Alpha-l-rhamnosidase mutant, method for constructing same, and use thereof

Abstract

This invention belongs to the fields of biocatalysis and protein engineering technology, and particularly relates to an α-L-rhamnosidase mutant, its construction method, and its applications. The α-L-rhamnosidase mutant is an α-L-rhamnosidase with the amino acid sequence shown in SEQ ID NO.1, where arginine at position 783 is mutated to alanine. The amino acid sequence of the α-L-rhamnosidase mutant is shown in SEQ ID NO.2. The mutant enzyme exhibits significantly enhanced catalytic activity, 4.56 times that of the wild-type enzyme. It can efficiently and specifically catalyze the conversion of naringin to prunine without the formation of byproducts. Compared to the wild-type enzyme, the mutant enzyme R783A shows a 3.56-fold increase in the conversion efficiency of naringin, demonstrating its promising application potential in the bioprocessing of prunine.

Description

Technical Field

[0001] This invention belongs to the fields of biocatalysis and protein engineering technology, and particularly relates to an α-L-rhamnosidase mutant, its construction method and application. Background Technology

[0002] α-L-rhamnosidase (EC 3.2.1.40) is a class of glycosidic hydrolases that specifically hydrolyze terminal α-L-rhamnosidic bonds. Its substrate range is broad, including flavonoid glycosides (such as naringin and rutin), terpenoid glycosides (such as ginsenosides), and other natural compounds containing α-L-rhamnosyl groups. This enzyme can act on various glycosidic bonds, including α-1,2, α-1,3, α-1,4, α-1,6, and α-1, showing significant application prospects in the food, pharmaceutical, and chemical industries, particularly in the preparation of bioactive substances, improving fruit juice quality, and enhancing the aroma of alcoholic beverages.

[0003] Pronin can be generated by the specific hydrolysis of naringin by α-L-rhamnosidase, and it lacks one rhamnosyl group compared to naringin. Compared to naringin, pronin has better solubility and exhibits various physiological activities, including cholesterol reduction, antioxidant activity, and antiviral activity. However, the low abundance of pronin in nature and the difficulty of direct extraction limit its large-scale acquisition. Currently, its preparation methods mainly include chemical extraction and enzymatic conversion. Among these, enzymatic hydrolysis of naringin to produce pronin has advantages such as mild conditions, high specificity, high conversion efficiency, and environmental friendliness, and therefore has attracted widespread attention.

[0004] The conversion of naringin to prunine via α-L-rhamnosidase is a highly efficient biosynthetic pathway. However, the catalytic efficiency of natural α-L-rhamnosidase still has significant room for improvement. To further optimize its industrial application performance, enzyme molecular modification using protein engineering techniques has become an important research direction in this field. In particular, employing rational design strategies such as site-directed mutagenesis to enhance the catalytic efficiency of α-L-rhamnosidase is of great significance for achieving efficient and large-scale enzymatic preparation of prunine. Summary of the Invention

[0005] The purpose of this invention is to provide an α-L-rhamnosidase mutant, its construction method, and its application. The α-L-rhamnosidase mutant has significantly higher enzyme activity than the wild type and a higher conversion rate in catalyzing the conversion of naringin to prunine.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] An α-L-rhamnosidase mutant, wherein the α-L-rhamnosidase mutant is an α-L-rhamnosidase with the amino acid sequence as shown in SEQ ID NO. 1, wherein the arginine (Arg / R) at position 783 is mutated to alanine (Ala / A), and the amino acid sequence of the α-L-rhamnosidase mutant is shown in SEQ ID NO. 2, and is named R783A.

[0008] Furthermore, the nucleotide sequence of the α-L-rhamnosidase is shown in SEQ ID NO. 3; the nucleotide sequence of the α-L-rhamnosidase mutant is shown in SEQ ID NO. 4.

[0009] Another object of the present invention is to provide a method for constructing an α-L-rhamnosidase mutant, wherein the α-L-rhamnosidase mutant is obtained by site-directed mutagenesis and screening, comprising the following steps:

[0010] S1. Using the gene encoding wild-type α-L-rhamnosidase as a template, a site-directed mutagenesis gene fragment was constructed using site-directed mutagenesis technology;

[0011] S2. The mutant gene is cloned into an expression vector to obtain a recombinant expression vector;

[0012] S3. Transform the recombinant expression vector into host cells to obtain recombinant bacteria;

[0013] S4. Cultivate and induce expression in the recombinant bacteria, and purify the α-L-rhamnosidase mutant from the bacterial cells.

[0014] Furthermore, the host cell is bacteria.

[0015] Furthermore, the host cell is Escherichia coli.

[0016] Furthermore, the carrier is a pET series carrier, preferably pET-28a(+).

[0017] Another object of the present invention is to provide an application of an α-L-rhamnosidase mutant in the catalytic conversion of naringin to prepare propranolol.

[0018] Furthermore, the catalytic reaction conditions for the α-L-rhamnosidase mutant in the conversion of naringin to proline are: pH 6.0-9.0 and temperature 50-90℃.

[0019] The advantages of this invention are:

[0020] This invention involves single- or multi-point mutations at sites “R386A, R477A, ​​E479A, D523A, Y587A, W590A, V596A, E782A, R783A, M792A, S530A” in the wild-type α-L-rhamnosidase. Through screening, an α-L-rhamnosidase mutant R783A with significantly enhanced enzyme activity was obtained, exhibiting 4.56 times the activity of the wild-type enzyme. This α-L-rhamnosidase mutant R783A specifically catalyzes the conversion of naringin to prunine, achieving a conversion rate of 98.3% without the formation of other byproducts. The α-L-rhamnosidase mutant R783A provided by this invention significantly improves catalytic efficiency, enhancing the enzyme's application potential in pharmaceuticals, chemicals, and food, particularly in the green biomanufacturing of high-value-added products such as prunine. Attached Figure Description

[0021] Figure 1 The results of SDS-PAGE analysis of wild-type α-L-rhamnosidase and its mutant enzymes are shown. In the figure, M: standard molecular weight protein, 1: wild type, 2: R477A, ​​3: R783A, 4: D523A, 5: E782A, 6: S530, 7: R477A, ​​8: V596A, 9: R386A, 10: Y587, 11: M792, 12: E479A.

[0022] Figure 2 This is a graph showing the relative activities of wild-type α-L-rhamnosidase and its mutant enzymes.

[0023] Figure 3 The results show the enzymatic properties of wild-type α-L-rhamnosidase and its mutant R783A. Among them, (a) shows the optimal reaction temperature of wild-type α-L-rhamnosidase and its mutant R783A; and (b) shows the optimal reaction pH of wild-type α-L-rhamnosidase and its mutant R783A.

[0024] Figure 4 The graph shows the conversion rate of naringin to prunine catalyzed by wild-type α-L-rhamnosidase and mutant R783A in Example 5.

[0025] Figure 5 HPLC chromatogram of the conversion of naringin to proline catalyzed by α-L-rhamnosidase R783A. Detailed Implementation

[0026] The present invention will now be described through illustrative specific embodiments, which do not limit the scope of the invention in any way. It should be noted, in particular, that all reagents used in the present invention are commercially available unless otherwise specified.

[0027] (a) The culture media involved in the examples are as follows:

[0028] LB liquid medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L;

[0029] LB solid medium: Add 2% agar powder to LB liquid medium.

[0030] (ii) Enzyme activity detection

[0031] Enzyme activity assay: The activity of α-L-rhamnoside was determined using p-nitrophenyl-α-L-rhamnoside (pNPR) as a substrate. At 50°C, 470 μL of phosphate buffer (100 mM, pH 7.0) was mixed with 10 μL of 10 mM pNPR and incubated for 2 min. 20 μL of enzyme solution was added to initiate the reaction, and the reaction was allowed to proceed accurately at 50°C for 5 min. Finally, 500 μL of 1 M sodium carbonate solution was added to terminate the reaction. The absorbance at 405 nm was measured immediately. The enzyme activity unit (U) is defined as the amount of enzyme required to catalyze the production of 1 μmol of p-nitrophenol per minute under the above assay conditions.

[0032] (III) Determination of the standard curve of p-nitrophenol

[0033] p-Nitrophenol standard solutions with concentrations of 0.1, 0.2, 0.5, 1.0, 1.5, and 2.0 mM were prepared, and the absorbance was measured at 405 nm. A standard curve was plotted with concentration on the x-axis and absorbance on the y-axis.

[0034] (iv) HPLC determination method for naringin and propranolol

[0035] The HPLC system used a C18 column (4.5 mm × 250 mm, 5 μm), with a mobile phase of 0.1% formic acid: acetonitrile = 7:3, a flow rate of 1 mL / min, a column temperature of 35 ℃, a detector wavelength of 270 nm, and an injection volume of 20 μL.

[0036] Example 1

[0037] Construction of wild-type strain of α-L-rhamnosidase

[0038] The α-L-rhamnosidase gene, synthesized after codon optimization, was cloned into the pET-28a(+) vector to obtain the recombinant plasmid pET-28a-α-Rha, whose amino acid sequence is shown in SEQ ID NO. 1. This plasmid was transformed into *E. coli* BL21(DE3) to obtain a wild-type recombinant strain. A single colony was inoculated into LB medium containing kanamycin (final concentration 50 μg / mL) and cultured overnight at 37°C as a seed culture. The seed culture was then transferred to fresh LB medium at a 1% inoculum and cultured at 37°C until the OD600 reached 0.6-0.8. IPTG was then added to a final concentration of 0.1 mM, and expression was induced at 20°C for 20 h. The bacterial cells were collected, resuspended in acid-water buffer (20 mM, pH 7.4), sonicated, and centrifuged to obtain the supernatant, which was the crude enzyme solution of the wild-type enzyme.

[0039] Example 2

[0040] Construction of α-L-rhamnosidase mutant

[0041] Based on the gene sequence of α-L-rhamnosidase, primer pairs for site-directed mutagenesis were designed, as shown in Table 1. Using the wild-type recombinant plasmid pET-28a-α-Rha as a template, site-directed mutagenesis was performed using a site-directed mutagenesis kit from TransGen Biotech Ltd. to obtain the mutant recombinant plasmid, and a mutant recombinant strain of α-L-rhamnosidase was constructed. The process included three steps: PCR amplification, digestion of the PCR product with DMT enzyme, and transformation.

[0042] Table 1. Mutant Primer Table

[0043]

[0044] (1) PCR amplification

[0045] The PCR reaction system and reaction process are as follows:

[0046] Table 2 PCR reaction system

[0047]

[0048] After thoroughly mixing the above reagents, PCR was performed. The PCR reaction program was as follows: pre-denaturation at 94℃ for 5 min; followed by 25 cycles: denaturation at 94℃ for 30 s, annealing at 65℃ for 30 s, extension at 72℃ for 1.5 min; after the cycles, final extension at 72℃ for 5 min; and finally storage at 4℃.

[0049] (2) Digest the PCR products with DMT enzyme

[0050] Add 1 μL of DMT enzyme to the PCR reaction solution obtained in step (1), mix gently, and react at 37°C for 1 h.

[0051] (3) Transformation

[0052] The digestion products were transformed into E. coli competent cells BL21(DE3), spread evenly on a solid medium containing kanamycin, and cultured overnight at 37°C. Clones were randomly selected for colony PCR identification and sequencing verification, and finally α-L-rhamnosidase mutants R386A, R477A, ​​E479A, D523A, Y587A, W590A, V596A, E782A, R783A, M792A, and S530A were obtained.

[0053] Example 3

[0054] Expression and purification of wild-type α-L-rhamnosidase and its mutant enzymes

[0055] Single colonies were inoculated into LB liquid medium containing kanamycin and activated overnight. Then, 1% of the colonies were inoculated into LB liquid medium and cultured with shaking at 37°C and 180 rpm for 3.5 h. Subsequently, IPTG was added to a final concentration of 0.1 mM, and the cells were cultured with shaking at 20°C and 180 rpm for 20 h to induce the expression of wild-type and mutant α-L-rhamnosidase. Cells were collected, resuspended in 20 mM phosphate-buffered saline (pH 7.4), and sonicated at low temperature for 20 min. The cell lysate was centrifuged at 4°C and 12,000 rpm for 30 min, and the supernatant was collected to obtain crude enzyme solutions of wild-type α-L-rhamnosidase and its mutant. The crude enzyme solution was filtered through a 0.45 μm filter membrane and then purified using an AKTA Pure protein purification system and a GE HisTrap HP nickel affinity chromatography column to obtain electrophoretically pure wild-type α-L-rhamnosidase and its mutant enzyme. SDS-PAGE analysis is shown below. Figure 1 As shown, the purified mutant enzyme has the same molecular weight as the wild-type enzyme, and both show a distinct band at approximately 106 kDa.

[0056] Example 4

[0057] Enzymatic properties of wild-type and mutant enzymes

[0058] (1) Enzyme activity assay

[0059] α-L-rhamnosidase activity assay: The total reaction volume was 1.2 mL, containing 50 mM phosphate buffer (pH 7.0), 3 mM pNPR substrate, and an appropriate amount of enzyme solution. After reacting for 3 min, the absorbance was measured at 405 nm. Each reaction was replicated. The relative enzyme activity of each mutant enzyme was calculated using wild-type enzyme activity as 100%. Results are as follows: Figure 2As shown, the activities of most mutant enzymes were increased to varying degrees compared with wild-type enzymes, with mutant R783A showing particularly outstanding activity, which was 4.56 times that of wild-type enzymes.

[0060] (2) Determination of the optimal reaction temperature

[0061] The activity of recombinant α-L-rhamnosidase was determined using the above method within a temperature range of 35-85℃. The total reaction volume was 1.2 mL, containing 50 mM phosphate buffer (pH 7.0), 3 mM pNPR substrate, and an appropriate amount of enzyme solution. After 3 min of reaction, the absorbance at 405 nm was measured, and parallel reactions were performed at each temperature point. The enzyme activity corresponding to the highest activity was defined as 100%, and the relative enzyme activity at each temperature was calculated. The results showed that the optimal reaction temperature for the wild-type enzyme was 60℃, while the optimal temperature for the mutant R783A increased to 80℃. The measurement results are as follows: Figure 3 As shown in (a).

[0062] (3) Determination of the optimal reaction pH

[0063] The recombinant enzyme activity was measured at 50°C in different pH buffers (50 mM, HAc-NaAc buffer for pH 3-6; phosphate buffer for pH 6-8; glycine-NaOH buffer for pH 9-10). The highest activity was defined as 100%, and the relative enzyme activity at each pH was calculated. The results showed that the optimal reaction pH for both the wild-type enzyme and its mutant R783A was 8.0. The measurement results are as follows: Figure 3 As shown in (b).

[0064] Example 5

[0065] α-L-rhamnosidase mutant catalyzes the conversion of naringin to prunine.

[0066] Pronin was prepared by catalyzing the conversion of naringin to wild-type α-L-rhamnosidase and its mutant R783A. The reaction was initiated by adding 2 mg / mL α-L-rhamnosidase or the mutant enzyme R783A to 2 mL of phosphate buffer (50 mM, pH 8.0) containing 20 mM naringin, and the reaction was carried out at 80 °C and 180 r / min. After 4 h of reaction, the results are as follows: Figure 4 As shown, the conversion rate of naringin catalyzed by wild-type α-L-rhamnosidase was 62.3%, while the conversion rate of naringin catalyzed by the mutant enzyme R783A was 98.7%. The high-performance liquid chromatography (HPLC) chromatogram of the naringin conversion process catalyzed by mutant R783A is shown below. Figure 5 As shown, the retention times of naringin and propranolol were 3.7 and 4.8 minutes, respectively.

Claims

1. An α-L-rhamnosidase mutant, characterized in that: The α-L-rhamnosidase mutant is an α-L-rhamnosidase with the amino acid sequence shown in SEQ ID NO. 1, in which arginine at position 783 is mutated to alanine. The amino acid sequence of the α-L-rhamnosidase mutant is shown in SEQ ID NO.

2.

2. The α-L-rhamnosidase mutant as described in claim 1, characterized in that: The nucleotide sequence of the α-L-rhamnosidase is shown in SEQ ID NO. 3; the nucleotide sequence of the α-L-rhamnosidase mutant is shown in SEQ ID NO.

4.

3. The application of the α-L-rhamnosidase mutant as described in claim 1 or 2 in the catalytic conversion of naringin to prepare proline.

4. The application according to claim 3, characterized in that: The catalytic reaction conditions for the α-L-rhamnosidase mutant in the conversion of naringin to proline are: pH 6.0-9.0 and temperature 50-90℃.

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