Alpha-l-rhamnosidase mutants and uses thereof

Abstract

The application relates to the technical field of genetic engineering, and discloses an alpha-L-rhamnosidase mutant and application thereof. The application improves the relative enzyme activity of alpha-L-rhamnosidase by carrying out single-point or multi-point mutation on the sites N325A, D328S, T598A, K341A, V337G, M340G, K266R, M268P, S270A, V723A, M677A, Y604L, L173A and L176A of wild-type alpha-L-rhamnosidase, and further improves the efficiency of converting icariin into icariside II. Especially, the single-point mutants N325A and V337G have excellent enzymatic properties, and when catalyzing icariin, the mutants N325A and V337G can specifically convert the substrate into icariside I without generating other by-products.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering, and particularly relates to an alpha-L-rhamnosidase mutant and application thereof. BACKGROUND

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

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

[0004] Alpha-L-rhamnosidase converts icariin into epimedoside I, which can be further converted into icaritin by hydrolysis of beta-D-glucosidase. How to improve the efficiency of alpha-L-rhamnosidase in catalyzing icariin is of great significance to the biological catalysis of icariin and the generation of icaritin. SUMMARY

[0005] In order to improve the efficiency of alpha-L-rhamnosidase in catalyzing icariin conversion, the present application provides an alpha-L-rhamnosidase mutant and application thereof.

[0006] The specific technical scheme of the present application is as follows:

[0007] In a first aspect, the present application provides an alpha-L-rhamnosidase mutant, which is characterized by the following single-point or multi-point combined mutation of the alpha-L-rhamnosidase with the amino acid sequence shown in SEQ ID NO. 1: N325A, V337G.

[0008] The present application is obtained by site single-point or multi-point mutation of wild-type alpha-L-rhamnosidase, and mutant screening, and it is found that mutation at N325A and V337G sites can improve the relative enzyme activity of alpha-L-rhamnosidase, and further improve the efficiency of conversion of icariin to icariside II. Especially, the single-point mutant N325A and V337G have excellent enzymatic properties, and when catalyzing icariin, the mutant N325A and V337G can specifically convert the substrate to icariside I without other by-products.

[0009] As preferred, the mutant is obtained by single-point mutation of the alpha-L-rhamnosidase with the amino acid sequence shown in SEQ ID NO. 1 at the following sites: N325A, V337G.

[0010] In a second aspect, the present application provides a coding gene of the mutant.

[0011] In a third aspect, the present application provides a recombinant expression vector of the coding gene.

[0012] The recombinant expression vector is a DNA molecule used for introducing an exogenous gene into a host cell for expression. Based on the coding gene of the alpha-L-rhamnosidase mutant provided by the present application, the recombinant expression vector can be constructed by connecting the nucleotide sequence of the coding gene of the alpha-L-rhamnosidase mutant of the present application to various vectors by conventional methods in the art. Various vectors in the art, such as various plasmids, bacteriophages or viral vectors, etc., connected to the nucleotide sequence of the nicotinamide mononucleotide adenylyltransferase mutant of the present application, should all belong to the protection scope of the present application.

[0013] Further preferably, the recombinant expression vector is a plasmid, a bacteriophage or a viral vector.

[0014] In a fourth aspect, the present application provides a genetically engineered bacterium carrying the coding gene.

[0015] In a fifth aspect, based on the above, the present application provides the use of the mutant or the genetically engineered bacterium in the preparation of icariside II.

[0016] In a sixth aspect, based on the above, the present application provides the use of the mutant or the genetically engineered bacterium in the preparation of icariin.

[0017] Compared with the prior art, the present application has the following technical effects:

[0018] The present application is through site "N325A, D328S, T598A, K341A, V337G, M340G, K266R, M268P, S270A, V723A, M677A, Y604L, L173A, L176A" single point or multi-point mutation of wild type alpha-L-rhamnosidase, mutant screening is carried out, it is found that the relative enzyme activity of alpha-L-rhamnosidase can be improved by mutation at N325A and V337G sites, and then the efficiency of converting icariin to icariside II is improved. Especially the single point mutant N325A and V337G have excellent enzymatic properties, and when catalyzing icariin, the mutant N325A and V337G can specifically convert the substrate to icariside I, and no other byproduct is generated. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 It is mutant recombinant strain SDS-PAGE analysis result.

[0020] Figure 2 It is Novosphingobium sp.GX9 alpha-L-rhamnosidase and icariin molecular docking result.

[0021] Figure 3 It is wild type alpha-L-rhamnosidase and alpha-L-rhamnosidase mutant relative enzyme activity chart in example 2.

[0022] Figure 4 It is the enzymatic property determination result of the alpha-L-rhamnosidase SPRHA2 mutant of the present application, wherein (a) is the determination of the optimum reaction temperature of the wild type recombinant alpha-L-rhamnosidase and its mutant; (b) is the determination of the optimum reaction pH of the wild type recombinant alpha-L-rhamnosidase and its mutant. DETAILED DESCRIPTION

[0023] The present application will be further described below in combination with examples. Those skilled in the art will be able to implement the present application based on these descriptions. In addition, the examples of the present application involved in the following description are generally only a part of the examples of the present application, not all examples. Therefore, based on the examples in the present application, all other examples obtained by those skilled in the art without creative labor shall belong to the scope of protection of the present application.

[0024] The present application example is the wild type alpha-L-rhamnosidase gene SPRHA2 shown as SEQ ID NO. 1, which is obtained by optimizing the codon preference of alpha-L-rhamnosidase gene from Novosphingobium sp.GX9, like Figure 2The figure shows the docking results of Novosphingobium sp. GX9 a-L-rhamnosidase and Icaritin molecule.

[0025] Example 1 Construction of a-L-rhamnosidase mutants

[0026] Construction of mutant enzyme recombinant expression vector:

[0027] The coding gene of wild-type a-L-rhamnosidase SPRHA2 as shown in SEQ ID NO. 1 was synthesized by gene synthesis, and a solubility tag SUMO was added to improve the solubility expression level, and was connected to the plasmid pET28a to construct the recombinant plasmid pET28a-SPRHA2-SUMO. The vector of plasmid pET28a-SPRHA2-SUMO (WT) was used as a template to construct a mutant enzyme recombinant expression vector using a kit from Genescript Biotech Co., Ltd., including three steps of PCR amplification, digestion of template plasmid DNA with Dpn I, and PCR product self-circularization.

[0028] (1) PCR amplification

[0029] According to the gene sequence of wild-type a-L-rhamnosidase and the mutation of the target site, the corresponding site-directed mutation primers were designed as shown in Table 1, and PCR amplification was performed using the vector of plasmid pET28a-SPRHA2-SUMO (WT) as a template. For example, the site-directed mutation primer corresponding to the site-directed mutation at position 325 is N325A-F and N325A-R. The PCR amplification conditions are 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; reaction stop, finally 25°C incubation. The mutation of the target site is single-point mutation at the following sites: N325A, D328S, T598A, K341A, V337G, M340G, K266R, M268P, S270A, V723A, M677A, Y604L, L173A, L176A.

[0030] Table 1

[0031]

[0032]

[0033] (2) Digestion of template plasmid DNA with Dpn I

[0034] 1 μL Dpn I was added to the PCR reaction solution obtained in step (1) and mixed gently, and incubated at 37°C for 1 hour to eliminate the original template.

[0035] (3) The digestion product obtained in step (2) is transformed into E. coli BL21 (DE3) to obtain the corresponding recombinant E. coli, which is coated on a plate containing kanamycin and cultured at 37°C overnight, and randomly selected clones are subjected to colony PCR identification and sequencing verification. The results show that the recombinant expression vector containing the α-L-rhamnosidase mutant gene is successfully transformed into the expression host E. coli BL21 (DE3). Figure 1 The mutant recombinant strain SDS-PAGE analysis results. The bacterial liquid verified by sequencing that the mutation is successful is added with glycerol and stored in a refrigerator at -80°C. Finally, the α-L-rhamnosidase mutants N325A, D328S, T598A, K341A, V337G, M340G, K266R, M268P, S270A, V723A, M677A, Y604L, L173A and L176A are obtained. Among them: the α-L-rhamnosidase mutant N325A is the wild type asparagine (N) at position 325 mutated to alanine (A); the α-L-rhamnosidase mutant D328S is the wild type aspartic acid (D) at position 328 mutated to serine (S); the α-L-rhamnosidase mutant T598A is the wild type threonine (T) at position 598 mutated to alanine (A); the α-L-rhamnosidase mutant K341A is the wild type lysine (K) at position 341 mutated to alanine (A); the α-L-rhamnosidase mutant V337G is the wild type valine (V) at position 337 mutated to glycine (G); the α-L-rhamnosidase mutant M340G is the wild type methionine (M) at position 340 mutated to glycine (G); the α-L-rhamnosidase mutant K266R is the wild type lysine (K) at position 266 mutated to arginine (R); the α-L-rhamnosidase mutant M268P is the wild type methionine (M) at position 268 mutated to proline (P); the α-L-rhamnosidase mutant S270A is the wild type serine (S) at position 270 mutated to alanine (A); the α-L-rhamnosidase mutant V723A is the wild type valine (V) at position 723 mutated to alanine (A); the α-L-rhamnosidase mutant M677A is the wild type methionine (M) at position 677 mutated to alanine (A); the α-L-rhamnosidase mutant Y604L is the wild type tyrosine (Y) at position 604 mutated to leucine (L); the α-L-rhamnosidase mutant L173A is the wild type leucine (L) at position 173 mutated to alanine (A); and the α-L-rhamnosidase mutant L176A is the wild type leucine (L) at position 176 mutated to alanine (A).

[0036] Example 2 Expression and purification of wild-type α-L-rhamnosidase and mutants

[0037] The method of Reference Example 1 was used to construct recombinant plasmid pET28a-SPRHA2-SUMO and the mutant plasmid obtained in Example 1, which were then transformed into E. coli BL21(DE3), respectively, and spread on LB solid medium containing kanamycin and incubated at 37°C overnight. Then single colonies were inoculated into LB liquid medium containing kanamycin and incubated at 37°C, 200 rpm for 2 h. Then isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 0.1 mM, and the expression of wild-type and mutant α-L-rhamnosidase was induced by incubation at 25°C, 180 rpm for 10 h. The cell culture of recombinant E. coli was centrifuged at 4°C, 8000 rpm for 10 min, and the wet bacteria were collected and stored at -20°C. The bacteria were resuspended in phosphate buffer at pH 7.0, and ultrasonically broken at low temperature for 30 min. The cell lysate was centrifuged at 4°C, 10000 rpm for 30 min, and the supernatant was collected to obtain crude enzyme solution of wild-type and mutant α-L-rhamnosidase, respectively.

[0038] The crude enzyme was filtered with a 0.45 μm filter membrane, and then purified by Ni SepharoseTM6 Fast Flow affinity chromatography column and nucleic acid protein detector, eluted with a buffer solution containing 50 mM NaH2PO4(pH 7.4), 300 mM sodium chloride and 250 mM imidazole, to obtain pure enzyme of wild-type and mutant α-L-rhamnosidase.

[0039] Example 3 Enzymatic property analysis of wild-type and mutant enzymes

[0040] (1) Determination of enzyme activity

[0041] Reaction system: 460 μL of 100 mmol / L citric acid-disodium hydrogen phosphate buffer (pH 7.0) was added to 20 μL of 10 mmol / L p-nitrophenyl-α-L- rhamnopyranoside (pNPR), incubated at the optimum temperature for 2 min, then 20 μL of diluted crude enzyme solution was added, and the reaction was carried out at 50°C for 5 min. After color development, 1 mol / L sodium carbonate solution was added to terminate the reaction. The absorbance value was determined at 405 nm. The results of determination of relative enzyme activity of wild-type and mutant α-L-rhamnosidase are shown in Table 1. Figure 3The relative enzyme activity of the wild type and the mutants was determined. The results showed that the relative enzyme activity of the mutants N325A and V337G was increased by 180% and 134% respectively, compared with the wild type, and the relative enzyme activity was greatly improved. The relative enzyme activity of other mutants was not improved or was reduced to different degrees. Therefore, the mutants N325A and V337G of the α-L-rhamnosidase can efficiently convert icariin to Icaririn.

[0042] The enzyme activity unit (U) is defined as: the amount of enzyme used to produce 1 μmol of p-nitrophenol per minute under the determination conditions.

[0043] (2) Determination of the optimum reaction temperature: The enzyme activity was determined at every 5°C in the range of 45-65°C. The buffer was 100 mmol / L citric acid-sodium hydrogen phosphate buffer (pH 7.0), and it was found that the optimum reaction temperature of the wild type recombinant α-L-rhamnosidase and the mutants was 50°C. The determination results are shown in Table 2. Figure 4 a.

[0044] (3) Determination of the optimum reaction pH: The enzyme activity was determined at 50°C in 100 mmol / L citric acid-sodium hydrogen phosphate buffer at different pH (7.0-10.0), and it was found that the optimum reaction pH of the wild type recombinant α-L-rhamnosidase and the mutants was 9.0. The determination results are shown in Table 2. Figure 4 b.

[0045] Example 4 Process for preparing Icaritin by using the mutants N325A and V337G of the recombinant α-L-rhamnosidase The wild type and the mutants N325A and V337G of the recombinant α-L-rhamnosidase were used to convert icariin. The conversion system was as follows: the reaction temperature was 50°C, the pH was 9.0, the concentration of the substrate icariin was 4 g / L, 5 g / L of the recombinant α-L-rhamnosidase SPRHA2 or the mutant N325A or the mutant V337G was added, 50°C was used for reaction for 1 h, boiling was used for 5 min to terminate the reaction, the sample was filtered, and HPLC detection was performed. The conversion yield results are shown in Table 2.

[0046] Table 2

[0047] Alpha-L-rhamnosidase Icaritin production (g / L) Wild type 1.71 Mutant N325A 3.93 Mutant V337G 2.92

[0048] From the conversion experiment of the icariin substrate, compared with the WT, the mutant N325A and V337G can specifically convert the substrate to generate icariin, no other by-product is generated, and the yield of icariin can reach 3.93 g / L and 2.92 g / L. Therefore, the amino acid of the action of the alpha-L-rhamnose glycosidase and the icariin is subjected to site-directed mutagenesis, the alpha-L-rhamnose glycosidase mutant is obtained, especially the mutant N325A and V337G, has excellent enzymatic properties, so that the preparation process of icariin has the advantages of simple operation, low cost, environmental friendliness and the like, has a wide application prospect, and provides an important tool enzyme for the next step of producing icariin.

[0049] The raw materials and equipment used in the present application are common raw materials and equipment in the art, unless otherwise specified; the methods used in the present application are conventional methods in the art, unless otherwise specified.

[0050] The above description is only a preferred embodiment of the present application, and does not limit the present application in any way, and any simple modification, change and equivalent transformation of the above embodiment according to the technical essence of the present application still belong to the protection scope of the technical solution of the present application.

Claims

1. An α-L-rhamnosidase mutant, characterized in that: The following single-point mutations were performed on the α-L-rhamnosidase, whose amino acid sequence is shown in SEQ ID NO.1, to obtain N325A and V337G.

2. The coding gene of the mutant as described in claim 1.

3. A recombinant expression vector carrying the encoding gene as described in claim 2.

4. The recombinant expression vector as described in claim 3, characterized in that: The recombinant expression vector is a plasmid, bacteriophage, or viral vector.

5. Genetically engineered bacteria carrying the encoding gene as described in claim 2.

6. The use of the mutant as described in claim 1 in the preparation of icariin I.

7. The use of the mutant as described in claim 1 in the preparation of icariin.

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