Alpha-L-rhamnosidase mutant and application thereof

Abstract

The invention relates to the technical field of gene engineering, and discloses an alpha-L-rhamnosidase mutant and application thereof. According to the present invention, single-point or multi-point mutation of sites such as N325A, D328S, T598A, K341A, V337G, M340G, K266R, M268P, S270A, V723A, M677A, Y604L, L173A and L176A is performed on the wild type alpha-L-rhamnosidase, such that the relative enzyme activity of the alpha-L-rhamnosidase is improved, and the efficiency of converting icariin to generate icariside I is improved. Especially, single-point mutants N325A and V337G have excellent enzymatic characteristics, when the icariin is catalyzed, the mutants N325A and V337G can specifically convert a substrate to generate icariside I, and no other by-products are generated.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and particularly relates to an α-L-rhamnosidase mutant and its application. Background Art

[0002] α-L-rhamnosidase (EC 3.2.1.40) is an important glycoside hydrolase, which can specifically hydrolyze the α-1,2, α-1,3, α-1,4, α-1, α-1,6 glycosidic bonds of natural glycosides such as flavonoids, saponins, and terpene glycosides, release L-rhamnose, and produce new polysaccharides or glycoside compounds. According to the similarity of amino acid sequences, α-L-rhamnosidase can be divided into three families, namely GH 13, GH 78, and GH 106 families.

[0003] Epimedium is a perennial herb of the genus Epimedium in the Berberidaceae family. Its flavonoid components, including icariin, epimedin A, epimedin B, epimedin C, etc., have various pharmacological activities. Icariin has a wide range of therapeutic effects because it is an osteoprotective, neuroprotective, cardiovascular protective, anti-cancer, and immunoprotective agent, and can also affect reproductive function. Icariin II has important application prospects in the prevention and treatment of infectious diseases and the immunotherapy of tumors. Icaritin is the glycosyl hydrolysis product of icariin and is an isopentenyl-substituted flavonol with various pharmacological activities such as anti-tumor, neuroprotection, immunomodulation, regulation of bone metabolism, anti-inflammatory and antioxidant in vivo and in vitro. Some studies have shown that icaritin has a good anti-proliferative effect on primary liver cancer and is a typical advanced small molecule immunomodulatory drug.

[0004] α-L-rhamnosidase converts icariin into icariin II, and then through the hydrolysis of β-D-glucosidase, it can be further converted into icaritin. How to improve the catalytic efficiency of α-L-rhamnosidase on icariin is of great significance for the biocatalysis of icariin and the production of icaritin. Summary of the Invention

[0005] In order to improve the catalytic efficiency of α-L-rhamnosidase in converting icariin, the present invention provides an α-L-rhamnosidase mutant and its application.

[0006] The specific technical solution of the present invention is as follows: In the first aspect, the present invention provides an α-L-rhamnosidase mutant, which is characterized in that it is obtained by single-point or multi-point combination mutation at the following sites of α-L-rhamnosidase with the amino acid sequence shown in SEQ ID NO.1: N325A, V337G.

[0007] The present invention performs single-site or multi-site mutations at the sites of "N325A, D328S, T598A, K341A, V337G, M340G, K266R, M268P, S270A, V723A, M677A, Y604L, L173A, L176A" on wild-type α-L-rhamnosidase, screens mutants, and finds that mutations at the N325A and V337G sites can increase the relative enzyme activity of α-L-rhamnosidase, thereby improving the efficiency of its conversion of icariin to icariside I. In particular, the single-site mutants N325A and V337G have excellent enzymatic properties. When catalyzing icariin, the mutants N325A and V337G can specifically convert the substrate to produce icariside I without generating other by-products.

[0008] Preferably, the mutant is obtained by performing single-site mutations at the following sites on α-L-rhamnosidase with the amino acid sequence shown in SEQ ID NO.1: N325A, V337G.

[0009] In a second aspect, the present invention provides the coding gene of the above mutant.

[0010] In a third aspect, the present invention provides a recombinant expression vector of the above coding gene.

[0011] A recombinant expression vector is a DNA molecule used to introduce foreign genes into host cells for expression. Based on the α-L-rhamnosidase mutant coding gene provided by the present invention, the recombinant expression vector can be constructed by connecting the nucleotide sequence of the α-L-rhamnosidase mutant gene of the present invention to various vectors by conventional methods in the art. Various conventional vectors in the art, such as various plasmids, phages or viral vectors, etc., connecting the nucleotide sequence of the nicotinamide mononucleotide adenylyltransferase mutant of the present invention, should all fall within the protection scope of the present invention.

[0012] More preferably, the recombinant expression vector is a plasmid, phage or viral vector.

[0013] In a fourth aspect, the present invention provides a genetically engineered bacterium carrying the above coding gene.

[0014] In a fifth aspect, based on the above, the present invention provides the use of the above mutant or genetically engineered bacterium in the preparation of icariside I.

[0015] In a sixth aspect, based on the above, the present invention provides the use of the above mutant or genetically engineered bacterium in the preparation of icariin.

[0016] Compared with the prior art, the present invention has the following technical effects: The present invention performs single-point or multi-point mutations at the sites of "N325A, D328S, T598A, K341A, V337G, M340G, K266R, M268P, S270A, V723A, M677A, Y604L, L173A, L176A" on the wild-type α-L-rhamnosidase, screens mutants, and finds that mutations at the N325A and V337G sites can increase the relative enzyme activity of α-L-rhamnosidase, thereby improving its efficiency in converting icariin to icariside I. In particular, the single-point mutants N325A and V337G have excellent enzymatic properties. When catalyzing icariin, the mutants N325A and V337G can specifically convert the substrate to produce icariside I without generating other by-products. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Results of SDS-PAGE analysis of mutant recombinant strains.

[0018] Figure 2 Molecular docking results of Novosphingobium sp. GX9 α-L-rhamnosidase and icariin.

[0019] Figure 3 Relative enzyme activity graphs of wild-type α-L-rhamnosidase and α-L-rhamnosidase mutants in Example 2.

[0020] Figure 4 Results of the determination of the enzymatic properties of the α-L-rhamnosidase SPRHA2 mutant of the present invention. Among them, (a) is the determination of the optimal reaction temperature of wild-type recombinant α-L-rhamnosidase and its mutants; (b) is the determination of the optimal reaction pH of wild-type recombinant α-L-rhamnosidase and its mutants. DETAILED DESCRIPTION OF THE INVENTION

[0021] The present invention will be further described below in conjunction with embodiments. Those of ordinary skill in the art will be able to implement the present invention based on these descriptions. In addition, the embodiments of the present invention involved in the following description are usually only a part of the embodiments of the present invention, rather than all the embodiments. Therefore, all other embodiments obtained by those of ordinary skill in the art without creative efforts based on the embodiments of the present invention should fall within the scope of protection of the present invention.

[0022] The wild-type α-L-rhamnosidase gene SPRHA2 of the present invention as shown in SEQ ID NO.1 is obtained by optimizing the codon preference of the α-L-rhamnosidase gene from Novosphingobium sp. GX9, such as Figure 2As shown, it is the molecular docking result of Novosphingobium sp. GX9 α-L-rhamnosidase and icariin.

[0023] Example 1 Construction of α-L-rhamnosidase mutants Construction of the recombinant expression vector of the mutant enzyme: The coding gene of the wild-type α-L-rhamnosidase SPRHA2 shown in SEQ ID NO.1 was synthesized by gene synthesis, and the solubility-enhancing tag SUMO was added to improve its soluble expression level. It was ligated to the plasmid pET28a to construct the recombinant plasmid pET28a-SPRHA2-SUMO. Using the plasmid pET28a-SPRHA2-SUMO(WT) vector as a template, a kit from Tsingke Biotechnology Co., Ltd. was used to construct the recombinant expression vector of the mutant enzyme, which included three steps: PCR amplification, digestion of the template plasmid DNA with Dpn I, and self-cyclization of the PCR product.

[0024] (1) PCR amplification According to the gene sequence of the wild-type α-L-rhamnosidase and the mutations at the target sites, corresponding site-directed mutagenesis primers were designed as shown in Table 1, and PCR amplification was carried out using the plasmid pET28a-SPRHA2-SUMO(WT) vector as a template respectively. The corresponding site-directed mutagenesis primers were, taking the site-directed mutation at position 325 as an example, the corresponding site-directed mutagenesis primers were N325A-F and N325A-R. The PCR amplification conditions were as follows: 95°C, 5 min; 31 cycles (95°C, 30 s; 60°C, 30 s; 72°C, 4 min 30 s); 72°C, 5 min; the reaction stopped, and finally incubated at 25°C. Among them, the mutations at the target sites were single-point mutations at the following sites: N325A, D328S, T598A, K341A, V337G, M340G, K266R, M268P, S270A, V723A, M677A, Y604L, L173A, L176A.

[0025] Table 1 (2) Digestion of the template plasmid DNA with Dpn I 1 μL of Dpn I was added to the PCR reaction solution obtained in step (1), gently mixed, and reacted at 37°C for 1 hour to eliminate the original template.

[0026] (3) Self-cyclization of PCR products The digestion products obtained in step (2) were transformed into Escherichia coli BL21(DE3) to obtain the corresponding recombinant Escherichia coli, which was spread on a plate containing kanamycin and cultured overnight at 37 °C. Clones were randomly selected for colony PCR identification and sequencing verification. The results showed that the recombinant expression vector containing the α-L-rhamnosidase mutant gene was successfully transformed into the expression host Escherichia coli BL21(DE3). Figure 1 Figure 1 shows the SDS-PAGE analysis results of the mutant recombinant strains. The bacterial solution with successful mutation verified by sequencing was added with glycerol and stored at -80 °C in a refrigerator. Finally, α-L-rhamnosidase mutants N325A, D328S, T598A, K341A, V337G, M340G, K266R, M268P, S270A, V723A, M677A, Y604L, L173A, and L176A were obtained. Among them: α-L-rhamnosidase mutant N325A is the mutation of asparagine (N) at position 325 of the wild type to alanine (A); α-L-rhamnosidase mutant D328S is the mutation of aspartic acid (D) at position 328 of the wild type to serine (S); α-L-rhamnosidase mutant T598A is the mutation of threonine (T) at position 598 of the wild type to alanine (A); α-L-rhamnosidase mutant K341A is the mutation of lysine (K) at position 341 of the wild type to alanine (A); α-L-rhamnosidase mutant V337G is the mutation of valine (V) at position 337 of the wild type to glycine (G); α-L-rhamnosidase mutant M340G is the mutation of methionine (M) at position 340 of the wild type to glycine (G); α-L-rhamnosidase mutant K266R is the mutation of lysine (K) at position 266 of the wild type to arginine (R); α-L-rhamnosidase mutant M268P is the mutation of methionine (M) at position 268 of the wild type to proline (P); α-L-rhamnosidase mutant S270A is the mutation of serine (S) at position 270 of the wild type to alanine (A); α-L-rhamnosidase mutant V723A is the mutation of valine (V) at position 723 of the wild type to alanine (A); α-L-rhamnosidase mutant M677A is the mutation of methionine (M) at position 677 of the wild type to alanine (A); α-L-rhamnosidase mutant Y604L is the mutation of tyrosine (Y) at position 604 of the wild type to leucine (L); α-L-rhamnosidase mutant L173A is the mutation of leucine (L) at position 173 of the wild type to alanine (A); α-L-rhamnosidase mutant L176A is the mutation of leucine (L) at position 176 of the wild type to alanine (A).

[0027] Example 2 Expression and purification of wild-type α-L-rhamnosidase and mutants Referring to the method of Example 1, construct the recombinant plasmid pET28a-SPRHA2-SUMO and the mutant plasmid obtained in Example 1, then transform Escherichia coli E. coli BL21(DE3) respectively, and coat them on LB solid medium containing kanamycin, and culture overnight at 37°C. Subsequently, inoculate the single colony into LB liquid medium containing kanamycin, and culture with shaking at 37°C and 200 rpm for 2 h. Subsequently, add isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 0.1 mM, and culture with shaking at 25°C and 180 rpm for 10 h to induce the expression of wild-type and mutant α-L-rhamnosidase. Centrifuge the cell culture solution of recombinant Escherichia coli at 4°C and 8000 rpm for 10 min, collect the wet cells, and store them at -20°C for later use. Resuspend the cells with phosphate buffer at pH 7.0 and ultrasonically disrupt them at low temperature for 30 min. Centrifuge the cell lysate at 4°C and 10,000 rpm for 30 min, collect the supernatant, and obtain the crude enzyme solutions of wild-type α-L-rhamnosidase and its mutants respectively.

[0028] After filtering the crude enzyme through a 0.45 μm filter membrane, purify it through a Ni SepharoseTM 6 Fast Flow affinity chromatography column and a nucleic acid and protein detector, and elute it with a buffer solution containing 50 mM NaH 2 PO 4 (pH 7.4), 300 mM sodium chloride, and 250 mM imidazole to obtain pure enzymes of wild-type α-L-rhamnosidase and mutants.

[0029] Enzymatic property analysis of wild-type and mutant enzymes in Example 3 (1) Determination of enzyme activity Reaction system: Add 460 μL of 100 mmol / L citric acid-disodium hydrogen phosphate buffer (pH 7.0) to 20 μL of 10 mmol / L p-nitrophenyl-α-L-rhamnopyranoside (pNPR), incubate at the optimal temperature for 2 min, then add 20 μL of diluted crude enzyme solution, react at 50°C for 5 min, add 1 mol / L sodium carbonate solution to terminate the reaction after color development. Measure the absorbance value at 405 nm. The relative enzyme activity determination results of wild-type α-L-rhamnosidase and α-L-rhamnosidase mutants are as Figure 3As shown. From the relative enzyme activity determination results, it can be seen that for the α-L-rhamnosidase mutants N325A and V337G, compared with the wild type, their relative enzyme activities increased by 180% and 134% respectively, and the relative enzyme activities were greatly improved; while for other mutants, no increase or different degrees of decrease in relative enzyme activity were observed. Thus, it can be known that the α-L-rhamnosidase mutants N325A and V337G can efficiently convert icariin into icariside I.

[0030] The definition of enzyme activity unit (U) is: under the assay conditions, the amount of enzyme that produces 1 μmol of p-nitrophenol per minute is 1 enzyme activity unit.

[0031] (2) Determination of the optimum reaction temperature: In the range of 45 - 65 °C, at intervals of 5 °C, the enzyme activities were measured respectively. The buffer solution was 100 mmol / L citric acid - disodium hydrogen phosphate buffer (pH 7.0), and it was found that the optimum reaction temperature of the wild-type recombinant α-L-rhamnosidase and its mutants was 50 °C. The measurement results are as Figure 4 shown in Figure a.

[0032] (3) Determination of the optimum reaction pH: Under the conditions of 100 mmol / L citric acid - disodium hydrogen phosphate buffer with different pH values (7.0 - 10.0), the enzyme activities were measured at 50 °C respectively, and it was found that the optimum reaction pH of the wild-type recombinant α-L-rhamnosidase and its mutants was 9.0. The measurement results are as Figure 4 shown in Figure b.

[0033] Example 4 Process for producing icarisidin by the dominant mutants N325A and V337G of recombinant α-L-rhamnosidase The wild-type α-L-rhamnosidase and its mutant N325A and V337G engineering bacteria (the construction of the engineering bacteria was the same as in Example 2) were used for the conversion of icariin substrate. The conversion system was: the reaction temperature was 50 °C, pH 9.0, the substrate icariin concentration was 4 g / L, 5 g / L of recombinant α-L-rhamnosidase SPRHA2 or mutant N325A or mutant V337G was added, β-D-glucosidase was added, and after reacting at 50 °C for 1 h, the reaction was terminated by boiling for 5 min. After the sample was filtered through a membrane, it was detected by HPLC. The conversion yield results are shown in Table 2.

[0034] Table 2 α-L-rhamnosidase Production of icariin (g / L) Wild type 1.71 Mutant N325A 3.93 Mutant V337G 2.92 From the conversion experiment of icariin substrate, it can be seen that compared with WT, when catalyzing icariin, the mutants N325A and V337G can specifically convert the substrate to produce icariside I without the formation of other by-products, and the production yields of icariin can reach 3.93 g / L and 2.92 g / L. It can be seen therefrom that in the present invention, site-directed mutagenesis is carried out on the amino acids of α-L-rhamnosidase acting on icariin to obtain α-L-rhamnosidase mutants, especially the mutants N325A and V337G, which have excellent enzymatic properties, making the preparation process of icariside I have the advantages of simple operation, low cost, environmental friendliness, etc., and have broad application prospects, providing an important tool enzyme for the next step of producing icariin.

[0035] In the present invention, the raw materials and equipment used, unless otherwise specified, are all common raw materials and equipment in the art; the methods used in the present invention, unless otherwise specified, are all conventional methods in the art.

[0036] The above are only the preferred embodiments of the present invention, and do not impose any limitations on the present invention. Any simple modifications, changes and equivalent transformations made to the above embodiments according to the technical essence of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. An α-L-rhamnosidase mutant, characterized in that: The α-L-rhamnosidase with the amino acid sequence shown in SEQ ID NO.1 is subjected to single-point or multi-point combined mutation at the following sites: N325A, V337G.

2. An α-L-rhamnosidase mutant according to claim 1, characterized in that: The α-L-rhamnosidase with the amino acid sequence shown in SEQ ID NO.1 is subjected to single point mutations at the following sites: N325A, V337G.

3. A gene encoding the mutant according to any one of claims 1 to 2.

4. A recombinant expression vector carrying the coding gene as claimed in claim 3.

5. The recombinant expression vector according to claim 4, characterized in that: The recombinant expression vector is a plasmid, a phage or a virus vector.

6. A genetically engineered bacterium carrying the coding gene as claimed in claim 3.

7. Use of the mutant according to any one of claims 1 to 2 in the preparation of icariin I.

8. Use of the mutant according to any one of claims 1 to 2 in the preparation of icariin.

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