Novel thermophilic alpha-L-rhamnosidase
By mining and optimizing the thermophilic α-L-rhamnosidase YJ-Rha72 from the hot spring metagenome, the problems of low catalytic efficiency and poor stability of existing enzymes were solved, and efficient and stable conversion of rutin to isoquercetin was achieved, which is suitable for industrial applications under high temperature conditions.
Patent Information
- Application Number
- CN202510909666.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-02
- Publication Date
- 2025-09-16
AI Technical Summary
Existing α-L-rhamnosidase has low catalytic efficiency, insufficient selectivity, and poor thermal stability, making it difficult to meet the needs of industrial production, resulting in high cost and low purity for the conversion of rutin to isoquercetin.
The thermophilic α-L-rhamnosidase YJ-Rha72 was extracted from the hot spring metagenome of Yuanjiang County, Yunnan Province. Its amino acid sequence was optimized and heterologously expressed in Escherichia coli. Purification by Ni-NTA affinity chromatography obtained an efficient and stable α-L-rhamnosidase suitable for biotransformation under high temperature conditions.
The efficient hydrolysis of rutin to produce isoquercetin was achieved at 75°C, with a conversion rate of up to 100%, which significantly improved the thermal stability and selectivity of the enzyme and reduced industrial production costs.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of enzyme gene engineering and biochemical engineering, and particularly relates to a novel thermophilic alpha-L-rhamnosidase. Background Art
[0002] α-L-Rhamnosidase (α-L-Rhamnosidase, EC 3.2.1.40) is a class of enzymes that can catalyze the hydrolysis of terminal L-rhamnose in glycosides such as flavonoids, terpenes, and saponins, including α-1, α-1, 2, α-1, 3, α-1, 4, and α-1, 6 glycosidic bonds, such as rutin, naringin, hesperidin, and neohesperidin. α-L-Rhamnosidase was first discovered in celery seeds in 1938, and subsequently found in bacteria, fungi, animals, and other organisms. Among them, enzymes derived from microorganisms have become a research hotspot because they are easy to produce on a large scale, such as those from Bacillus velezensis, Lactobacillus, Bacillus α-L-rhamnosidase from bacteria such as sp., and from Aspergillus niger, Aspergillus aculeatus, Pichia pastoris α-L-rhamnosidases from fungi such as α-L-rhamnosidases. Currently, about 60 α-L-rhamnosidases have been characterized (https: / / www.cazy.org / Home.html).
[0003] α-L-rhamnosidase can be used to debitter citrus juice, enhance the flavor of wine, and produce isoquercetin, prunin, rhamnose, etc. It has important value in the cosmetics, food, and pharmaceutical industries. Among them, the preparation of isoquercetin by hydrolysis of rutin by α-L-rhamnosidase is an important application direction.
[0004] Rutin, also known as rutin, is a flavonoid glycoside compound (quercetin-3-O-rutinoside) widely found in plants. It exhibits significant antioxidant, anti-inflammatory, anti-tumor, and cardiovascular protective activities. However, the rutin molecule's rutinosyl group (a disaccharide composed of glucose and rhamnose) results in low water solubility and bioavailability, severely limiting its application in medicine, food, and cosmetics.
[0005] Currently, the industrial conversion of rutin into more active monosaccharides (such as isoquercetin) or quercetin is primarily accomplished through acid hydrolysis or chemical methods. However, these reactions require harsh conditions such as strong acid and high temperature, which can lead to structural damage to isoquercetin and quercetin, resulting in low yields. They also generate large amounts of acidic wastewater, resulting in high treatment costs and environmental pollution. Furthermore, they produce byproducts (such as quercetin dimers), which affect purity and lead to poor specificity.
[0006] Enzymatic hydrolysis, with its mild conditions, high specificity, and environmental friendliness, has become an ideal alternative. α-L-rhamnosidase specifically hydrolyzes the terminal α-1,6-glycosidic bond of rutin, releasing rhamnose to produce isoquercitrin (quercetin-3-O-glucoside), which has a bioavailability over 10 times that of rutin and exhibits both higher stability and pharmacological activity. However, existing α-L-rhamnosidases have low catalytic efficiency, with specific activities for rutin generally less than 50 U / mg, making industrial production expensive. Furthermore, they suffer from insufficient substrate selectivity, with some enzymes also hydrolyzing the glucosidic bond to form free quercetin, reducing the yield of the target product, isoquercitrin. Furthermore, the enzymes have poor thermal stability and a narrow pH tolerance, making them difficult to adapt to industrial reaction systems.
[0007] Therefore, developing an efficient, highly selective, and highly stable α-L-rhamnosidase and its application process to achieve the green and efficient conversion of rutin to isoquercetin is of great significance to enhancing the added value of flavonoids and promoting the high-value utilization of natural products. Summary of the Invention
[0008] The first object of the present invention is to provide a thermophilic α-L-rhamnosidase YJ-Rha72, the amino acid sequence of which is shown in SEQ ID No. 1, which was mined from a hot spring metagenome in Yuanjiang County, Yunnan Province, and was isolated from Escherichia coli. Escherichia coli The enzyme was heterologously expressed in BL21 and sequence identity analysis showed that it was a novel α-L-rhamnosidase.
[0009] A second object of the present invention is to provide an amino acid sequence encoding the α-L-rhamnosidase YJ-Rha72, which includes an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID No. 1, or an amino acid sequence having at least 95% identity with the amino acid sequence shown in SEQ ID No. 2, or an amino acid sequence having at least 95% identity with the amino acid sequence shown in SEQ ID No. 3, or an amino acid sequence having at least 89% identity with the amino acid sequence shown in SEQ ID No. 4, or an amino acid sequence having at least 95% identity with the amino acid sequence shown in SEQ ID No. 5, or an amino acid sequence having at least 94% identity with the amino acid sequence shown in SEQ ID No. 6.
[0010] The present invention claims to protect the amino acid sequence of the α-L-rhamnosidase YJ-Rha72.
[0011] The present invention uses metagenomic sequencing of hot spring samples from Yuanjiang County, Yunnan Province, and obtains α-L-rhamnosidase by binning, assembly, and annotation. YJ-Rha72The gene was optimized and synthesized based on the codon table of Escherichia coli, and then recombined into the pET-28a plasmid to obtain a gene containing α-L-rhamnosidase. YJ-Rha72 The recombinant plasmid of the gene is transformed into the host bacteria E coli Heterologous expression was performed in BL21 cells. The cells were induced with 0.5 mM IPTG at 16°C for 20 h to obtain bacterial cells. The cells were then broken and purified using a Ni-NTA affinity chromatography column. SDS-PAGE electrophoresis was used to detect whether pure α-L-rhamnosidase YJ-Rha72 was obtained. Effects of the Invention
[0012] The thermophilic α-L-rhamnosidase YJ-Rha72 provided herein has the ability to hydrolyze rhamnosides containing α-1, α-1, 2, and α-1, 6 glycosidic bonds, such as pNPR, rutin, naringin, hesperidin, and neohesperidin. Rutin hydrolysis is particularly active, with 1 mM rutin converted to 1 mM rhamnose in 20 minutes. The enzyme has an optimal reaction temperature of 75°C and an optimal pH of 6.0. This may facilitate its application in industrial applications requiring high temperatures, such as the bioconversion of rhamnosides such as rutin and naringin, as well as for debittering citrus juice and enhancing wine flavor. BRIEF DESCRIPTION OF THE DRAWINGS
[0013] Figure 1 This is the SDS-PAGE electrophoresis result of the purification of α-L-rhamnosidase YJ-Rha72 in Example 2. Marker represents protein standard; Vector represents E. coli BL21 (DE3) cell extract sample; CE represents the crude enzyme solution in Example 1; PE represents the purified enzyme solution in Example 2.
[0014] Figure 2 This is the optimal pH diagram of α-L-rhamnosidase YJ-Rha72 in Example 3.
[0015] Figure 3 This is the optimum temperature diagram of α-L-rhamnosidase YJ-Rha72 in Example 3.
[0016] Figure 4 This is a graph showing the substrate specificity of α-L-rhamnosidase YJ-Rha72 in Example 3. pNPR represents p-nitrophenyl-α-L-rhamnopyranoside, pNPG represents p-nitrophenyl-β-D-glucopyranoside, Naringin represents naringin, Hesperidin represents hesperidin, Neohesperidin represents neohesperidin, and Rutin represents rutin.
[0017] Figure 5This is a graph showing the conversion rate of α-L-rhamnosidase YJ-Rha72 hydrolyzing rutin to prepare isoquercetin in Example 4. DETAILED DESCRIPTION
[0018] The present invention will be further described below in conjunction with the accompanying drawings and examples to facilitate understanding of the present invention by those skilled in the art. However, the present invention is not limited in any way, and any changes or improvements made based on the teachings of the present invention fall within the scope of protection of the present invention. In the present embodiments, the methods are operated according to conventional methods unless otherwise specified.
[0019] The present invention is a novel thermophilic α-L-rhamnosidase YJ-Rha72, which includes an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID No. 1, or an amino acid sequence having at least 95% identity with the amino acid sequence shown in SEQ ID No. 2, or an amino acid sequence having at least 95% identity with the amino acid sequence shown in SEQ ID No. 3, or an amino acid sequence having at least 89% identity with the amino acid sequence shown in SEQ ID No. 4, or an amino acid sequence having at least 95% identity with the amino acid sequence shown in SEQ ID No. 5, or an amino acid sequence having at least 94% identity with the amino acid sequence shown in SEQ ID No. 6.
[0020] The theoretical molecular weight of the novel thermophilic α-L-rhamnosidase YJ-Rha72 is 118.01 kDa.
[0021] The optimum temperature of the novel thermophilic α-L-rhamnosidase YJ-Rha72 is 75° C., and the optimum pH is 6.0.
[0022] Example 1 α-L-rhamnosidase YJ-Rha72 E. coli Heterologous expression in BL21 1. α-L-rhamnosidase YJ-Rha72 Gene mining Sediment samples were collected from Reshuitang Hot Spring in Yuanjiang County, Yunnan Province. Microbial genomic DNA was extracted and subjected to metagenomic DNA second-generation sequencing. Sequencing results were quality-controlled, and the quality-control data were binned, assembled, and annotated to obtain amino acid sequences encoding amino acid sequences similar to known α-L-rhamnosidases (e.g., sequences with more than 20% sequence identity and an expected value (E-value) of 1e 20 The open reading frame (ORF) of the amino acid sequence of the nucleotide ... YJ-Rha72 α-L-rhamnosidase YJ-Rha72The gene was optimized and synthesized by Sangon Biotech Co., Ltd. based on the codon table of E. coli, and the recombinant plasmid vector pET-28a- YJ- Rha72 , a histidine tag was added to the C-terminus. The recombinant plasmid was transformed into E coli JM109 preservation and copying, α-L-rhamnosidase YJ-Rha72 E. coli Expression in BL21.
[0023] 2. Plasmid Extraction Containing recombinant plasmid E coli JM109 was cultured in LB medium for 12-16 h, and α-L-rhamnosidase was extracted using the SanPrep column-type plasmid DNA miniprep kit according to the instructions. YJ-Rha72 The recombinant plasmid pET-28a of the gene.
[0024] 3. Conversion Take 20 μL of recombinant plasmid and add it to the ice water mixture. E. coli BL21 (DE3) competent cells were pipetted to mix thoroughly and then placed on ice for 30 min; heat-shocked in a 42°C water bath for 90 s, immediately removed from the ice bath for 5 min; 500 μL of antibiotic-free LB liquid medium was added and cultured at 37°C, 220 rpm for 30 min; the above bacterial solution was mixed thoroughly and 50 μL was evenly spread on an LB plate containing kanamycin sulfate (final concentration 50 μg / mL); after inoculation, the plate was first cultured upright at 37°C for 30 min, then inverted for 12 h-16 h to obtain the recombinant plasmid. E. coli BL21(DE3) expression strain.
[0025] 4. Inducible expression of α-L-rhamnosidase YJ-Rha72 Pick from LB plate E. coli BL21 monoclonal cells were inoculated into fresh LB liquid medium containing resistance (final concentration of kanamycin was 50 μg / mL) and cultured at 37°C and 220 rpm for 12-16 h to obtain recombinant plasmids. E. coli BL21 seed solution.
[0026] 5. Seed solution preparation Inoculate the seed solution into 300 mL of fresh LB liquid medium containing resistance at a 2% inoculum volume (6 mL) and culture at 37°C, 220 rpm until the bacterial solution OD 600 The concentration of the microorganisms was 0.4-0.6, IPTG (final concentration 0.5 mM) was added, cultured at 16°C and 180 rpm for 20 h, centrifuged at 4000 rpm / min for 30 min, washed the cells 2-3 times, collected the cells, and frozen at -20°C until broken.
[0027] 6. Obtaining Crude Enzyme Solution Resuspend the cells at a concentration of 1 g per 5 mL of nondenaturing lysis buffer, add lysozyme, mix thoroughly (final concentration 0.5 mg / mL), and incubate on ice for 30 min. Disrupt the cells using a sonicator at 40 W for 30 min (2 s of sonication followed by 4 s of rest). Centrifuge the cell lysate at 8000 rpm for 15 min, and collect the supernatant. Incubate the supernatant at 70°C for 2 h and centrifuge it at 8000 rpm for 15 min. This supernatant is the crude enzyme solution.
[0028] Example 2 Purification of α-L-rhamnosidase YJ-Rha72 The enzyme was purified using a Ni-NTA prepacked column (prepacked gravity column purification kit: BBI) using the histidine tag of α-L-rhamnosidase YJ-Rha72, and its purity was determined by denaturing SDS-PAGE electrophoresis. The specific steps are as follows: 1. Take a Ni-NTA prepacked column of corresponding specifications according to the required loading capacity and let the storage buffer flow out by gravity.
[0029] 2. Equilibrate the column with twice the column volume of Binding / Wash Buffer and use a flow rate of 0.5-1 mL / min to allow the Buffer to slowly drain from the resin.
[0030] 3. Prepare the sample solution by mixing the protein extract with Binding / Wash Buffer at a ratio of 1:1 so that the total volume of the sample solution is twice the column volume.
[0031] 4. Add the sample solution to the column and collect the flow-through into a centrifuge tube. If there is excess sample, re-apply the sample. Re-circulating the sample once can improve the binding force between the sample and the filler.
[0032] 5. Wash the column with two column volumes of Binding / Wash Buffer and collect the flow-through. Repeat this step in a new collection tube until the absorbance of the flow-through at 280 nm is close to the baseline.
[0033] 6. Elute the histidine-tagged protein from the column with twice the column volume of Elution Buffer. Repeat this step twice and store the eluate separately until the absorbance of the eluate at 280 nm is close to the baseline.
[0034] 7. Column material post-treatment: Elute the column material with 5 volumes of Elution Buffer, then equilibrate the column material with 5 volumes of Binding / Wash Buffer, and finally wash the column material with 5 volumes of ddH2O. Add 20% ethanol protective solution and store at 2-8°C.
[0035] 8. Use SDS-PAGE to determine whether α-L-rhamnosidase YJ-Rha72 is purified using Ni-NTA prepacked columns.
[0036] SDS-PAGE results showed that the purified enzyme solution showed a single band with a molecular weight close to 100 kDa ( Figure 1 ), which is close to the theoretical molecular weight of YJ-Rha72 (118.01 kDa). This indicates that pure α-L-rhamnosidase YJ-Rha72 was obtained by purification via Ni-NTA prepacked column.
[0037] Example 3 Enzymatic Characteristics of α-L-rhamnosidase YJ-Rha72 The activity of α-L-rhamnosidase YJ-Rha72 was determined using the dinitrosalicylic acid method (DNS). Specifically, 100 μL of the reaction mixture was incubated in a PCR tube at 75°C, pH 6.0 for 30 min, followed by a 5-min ice bath (to terminate the reaction). 100 μL of DNS was added and mixed, followed by a 10-min incubation at 90°C for color development, followed by a 5-min ice bath. OD was measured using a microplate reader. 540 , and then calculate the enzyme activity. The reaction mixture consists of 100 mM citric acid-sodium hydrogen phosphate buffer, 1 mM rutin, and 25 μL of the enzyme solution from Example 1. One unit of enzyme activity (U) is defined as the amount of enzyme required to hydrolyze 1 μmol of rhamnose per minute at 75°C and pH 6.0.
[0038] Optimal pH of α-L-rhamnosidase YJ-Rha72 The optimal pH (3.6-9) of YJ-Rha72 was determined in 100 mM buffer: citric acid-sodium hydrogen phosphate buffer (pH 3.6-7.5), Tris-HCl (pH 7.5-9).
[0039] The results showed that the optimum pH of α-L-rhamnosidase YJ-Rha72 was 6.0, and its activity exceeded 74% in the pH range of 5-7.5. Figure 2 shown.
[0040] 2 Optimum temperature of α-L-rhamnosidase YJ-Rha72 The optimal temperature (30-95°C) was determined in 100 mM citric acid-sodium hydrogen phosphate buffer, pH 6.0. The enzyme was heated in a water bath at 30-95°C for 10 min, and the relative enzyme activity was calculated.
[0041] The results showed that the optimum temperature of α-L-rhamnosidase YJ-Rha72 was 75°C and it was active at 50-85°C (residual activity exceeded 34.9%). Figure 3 shown.
[0042] 3 Substrate specificity of α-L-rhamnosidase YJ-Rha72 In order to explore the substrate specificity of YJ-Rha72, activity tests were carried out using 1 mM pNPG, pNPR, naringin, hesperidin, neohesperidin, and rutin as substrates.
[0043] The results showed that α-L-rhamnosidase YJ-Rha72 could hydrolyze pNPR, naringin, hesperidin, neohesperidin, rutin and other substances, and the activity of hydrolyzing rutin was the highest. Figure 4 shown.
[0044] Example 4 Preparation of Isoquercetin by Hydrolysis of Rutin with α-L-Rhamnosidase YJ-Rha72 To determine the efficiency of α-L-rhamnosidase YJ-Rha72 in converting rutin to isoquercetin, the amount of reducing sugar (rhamnose) produced within 2 h was measured.
[0045] The results showed that 1 mM of rutin could produce 1 mM reducing sugar after reacting with α-L-rhamnosidase YJ-Rha72 for 20 min, indicating that the conversion rate of rutin by α-L-rhamnosidase YJ-Rha72 was about 100%. Figure 5 shown.
Claims
1. A novel α-L-rhamnosidase or a functional analogue thereof, comprising an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID No. 1, or an amino acid sequence having at least 95% identity with the amino acid sequence shown in SEQ ID No. 2, or an amino acid sequence having at least 95% identity with the amino acid sequence shown in SEQ ID No. 3, or an amino acid sequence having at least 89% identity with the amino acid sequence shown in SEQ ID No. 4, or an amino acid sequence having at least 95% identity with the amino acid sequence shown in SEQ ID No. 5, or an amino acid sequence having at least 94% identity with the amino acid sequence shown in SEQ ID No.
6.
2. A DNA sequence encoding the α-L-rhamnosidase or a functional analogue thereof according to any one of claim 1.
3. A recombinant expression vector containing the DNA sequence according to claim 2.
4. A host cell transformed, transduced or transfected with the recombinant expression vector according to claim 3.
5. Use of the DNA sequence according to claim 2 in the preparation of α-L-rhamnosidase.