α-L-rhamnosidase BtRha78A mutant and its preparation method and application

Abstract

This invention relates to the fields of enzyme engineering and genetic engineering, specifically to the α-L-rhamnosidase BtRha78A mutant, its preparation method, and its applications. The amino acid sequence of the mutant differs from the BtRha78A enzyme shown in SEQ ID NO.1 by a single amino acid mutation: the phenylalanine at position 44 is mutated to aspartic acid or serine. The mutant of this invention exhibits higher catalytic efficiency in a shorter time, providing theoretical guidance for the industrial-scale production of isoquercetin from the large-scale hydrolysis of rutin.

Description

Technical Field

[0001] The invention relates to the fields of enzyme engineering and genetic engineering, and in particular to an alpha-L-rhamnosidase BtRha78A mutant and a preparation method and application thereof. Background Art

[0002] α-L-rhamnosidase is an important biotechnological enzyme that can hydrolyze L-rhamnose at the end of most natural products, such as rutin, hesperidin, ginsenosides, etc. It is widely distributed in bacteria, fungi, plants, animal tissues, etc. It is used in industry, medicinal pretreatment and food production, for example, it can debitter fruit juice, enhance grape juice and improve wine aroma by deglycosylation of terpenes, etc. Rutin, derived from plants such as Sophora japonica and Tartary Buckwheat, belongs to flavonols and is a disaccharide glycoside of quercetin. It has a wide range of pharmacological activities, including antioxidant, antihypertensive, anticancer, anti-influenza and anti-inflammatory effects. Isoquercetin also has this pharmacological activity, but because of the presence of glycosides, it has the advantages of improving compound stability, increasing water solubility, reducing toxic side effects, and improving drug specificity and targeting, which has attracted the attention of more and more scholars. However, there are still a lot of technical barriers in the large-scale industrial and food production of α-L-rhamnosidase. Its application is limited by factors such as the low activity of natural α-L-rhamnosidase and unsuitable reaction conditions.

[0003] With the continuous rise of protein engineering technology, the protein sequence has been modified and edited, and the desired enzyme has been molecularly modified to finally achieve our target enzyme. At present, molecular modification includes directed evolution, rational design and semi-rational design. Directed evolution is to introduce randomly distributed mutations at a low frequency in a gene of interest, and then select mutant proteins with the desired properties. Directed evolution can enable relatively rapid engineering of enzymes without the need for in-depth understanding of the structure-function relationship. The main limitation is that a high-throughput screening method must be developed. Unlike directed evolution, rational design requires an in-depth understanding of the sequence, structure and functional relationship of the modified enzyme, and finally determines the target of the desired mutation, and performs site-directed mutagenesis to study its properties. The latest progress uses a combination of directed evolution and rational design, namely semi-rational design, to make up for the above defects. Simply put, this method uses protein sequence and structural information, as well as related calculations, to construct a smaller but intelligent mutation library to target specific residues. Focusing on specific amino acid positions will lead to a significant reduction in library size, thereby screening excellent mutants.

[0004] The patent number is CN202010653391.2, and its name is a kind of α-rhamnosidase, its encoding gene, its expression and application, which involves a high temperature resistant and thermostable α-rhamnosidase, which can almost completely convert rutin into isoquercetin under suitable conditions, and its catalytic activity is still limited, but it does not involve molecular modification technology; the patent number is CN202110692714.3, and its name is a kind of rhamnosidase mutant and its preparation method and application, which semi-rational design of α-L-rhamnosidase obtains highly active combined mutants, and is not used for the large-scale preparation of isoquercetin. Therefore, it is a meaningful research direction to semi-rational design of α-L-rhamnosidase BtRha78A to obtain an excellent α-L-rhamnosidase mutant with high efficiency in hydrolyzing rutin to produce isoquercetin. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides an α-L-rhamnosidase BtRha78A mutant and a preparation method and application thereof.

[0006] In a first aspect, the present invention provides an α-L-rhamnosidase BtRha78A mutant, the amino acid sequence of which has an amino acid mutation with the BtRha78A enzyme shown in SEQ ID NO.1, wherein the phenylalanine at position 44 is mutated to aspartic acid or serine.

[0007] In a second aspect, the present invention provides a DNA molecule encoding the α-L-rhamnosidase BtRha78A mutant.

[0008] Furthermore, the DNA molecule has any one of the nucleotide sequences shown in a1-a3:

[0009] a1, the nucleotide sequence shown in SEQ ID NO.2 or SEQ ID NO.3;

[0010] a2, the complementary sequence of the nucleotide sequence in a1;

[0011] a3. A nucleotide sequence encoding the α-L-rhamnosidase BtRha78A mutant, which is different from a1 and a2 due to the degeneracy of the genetic code.

[0012] In a third aspect, the present invention provides a recombinant expression vector having the DNA molecule.

[0013] In a fourth aspect, the present invention provides a host cell comprising the recombinant expression vector.

[0014] In a fifth aspect, the present invention provides a method for preparing the α-L-rhamnosidase BtRha78A mutant, comprising the following steps: inducing the host cell to express the α-L-rhamnosidase BtRha78A mutant, and isolating and purifying the expressed α-L-rhamnosidase BtRha78A mutant.

[0015] In a sixth aspect, the present invention provides the use of the α-L-rhamnosidase BtRha78A mutant in catalyzing rutin to prepare isoquercetin.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] 1. The present invention uses PyMOL to analyze the amino acids at the entrance of the substrate channel of α-L-rhamnosidase BtRha78A, determines the 44th as the mutation target, and semi-rationally designs a new enzyme, thereby obtaining an excellent mutant with higher catalytic efficiency in a short time, which provides theoretical guidance for the industrialization of large-scale hydrolysis of rutin to prepare isoquercetin.

[0018] 2. The whole cell and pure enzyme were used to catalyze the reaction of rutin, and the mutants were reacted at 37°C and pH 6.5. Under the whole cell condition, when the reaction time was 60 min, the conversion rate of WT was 33.9%, and the conversion rates of F44S and F44D were 45.8% and 33.1% higher than that of WT, respectively. Under the pure enzyme condition, when the reaction time was 20 min, the conversion rate of WT was only 10.7%, while the conversion rates of F44S and F44D were increased by 2.55 times and 1.38 times, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 are the amino acid residues at the substrate channel and its entrance in Example 1.

[0020] Figure 2 The relative activity of the whole cells of the alanine scanning mutant in Example 1 on rutin catalysis.

[0021] Figure 3 This is the nucleic acid electrophoresis diagram of the whole plasmid PCR product in Example 2.

[0022] Figure 4 This is a flow chart of the catalytic reaction in Example 2.

[0023] Figure 5 This is the standard curve of rutin and isoquercetin in Example 2.

[0024] Figure 6 This is the evaluation of the rutin hydrolysis activity of the wild-type and mutant whole cells in Example 2.

[0025] Figure 7This is the SDS-PAGE of the target protein after the wild type and mutants were purified in Example 3. Lane 1 in the figure represents Maker, and lanes 2-5 represent the target proteins after WT, F44A, F44D, and F44S purification, respectively.

[0026] Figure 8 This is the time curve of rutin catalysis by the high-activity mutant under pure enzyme conditions in Example 3.

[0027] Fig. 9 The catalytic activity of the excellent mutant on rutin under the pure enzyme condition in Example 3 when the reaction time is 20 min. DETAILED DESCRIPTION

[0028] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0029] The plasmid pET-28a-BtRha78A containing α-L-rhamnosidase BtRha78A (amino acid sequence as shown in SEQ ID NO.1) involved in the following examples is preserved in the applicant's laboratory and is disclosed in the document "Characterization of aglycoside hydrolase family 78a-L-rhamnosidase from Bacteroides thetaiotaomicron VPI-5482 and identification of functional residues".

[0030] Example 1: Determination of mutation site of α-L-rhamnosidase BtRha78A

[0031] Pymol was used to study the spatial structure of α-L-rhamnosidase BtRha78A (PDB: 3CIH) from human intestinal bacteria Bacteroides thetaiotaomicron VPI-5482, and the amino acid residues at the substrate channel and its entrance were analyzed. 17 amino acid residues were identified as mutation targets, namely E39, F44, P45, P46, F47, W48, E233, T379, I380, W435, V436, F437, V438, D439, Y610, G611, and R612. The protein structure is shown in Figure 1 shown.

[0032] Alanine scanning mutants with 17 mutation sites were constructed and after heterologous expression, whole cells catalyzed rutin to produce isoquercetin, and the relative activities of the 17 mutants were compared with those of the WT, as shown in Figure 2. Figure 2 As shown, it was found that only F44A had a relative activity that was 32.8% higher than WT, and the relative activities of the other sites were all lower than that of the wild type. We believe that this site has a high degree of tolerance to mutation and has high plasticity, so we chose the F44 site for site-directed saturation mutagenesis.

[0033] Example 2: Preparation and catalytic reaction of α-L-rhamnosidase BtRha78A mutant

[0034] 1. Construction of F44 site-directed saturation mutant library

[0035] (1) Primer design is shown in Table 1;

[0036] Table 1 Primers for site-directed saturation mutagenesis of F44

[0037]

[0038] (2) Plasmid pET-28a-BtRha78A WT A small amount of extraction was performed (refer to the instructions of the SanPrep column plasmid DNA small amount extraction kit). The whole plasmid PCR was performed using the plasmid pET-28a-BtRha78A as a template. The PCR system and PCR program are shown in Tables 2 and 3;

[0039] Table 2 Full plasmid PCR system

[0040]

[0041] Table 3 Whole plasmid PCR program

[0042]

[0043] (3) Nucleic acid electrophoresis: PCR products were identified by 1% agarose gel electrophoresis, with DL10000 DNA Marker as the standard molecular weight. The nucleic acid electrophoresis diagram is shown in Figure 3 As shown, the product bands are between 7000-10000 bp, indicating that the whole plasmid PCR amplification is successful.

[0044] (4) Digestion of template: Add 1 μL QuickCut Dpn I to the PCR product and place in a 37°C metal bath to digest the template for 2 h;

[0045] (5) The above PCR products, wild-type plasmid and BtRha78A-F44A plasmid (as a positive control) were transformed into Escherichia coli BL21 (DE3) competent cells and cultured in a 37°C incubator for 12 h.

[0046] 2. Screening of F44 site-directed saturation mutant library and preparation of α-L-rhamnosidase BtRha78A mutant

[0047] (1) Initial screening:

[0048] Pick single clones: add 200 μL 2×YT liquid culture medium containing kanamycin to each well of the master plate, pick 252 clones with a sterilized toothpick, each 96-well master plate (3 in total) contains 6 wild-type, 4 positive controls and 2 blank controls, and culture at 37°C with constant temperature shaking at 150 rpm overnight (about 15 h);

[0049] Transfer and storage of bacterial solution: add 160 μL of 2×YT liquid culture medium containing kanamycin to each well of the daughter plate, then take 20 μL of bacterial solution from the mother plate and add it to the corresponding daughter plates. The daughter plates are cultured at 37°C and 150 rpm constant temperature shaking for about 5.5 h until the bacterial density OD600 = 1.0. Add 50 μL of 70% glycerol to each well of the mother plate and temporarily store in a -40°C refrigerator;

[0050] Induce expression: add 20 μL of 5 mM IPTG to each well of the daughter plate, and induce expression at 16°C, 150 rpm overnight (about 15 h);

[0051] Screening reaction: centrifuge the induced expression bacterial solution at 3700rpm for 30min, discard the supernatant, add 100μL premix to each well (the premix contains: rutin (final concentration of 1mM), β-D-glucosidase TnBgl1A-DM pure enzyme solution (final concentration of 0.02mg / mL) and pH 6.5 50mM PB buffer), mix by pipetting, react at 37℃, 650rpm for 2h (α-L-rhamnosidase reaction), react in an oven at 80℃ for 30min (β-D-glucosidase reaction), add 100μL methanol to each well to terminate the reaction, centrifuge at 3700rpm for 30min (to remove bacteria and debris), take 60μL of the supernatant and add it to the detection plate containing 140μL pH 10.0 50mM sodium carbonate (quercetin in alkaline pH 10.0, self-oxidized to form polymers, with a maximum absorption peak at 320nm, known as the characteristic peak of quercetin), and allowed to stand at room temperature for 30min, and the absorbance value at 320nm was detected by an enzyme marker.

[0052] Initial screening results: After the initial screening, 75 mutants were selected from 252 mutants for rescreening.

[0053] (2) Rescreening:

[0054] The clones obtained from the primary screening were transferred from the mother plate to 5 mL 2×YT liquid medium containing kanamycin at a 1% inoculum, cultured at 37°C, 180 rpm until OD600 = 1.0, and 5 μL 0.5 M IPTG was added to induce expression overnight at low temperature for 12-14 h, and the bacteria were collected;

[0055] Screening reaction: Each group of reactions were repeated three times. The experimental process is as follows Figure 4 shown.

[0056] Results of rescreening: 75 mutants were rescreened, and 36 mutants with catalytic activity 15% higher than that of the wild type were selected for final screening.

[0057] 3. Final screening

[0058] (1) Mutant induced expression

[0059] The mutant strains obtained by repeated screening were inoculated with 1% inoculum into 5mL 2×YT liquid culture medium containing kanamycin (final concentration of 50ng / mL) for strain activation, and cultured at 37°C, 210rpm for 8-9h; the activated seed liquid was transferred to 100mL 2×YT liquid culture medium containing kanamycin at 1% inoculum, and cultured at 37°C, 180rpm to OD600=1.0; IPTG (final concentration of 0.5mM) was added, and expression was induced overnight at 16°C, 180rpm for 12-14h; the bacterial solution was centrifuged at 4°C, 8000rpm for 10min, and the bacteria were collected.

[0060] (2) Mutant catalytic reaction

[0061] The hydrolysis reaction of mutant whole cells to rutin was as follows Figure 4 shown.

[0062] (3) Quantitative analysis of isoquercetin was performed using HPLC, and the chromatographic conditions were as follows:

[0063] High performance liquid chromatography: 1525 / 2487 high performance liquid chromatography system (Waters China Co., Ltd.)

[0064] Chromatographic column: Hypersil OSD2-C18 chromatographic column (Dalian Yilite Analytical Instrument Co., Ltd.)

[0065] Detected substances: Rutin and isoquercetin

[0066] Detection wavelength: 260nm

[0067] Run time: 16 minutes

[0068] Flow rate: 1mL / min

[0069] Mobile phase: 0.5% glacial acetic acid: acetonitrile = 84:16

[0070] Sample volume: 10 μL

[0071] (4) Preparation of HPLC standard curve

[0072] The concentration and peak area standard curves were prepared with rutin and isoquercetin standards to calculate substrate concentration, product concentration and conversion rate. The standard samples of rutin and isoquercetin were prepared with methanol solution in the ratios of 0.20mM: 0.00mM, 0.18mM: 0.02mM, 0.16mM: 0.04mM, 0.14mM: 0.06mM, 0.12mM: 0.08mM, 0.10mM: 0.10mM, 0.08mM: 0.12mM, 0.06mM: 0.14mM, 0.04mM: 0.16mM, 0.02mM: 0.18mM, 0.00mM: 0.20mM, and after passing through a 0.22μm organic filter membrane, HPLC was used for detection. Figure 5 As shown, the standard curve of rutin is y=9.1x, and the standard curve of isoquercetin is y=8.2x.

[0073] Final screening results: 17 mutants with catalytic activity 10% higher than the wild type were selected and sequenced to obtain their sequences, and the excellent mutants F44D (encoding gene as shown in SEQ ID NO.2) and F44S (encoding gene as shown in SEQ ID NO.3) of α-L-rhamnosidase BtRha78A were prepared. Figure 5 The conversion rate of rutin and isoquercetin was calculated by standard curve, such as Figure 6 As shown, the conversion rates of WT, F44A, F44D and F44S were 33.9%, 54.0%, 67.0% and 79.7%, respectively. F44A, F44D and F44S increased the rutin conversion rate by more than 20%, among which F44S and F44D were 45.8% and 33.1%, respectively.

[0074] Example 3: Preparation of reaction time curves of excellent mutants (F44D and F44S) for rutin catalytic activity

[0075] 1. Expression and purification of BtRha78A wild type and its mutants:

[0076] (1) Expression of wild-type BtRha78A and its mutants, the same as the heterologous expression step in Example 2;

[0077] (2) The collected bacterial sludge was resuspended in pH 8.0 50 mM PB buffer at a ratio of 1:12 (w / v), then sonicated for 50 min, centrifuged at 13000 rpm for 30 min, and the supernatant was collected for nickel column affinity purification;

[0078] (3) Affinity purification The purification steps are as follows:

[0079] Table 4 Affinity purification purification steps

[0080]

[0081] In a 4°C chromatography cabinet, the affinity-purified target protein was dialyzed twice against 2L pH 8.0 50mM phosphate buffer, then dispensed into Eppendorf tubes, and the protein concentration was determined using an ultra-micro UV-visible spectrophotometer NanoDrop 2000. The purity of the target protein was detected by SDS-PAGE electrophoresis and temporarily stored in a -20°C refrigerator.

[0082] (4) Detect the purity of the target protein by SDS-PAGE electrophoresis. The electrophoresis results are as follows: Figure 7 As shown, the sizes of the four proteins were consistent with the theoretical molecular weights, and the purity was over 95%.

[0083] 2. Effects of BtRha78A wild type and its mutants on rutin catalytic activity at different reaction times

[0084] Determination of reaction time curve of enzymatic hydrolysis of rutin by high-activity mutants: The hydrolysis reaction process refers to the catalytic reaction of Example 2, the bacterial suspension is replaced with 1 mg / mL pure enzyme solution, and the catalytic activity of the wild type and mutants on rutin at different times within 0-80 min is determined, and three parallel reactions are performed in each group.

[0085] The reaction time curve of rutin hydrolysis by the highly active mutant under pure enzyme conditions, such as Figure 8 As shown, within 0-80 min, the conversion rates of the wild type and mutants showed an upward trend with increasing reaction time.

[0086] Under pure enzyme conditions, Fig. 9 As shown, when the reaction time was 20 min, the conversion rate of WT was only 10.7%, while the conversion rates of F44S and F44D increased by 2.55 times and 1.38 times, respectively.

[0087] In summary, the present invention molecularly modifies α-L rhamnosidase BtRha78A, semi-rationally designs the amino acids at the entrance of the substrate channel, and obtains excellent mutants with short reaction time and high catalytic efficiency through high-throughput screening. The natural flavonoid disaccharide rutin derived from the cheap raw materials of buckwheat and Sophora japonica isoquercetin is efficiently prepared by biotransformation, thereby laying a foundation for the industrial production and application of isoquercetin.

[0088] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0089] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. An α-L-rhamnosidase BtRha78A mutant, characterized in that The protein is obtained by mutating the phenylalanine at position 44 of the BtRha78A enzyme shown in SEQ ID NO.1 to aspartic acid or serine.

2. A DNA molecule encoding the α-L-rhamnosidase BtRha78A mutant according to claim 1.

3. The DNA molecule according to claim 2, characterized in that The nucleotide sequence of the DNA molecule corresponding to the α-L-rhamnosidase BtRha78A mutant obtained by mutating the phenylalanine at position 44 of the BtRha78A enzyme shown in SEQ ID NO.1 to aspartic acid is shown in SEQ ID NO.2, and the nucleotide sequence of the DNA molecule corresponding to the α-L-rhamnosidase BtRha78A mutant obtained by mutating the phenylalanine at position 44 of the BtRha78A enzyme shown in SEQ ID NO.1 to serine is shown in SEQ ID NO.

3.

4. A recombinant expression vector comprising the DNA molecule of claim 3.

5. An engineered Escherichia coli bacteria comprising the recombinant expression vector according to claim 4.

6. The method for preparing the α-L-rhamnosidase BtRha78A mutant according to claim 1, characterized in that: The method comprises the following steps: inducing the Escherichia coli engineered bacteria described in claim 5 to express an α-L-rhamnosidase BtRha78A mutant, and isolating and purifying the expressed α-L-rhamnosidase BtRha78A mutant.

7. Use of the α-L-rhamnosidase BtRha78A mutant according to claim 1 in catalyzing the preparation of isoquercetin from rutin.

Images