produce derhamnosylated flavonoids isoquercetin and icariin, respectively, and shows a higher conversion rate at 100°C, indicating that the α-rhamnosidase mutant has good application prospects in the selective biotransformation of flavonoid glycosides to prepare derhamnosylated flavonoids under high temperature conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a comparison chart of the optimal reaction temperatures of the rhamnosidase mutant IgRha M24-5 and the parent enzyme;

[0023] Figure 2 This is a comparison chart of the temperature stability of the rhamnosidase mutant IgRha M24-5 and the parent enzyme;

[0024] Figure 3 The biotransformation pathways and enzymatic conversion reaction routes of rutin and epimedin C (a. the products and reaction pathways obtained by biotransformation of epimedin 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 4HPLC detection diagram of the conversion of epimedin C products by α-rhamnosidase mutant IgaRhaM24-5 and parent enzyme respectively (STD is standard substrate epimedin C, icariin and icariin I; reaction time is 60 min);

[0026] Figure 5 HPLC detection chart of the products obtained by converting rutin by α-rhamnosidase mutant IgaRhaM24-5 and parent enzyme respectively (STD is standard substrate rutin and isoquercetin; reaction time is 60 min). DETAILED DESCRIPTION

[0027] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. Unless otherwise specified in the following embodiments, the technical means used are conventional means well known to those skilled in the art.

[0028] Example 1

[0029] 1. Recombinant expression of parental IgRha

[0030] The α-rhamnosidase IgRha from thermophilic archaea (Ignisphaera sp.) (NCBI accession number MEM1526583.1) was used as a parent, and its amino acid sequence is shown in SEQ ID NO. 1. The gene sequence of the artificially synthesized α-rhamnosidase 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, placed in a shaker at 37°C, 180 rpm, and rejuvenated for 60 minutes; after rejuvenating, the bacterial solution was spread on an LB plate containing 50 μg / mL kanamycin and incubated at 37°C Invert and culture overnight; pick a single colony on the plate, inoculate it into 30mL LB medium (250mL conical flask) containing 50μg / mL kanamycin, and culture it at 37℃, 220rpm without adding inducer for 12 hours; collect the bacteria at 12000rpm, 4℃, 10min, wash the collected bacteria twice with 1×PBS buffer solution, and use the washed bacteria (wet bacteria) directly for screening, or store them in a -80℃ refrigerator for later use.

[0031] 2. Construction of α-rhamnosidase mutants

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

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

[0040] The PCR reaction program was as follows: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 20 s, annealing temperature set according to the mutant primer for 60 s, extension at 60°C for 3 min, 28 cycles; extension at 60°C for 10 min; and storage of PCR products at 12°C.

[0041] After the PCR product was purified and recovered by 0.8% agarose gel electrophoresis, it was double digested with NcoI and XhoI, mixed with the pET-28a plasmid fragment that was double digested with NcoI and XhoI and purified by 0.8% agarose gel electrophoresis, added with T4 ligase, reacted at 16°C for 4-16 hours, and transformed into E. coli TOP10F' competent cells. The bacterial solution after recovery culture was spread on LB medium plates containing 50μg / mL kanamycin and inverted and cultured at 37°C overnight. According to the number of transformants, 1-50 transformants were selected for transformation efficiency verification.

[0042] According to the results of the previous round of transformant screening, positive mutant transformants with significantly improved transformation performance (Escherichia coli colonies dipped with sterile toothpicks were added to the error-prone PCR reaction system) were selected as templates, and error-prone PCR was performed on the basis of one or more transformants in the previous round using the same method as mentioned above to obtain mutants with more mutation points.

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

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

[0045] A single colony (50) of the transformant randomly selected from the plate was added to a centrifuge tube (10 mL) containing 5 mL of LB liquid culture medium, and cultured at 37°C and 220 rpm without adding an inducer for 12 hours. Then, 50 μL of substrate rutin was added to a final concentration of 1 g / L, and the mixture was reacted at 85-100°C for 1 hour. 500 μL of the transformation product was mixed with 500 uL of methanol, centrifuged at 12000 rpm at 4°C for 5 min, filtered with a 0.22 μm organic filter membrane, and the content was detected by HPLC. The substrate conversion rate (%) = the molar concentration of isoquercetin in the product / the molar concentration of initial rutin in the substrate × 100%.

[0046] The specific screening process is:

[0047] Taking the a-level screening as an example, the parent gene IgRha is used as a template, after the mutant transformants are obtained by the error-prone PCR in the previous step, rutin is used as a screening substrate, the mutant transformants are reacted at 85°C for 1 hour, and the transformation products are analyzed by HPLC. The first-round PCR positive transformants 1-27 and 1-39 (wherein, for example, 1-27 refers to the No. 27 transformant in the first-round PCR transformants) are obtained; when the number of positive transformants obtained in the transformants obtained by the error-prone PCR in the first round is less than 5, continue to use the parent gene Using IgRha as a template, a second round of error-prone PCR was performed and screened to obtain the second-round positive transformant 2-17 (wherein, for example, 2-17 refers to transformant No. 27 among the second-round error-prone PCR transformants. The same below); no positive transformants meeting the screening conditions were screened out in the third round, and then the fourth-round positive transformants 4-9 were obtained in turn; the fifth-round positive transformants 5-41 and 5-28 were obtained in turn; when the total number of positive transformants obtained by the a-level screening error-prone PCR was ≥5, the next level of screening was performed, and the positive transformants obtained at this level were used as templates for the next round of error-prone PCR.

[0048] Taking the b-level screening as an example, the 6 positive transformants 1-27, 1-39; 2-17; 4-9; 5-41, 5-28 obtained in the a-level screening were used as templates to perform the sixth round of error-prone PCR, using rutin as the screening substrate, reacting the mutant transformants at 85°C for 1 hour, and then analyzing the transformation products by HPLC. The sixth round of positive transformants 6-20, 6-22, and 6-44 were obtained under the condition of rutin conversion rate ≥ 80%, and the seventh round of error-prone PCR and screening were continued to obtain positive transformants 7-14 and 7-33. When the total number of positive transformants obtained by the b-level screening error-prone PCR is greater than ≥ 5, the next level of screening is performed, and the positive transformants obtained at this level are used as templates for the next round of error-prone PCR.

[0049] The results are shown in Table 1. Similarly, according to the screening conditions in Table 1, the 8-level screening conditions were finally passed, and 24 rounds of error-prone PCR screening were performed. After the 24th round of error-prone PCR, the transformants obtained were reacted at 95°C for 1 hour and then screened with a rutin conversion rate of ≥80%, and the mutant recombinant strain E. coli-IgRha M24-5 that can efficiently convert rutin at 95°C was obtained.

[0050] Table 1 Activity test results of α-rhamnosidase mutants

[0051]

[0052] 4. Purification of target protein

[0053] 20 μL of the parent α-rhamnosidase IgRha and the α-rhamnosidase mutant recombinant strain E. coli-IgRha M24-5 were taken respectively, and 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 bacterial solution was added to 350 mL of 200 mL LB medium containing 50 μg / mL kanamycin antibiotics, and cultured at 37°C and 180 rpm for 14 h; the culture solution was collected by centrifugation at 6000 rpm and 4°C for 10 min, washed twice with 1×PBS buffer solution, and the obtained wet bacteria were stored at -80°C for use; after induction of expression, 10 g (wet bacteria): 100 mL equilibrium buffer (500 mM NaCl, 20 mM imidazole, 20 mM The cells were resuspended in Tris-HCl buffer, pH = 7.2, and ultrasonically disrupted in an ice bath, on for 1 s, off for 2 s, for a total of 30 min; the cell disruption solution was centrifuged at 12000 rpm, 4°C for 15 min, and the supernatant was filtered with a 0.22 μm filter membrane for subsequent purification.

[0054] Nickel column affinity chromatography purification method: The purification column is a 5mL pre-packed column. The entire purification system is first balanced with an equilibrium buffer (500mM NaCl, 20mM imidazole, 20mM Tris-HCL buffer, pH=7.2), and then the sample is loaded at a flow rate of 1mL / min. The unattached impurities are further removed with an equilibrium buffer (500mM NaCl, 20mM imidazole, 20mM Tris-HCL buffer, pH=7.2), and then gradient elution is performed with an elution buffer (400mM NaCl, 500mM imidazole, 20mM Tris-HCL buffer, pH=7.2); the target protein is eluted at an imidazole concentration of approximately 250mM-300mM, the target protein is collected, and concentrated and desalted with an ultrafiltration tube (cutoff 50KDa) to a final volume of 1mL, and the protein solution is finally packaged and stored at -80°C for standby use;

[0055] The amino acid sequence of the α-rhamnosidase IgRha M24-5 mutant is shown in SEQ ID NO.2, which is a mutant of α-rhamnosidase parent amino acid sequence SEQ ID NO.1 with a total of 268 amino acid residue point mutations and 2 frameshift mutations adding 14 amino acid residues. The nucleotide sequence of its encoding gene is shown in SEQ ID NO.3.

[0056] Example 2

[0057] The reaction system is as follows: add 85 μL of 100 mmol / L citric acid-sodium hydrogen phosphate buffer (pH 7.0) to 5 μL of 20 mmol / L p-nitrobenzene-α-L-rhamnoside (pNPR), incubate at 100°C for 3 min, then add 2-10 μg of enzyme and react for 10 min, then add 600 μL of 1 mol / L sodium carbonate solution to terminate the reaction. After color development, the absorbance is measured at 405 nm. The specific enzyme activity unit (U) is defined as: under the optimal enzyme reaction conditions, the amount of enzyme required to hydrolyze the substrate to produce 1 μmol of product p-nitrophenol per minute per 1 mg of enzyme is 1 specific enzyme activity unit (U / mg).

[0058] 1. Optimum reaction temperature

[0059] The specific enzyme activity was measured at 5°C intervals within the range of 70-100°C. The buffer was 50 mmol / L citric acid-sodium hydrogen phosphate buffer, pH 7.0.

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

[0061] 2. Temperature stability

[0062] Under pH 7, the enzyme was kept at 80°C, 90°C, 95°C and 100°C for 60 min respectively, and the specific enzyme activity was measured. The specific enzyme activity without keeping warm (stored at 4°C) was taken as 100%.

[0063] The results are as follows Figure 2 As shown, the residual enzyme activity of the α-rhamnosidase mutant IgRha M24-5 was higher than 85% when incubated at 95°C for 1 hour, and was almost not inactivated when incubated at 90°C for 1 hour, but the parent enzyme lost significant enzyme activity when incubated at greater than 80°C for more than 1 hour.

[0064] 3. Activity of α-rhamnosidase in selective conversion of flavonoid glycosides

[0065] Standard products of flavonoid glycosides such as rutin, isoquercetin, epimedin C and icariin were purchased from Chengdu Munster Biotechnology Co., Ltd.

[0066] 1) The conversion pathway of α-rhamnosidase selectively converting epimedin C to icariin Figure 3 As shown in a.

[0067] The final concentration of epimedin C in the 50 μL reaction system was 10 g / L, 5 μL of buffer (500 mmol / L citric acid-sodium hydrogen phosphate buffer, pH 7) was added to the reaction system to a final concentration of 50 mmol / L, the amount of parent enzyme IgRha and mutant enzyme IgRhaM24-5 added was 47 μg, and samples were taken after reacting at 80°C and 95°C for 60 min, and 1 volume of methanol was added to terminate the reaction. The changes in product content were quantitatively analyzed by HPLC.

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

[0069] 2) The hydrolysis route of α-rhamnosidase selectively converting rutin to prepare isoquercetin is shown in the figure 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 citric acid-sodium hydrogen phosphate buffer, pH 7) was added to the reaction system to a final concentration of 50 mmol / L, the amount of parent enzyme IgRha and mutant enzyme IgRhaM24-5 added was 56 μg, and samples were taken after reacting at 80°C and 95°C for 60 min, and 1 volume of methanol was added to terminate the reaction. The 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 amount, the mutant enzyme IgRha M24-5 can almost completely convert high-concentration substrate concentration flavonoid glycosides (10 g / L) into de-rhamnosyl products at 95°C. Compared with the parent enzyme, the mutant enzyme shows excellent thermal stability and catalytic activity at 95°C. The α-rhamnosidase mutant can better catalyze rutin and epimedin C to generate de-rhamnosyl flavonoids isoquercetin and icariin, respectively, and shows a higher conversion rate under high temperature conditions, indicating that the α-rhamnosidase mutant has good application prospects in the selective biotransformation of flavonoid glycosides to prepare de-rhamnosyl flavonoids under high temperature conditions.

[0073] The technical solutions provided by the embodiments of the present application are introduced in detail above. Specific examples are used in this article to illustrate the principles and implementation methods of the embodiments of the present application. The description of the above embodiments is only applicable to help understand the principles of the embodiments of the present application. At the same time, for general technicians in this field, according to the embodiments of the present application, there will be changes in the specific implementation methods and application scope.

Claims

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

2.

2. The α-rhamnosidase mutant according to claim 1, characterized in that The amino acid sequence of the α-rhamnosidase mutant is based on the α-rhamnosidase parent amino acid sequence SEQ ID NO.1, with a total of 268 amino acid residue point mutations and 2 frameshift mutations adding 14 amino acid residues.

3. A recombinant strain E. coli-IgRha M24-5 containing the α-rhamnosidase mutant according to claim 1.

4. Use of the α-rhamnosidase mutant according to claim 1 in regulating the selective conversion efficiency of flavonoids.

5. Use of the α-rhamnosidase mutant according to claim 1 in regulating the selective conversion efficiency of flavonoid glycosides.

6. Use of the α-rhamnosidase mutant according to claim 1 in regulating the selective conversion of epimedin C to prepare icariin.

7. Use of the α-rhamnosidase mutant according to claim 1 in regulating the selective conversion of rutin to prepare isoquercetin.

8. Application of the recombinant strain E. coli-IgRha M24-5 containing an α-rhamnosidase mutant in regulating the selective conversion of epimedin C to prepare icariin.

9. Application of the recombinant strain E. coli-IgRha M24-5 containing an α-rhamnosidase mutant in regulating the selective conversion of rutin to produce isoquercetin.

10. The use according to any one of claims 8 or 9, characterized in that the step include: 1) preparing a recombinant plasmid encoding an α-rhamnosidase mutant; 2) transforming the recombinant plasmid into Escherichia coli to obtain an α-rhamnosidase mutant strain; 3) Cultivating and screening a recombinant strain E. coli-IgRha M24-5 that has improved selective conversion efficiency for epimedin C or rutin and is used to prepare icariin or isoquercetin.

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