High active state yhhw protein and its application as flavonol 2,4-dioxygenase in catalyzing degradation of flavonol compounds
Patent Information
- Application Number
- CN202610737219.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-05-27
- Publication Date
- 2026-08-21
AI Technical Summary
[0003]现有技术的不足之处在于：YhhW蛋白在常规表达纯化条件下呈现较低的催化活性，其是否能够以高活性状态发挥2,4-双加氧酶功能，以及如何获得该高活性状态，迄今未见报道
[0021]与现有技术相比，本发明提供的一种高活态YhhW蛋白及其作为黄酮醇2,4-双加氧酶在催化降解黄酮醇类化合物中的应用，本发明在实验过程中制备得到的YhhW蛋白存在两种不同的活性状态，通过优化纯化与筛选方法成功获得了高活态YhhW蛋白。该高活态蛋白能够高效催化黄酮醇类底物发生C环2位和4位特异性断裂，生成特征缩酚酸中间体并释放一氧化碳，其催化效率显著优于常规低活态YhhW蛋白，为黄酮醇类化合物的酶法降解提供了全新的高效生物催化剂。
Smart Images
Figure CN122609650A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, specifically to a highly active YhhW protein and its application as a flavonol 2,4-dioxygenase in the catalytic degradation of flavonol compounds. Background Technology
[0002] Flavonols (such as kaempferol and quercetin) are widely found in fruits, vegetables, and traditional Chinese medicines, possessing various pharmacological activities including antioxidant, anti-inflammatory, and anticancer properties, and have significant application value in food nutrition and pharmaceutical health fields. The degradation and metabolism of flavonols depend on an oxidase called quercetin 2,4-dioxygenase (QueD), which catalyzes the specific cleavage of the flavonol C ring at positions 2 and 4, generating phenolic acid intermediates and releasing carbon monoxide. Currently known QueD compounds mainly originate from fungi (such as Aspergillus japonicus) and bacteria (such as Bacillus subtilis and Streptomyces), and their active centers contain Cu, respectively. 2+ Fe 2+ or Ni 2+ Metal ions. YhhW is a bicupin family protein found in the human gut Escherichia coli K12 strain. Previous studies have shown that it has quercetin-like activity, but its catalytic efficiency is low, and its catalytic mechanism has not been fully elucidated.
[0003] The limitation of existing technologies lies in the fact that the YhhW protein exhibits low catalytic activity under conventional expression and purification conditions. Whether it can function as a 2,4-dioxygenase in a highly active state, and how to achieve this highly active state, remains unreported. This restricts the application of the YhhW protein in studies of flavonol degradation and gut microbiota metabolic regulation.
[0004] In response to this, this application proposes a highly active YhhW protein and its application as a flavonol 2,4-dioxygenase in the catalytic degradation of flavonol compounds, in order to solve the above-mentioned problems. Summary of the Invention
[0005] The purpose of this invention is to provide a highly active YhhW protein and its application as a flavonol 2,4-dioxygenase in the catalytic degradation of flavonol compounds, in order to solve the problems in the prior art where the YhhW protein exhibits low catalytic activity under conventional expression and purification conditions, whether it can perform the 2,4-dioxygenase function in a highly active state, and how to obtain such a highly active state.
[0006] To achieve the above objectives, the present invention provides the following technical solution:
[0007] In a first aspect, this application provides a method for the catalytic degradation of flavonols by a highly active YhhW protein, comprising the following steps:
[0008] The gene encoding the YhhW protein was introduced into host cells, and after induction of expression, the bacterial cells were collected and broken down to obtain a crude extract containing the YhhW protein.
[0009] The crude extract was subjected to nickel column affinity chromatography, and impurities were eluted sequentially with buffers containing 10 mM, 20 mM, and 50 mM imidazole. The eluted fraction with flavonol degradation activity was then collected after elution with buffer containing 300 mM imidazole to obtain highly active YhhW protein.
[0010] The highly active YhhW protein was mixed with flavonol substrates in an oxygen-containing buffer system and subjected to an oxidative cleavage reaction at 25°C to 37°C, which caused the 2- and 4-position bonds of the flavonol C ring to break, generating condensed phenolic acid intermediates and releasing carbon monoxide.
[0011] The reaction was terminated by adding a 5% (v / v) formic acid acetonitrile solution to the reaction system. The enzyme protein was removed by ultrafiltration, the filtrate was collected, and the condensed phenolic acid intermediate or its hydrolysis product was obtained.
[0012] Furthermore, when collecting the elution fraction with flavonol degradation activity, kaempferol is used as the substrate, and the substrate consumption rate is monitored at a wavelength of 368 nm. The elution fraction with a substrate absorbance decrease of more than 90% per unit time is selected as the highly active YhhW protein.
[0013] Furthermore, in the oxidative cleavage reaction, the molar ratio of highly active YhhW protein to substrate is 1:25 to 1:50, the buffer system is ammonium acetate buffer or PBS buffer at pH 7.5, and the reaction time is 10 to 20 minutes.
[0014] Furthermore, the phenolic acid intermediate is 2,4-dihydroxy-6-((4-hydroxybenzoyl)oxy)benzoic acid KIP or 2-((3,4-dihydroxybenzoyl)oxy)-4,6-dihydroxybenzoic acid (QIP), and is monitored and quantified in real time by high performance liquid chromatography-mass spectrometry-HPLC-MS in negative ion mode with characteristic ion peaks at m / z 289 or m / z 305.
[0015] Furthermore, the amount of carbon monoxide released was detected in real time using a portable infrared carbon monoxide analyzer, and the amount of CO generated by the known QueD enzyme catalyzing the same substrate was used as a positive control to verify that the catalytic mechanism of the highly active YhhW protein belongs to the 2,4-dioxygenase type.
[0016] Furthermore, the enzyme kinetic parameters of the highly active YhhW protein for flavonol substrates were determined by fitting the Michaelis-Menten equation:
[0017] Michaelis constant K when quercetin is used as a substrate mThe value was 9.49 ± 0.75 μM.
[0018] When kaempferol is used as a substrate, K m The value was 7.97 ± 1.41 μM.
[0019] Furthermore, after the reaction is terminated, the enzyme protein is removed by centrifugation at 4°C and 4000 g using a 10 kDa ultrafiltration tube, and the filtrate is collected for subsequent product separation or analysis.
[0020] Secondly, this application provides the application of the highly active YhhW protein prepared by the method described in the first aspect in the catalytic degradation of flavonols.
[0021] Compared with existing technologies, this invention provides a highly active YhhW protein and its application as a flavonol 2,4-dioxygenase in the catalytic degradation of flavonols. The YhhW protein prepared in this invention exhibits two different activity states during the experimental process. Through optimized purification and screening methods, a highly active YhhW protein was successfully obtained. This highly active protein can efficiently catalyze the specific cleavage of flavonol substrates at the C2 and C4 positions, generating characteristic phenolic acid intermediates and releasing carbon monoxide. Its catalytic efficiency is significantly superior to that of conventional low-activity YhhW protein, providing a novel and highly efficient biocatalyst for the enzymatic degradation of flavonols. Attached Figure Description
[0022] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.
[0023] Figure 1 SDS-PAGE electrophoresis image of the protein provided in the embodiments of the present invention;
[0024] Figure 2 The ultraviolet spectrum (A) and fluorescence emission spectrum (B) of kaempferol catalyzed by YhhW and YhhW* provided for embodiments of the present invention;
[0025] Figure 3 This is a substrate spectrum of kaempferol catalyzed by protease, provided in an embodiment of the present invention.
[0026] Figure 4 Chromatogram (A) and mass spectrum (B) of the reaction of hPirin, YhhW*, and QueD catalyzing kaempferol provided in the embodiments of the present invention;
[0027] Figure 5Comparison of secondary mass spectra of YhhW* (A), QueD (B) enzymatic reactants and KIP (C) provided in embodiments of the present invention;
[0028] Figure 6 This is a CO diagram showing the release of kaempferol catalyzed by YhhW* and QueD, provided in an embodiment of the present invention.
[0029] Figure 7 Standard curves for QIP and KIP provided for embodiments of the present invention; (A) QIP; (B) KIP;
[0030] Figure 8 Kinetic equation curves (Hill equations) for two flavonol substrates provided in embodiments of the present invention: (A1) YhhW*-kaempferol; (A2) YhhW*-quercetin; (B1) QueD-kaempferol; (B2) QueD-quercetin;
[0031] Figure 9 The ultraviolet spectrum of the product over time is provided in the embodiments of the present invention. Detailed Implementation
[0032] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.
[0033] As attached Figure 1 To be continued Figure 9 As shown:
[0034] Example 1:
[0035] Preparation of highly active YhhW protein:
[0036] 1.1 Construction of the expression carrier:
[0037] The gene encoding the YhhW protein of *E. coli* strain K12 was amplified by PCR, then cloned into the pET-28a(+) vector by double digestion with NdeI and XhoI to construct the recombinant expression plasmid pET-28a-YhhW. After sequencing verification, it was transformed into *E. coli* BL21(DE3) competent cells.
[0038] 1.2 Induced Expression:
[0039] Single colonies were picked and inoculated into LB liquid medium containing 50 μg / mL kanamycin, and cultured overnight at 37°C with shaking at 220 rpm. They were then transferred at a 1:100 ratio to TB medium (12 g / L tryptone, 24 g / L yeast extract, 4 mL / L glycerol, and TB salt solution) and cultured at 37°C until OD reached. 600The concentration was 0.6–0.8. 0.5 mM IPTG and 0.5 mM NiCl2 were added to a final concentration, and expression was induced at 25°C and 160 rpm for 18–20 hours.
[0040] 1.3 Cell collection and disruption:
[0041] Collect bacterial cells by centrifugation at 4℃ and 8000 rpm for 10 minutes, and wash twice with buffer (50 mM K2HPO4·3H2O, 50 mM KH2PO4, 10% glycerol, pH 7.5).
[0042] The bacterial cells were resuspended in buffer at a ratio of 1:10 (w / v), and lysozyme was added to a final concentration of 0.2 mg / mL. The mixture was then sonicated on ice (250 W, 2 seconds on, 3 seconds off, total time 30 minutes). The mixture was centrifuged at 12000 rpm for 1 hour at 4°C. The supernatant was then filtered through a 0.45 μm filter to obtain the crude extract.
[0043] 1.4 Purification by nickel column affinity chromatography:
[0044] The crude extract was loaded onto a Ni-NTA column pre-equilibrated with buffer at a flow rate of 1 mL / min. Impurities were eluted sequentially with 100 mL each of buffers containing 10 mM, 20 mM, and 50 mM imidazole (flow rate 2 mL / min). The target protein was then eluted with buffer containing 300 mM imidazole, and the elution peak was collected (detected at 280 nm UV). The collected solution was transferred to a 10 kDa ultrafiltration tube and concentrated to approximately 500 μL by centrifugation at 4°C and 4000 rpm. The solution was then washed three times with 5 mL of buffer. Protein concentration was determined, aliquoted, and flash-frozen in liquid nitrogen for storage at -80°C. Figure 1 As shown, SDS-PAGE electrophoresis revealed that the protein exhibited a single main band at approximately 28 kDa, with a purity >95%.
[0045] 1.5 Activity screening of highly active YhhW protein:
[0046] The purified protein was reacted with 50 μM kaempferol as a substrate in 50 mM ammonium acetate buffer (pH 7.5) at 37°C for 10 minutes. The wavelengths from 320 to 550 nm were scanned using a UV-Vis spectrophotometer. Figure 2 As shown, if the substrate absorption peak at 368 nm decreases rapidly (>90% decrease within 10 minutes) and no characteristic absorption peak at 430 nm is generated, it is identified as a highly active YhhW protein (named YhhW*). Figure 3 As shown, if an absorption peak of 430 nm appears, it indicates low-activity YhhW protein.
[0047] Example 2:
[0048] Highly active YhhW protein-catalyzed degradation of kaempferol and product characterization:
[0049] 2.1 Construction of the reaction system:
[0050] Add the following to a 1.5 mL centrifuge tube in sequence: 465 μL of 50 mM ammonium acetate buffer (pH 7.5), 25 μL of 1 mM kaempferol methanol solution (final concentration 50 μM), and 10 μL of 2 μM high-activity YhhW protein (final concentration 0.04 μM). Mix well and incubate at 25°C for 10 minutes.
[0051] 2.2 Reaction Termination and Sample Preparation:
[0052] The reaction was terminated by adding 500 μL of 5% (v / v) formic acid acetonitrile solution and vortexing to mix. The reaction solution was transferred to a 10 kDa ultrafiltration tube, centrifuged at 4000 rpm for 10 minutes at 4 °C, and the lower filtrate was collected and filtered through a 0.22 μm organic phase filter membrane before analysis.
[0053] 2.3 High-performance liquid chromatography-mass spectrometry (HPLC-MS) analysis:
[0054] Chromatographic conditions: ChromaiLotus C18 column (4.6×250 mm, 5 μm); mobile phase A was 0.1% formic acid in water, and mobile phase B was 0.1% formic acid in methanol; gradient elution program: 0–2 min, 20%–30% B; 2–3.5 min, 30%–50% B; 3.5–7 min, 50%–70% B; 7–18 min, 70% B; 18–20 min, 70%–20% B; 20–25 min, 20% B; flow rate 1 mL / min; column temperature 18℃; injection volume 10 μL.
[0055] Mass spectrometry conditions: electrospray ionization source, negative ion mode, scan range m / z 100~600.
[0056] like Figure 4 As shown, the highly active YhhW protein catalyzed the kaempferol reaction, resulting in a new chromatographic peak at a retention time of 11.0 min. Figure 4 As shown in Figure A, it is consistent with the QueD catalytic product and the KIP standard. Figure 4 As shown in Figure B, this peak corresponds to a quasi-molecular ion peak at m / z 289. Figure 5As shown, the fragment ion in the secondary mass spectrometry is m / z 151, which is in perfect agreement with the KIP standard. This indicates that the product is 2,4-dihydroxy-6-((4-hydroxybenzoyl)oxy)benzoic acid (KIP).
[0057] 2.4 Carbon monoxide (CO) detection:
[0058] Add 100 μM kaempferol and 4 μM high-activity YhhW protein (or 4 μM QueD as a positive control) to a 15 mL centrifuge tube and react at 25°C for 20 minutes. Insert the probe of a portable infrared carbon monoxide analyzer approximately 1 cm above the liquid surface to monitor CO concentration in real time. Figure 6 As shown, the amount of CO released by kaempferol catalyzed by the highly active YhhW protein is comparable to that of QueD, confirming that its catalytic mechanism belongs to the flavonol 2,4-dioxygenase type.
[0059] Example 3:
[0060] Referring to Example 2, the substrate was replaced with 50 μM quercetin, while other conditions remained the same. HPLC-MS analysis showed that the reaction produced a characteristic product with a retention time of 10.5 min, a quasi-molecular ion peak at m / z 305, and a secondary mass spectrometry fragment at m / z 151, consistent with the quercetin intermediate QIP. CO release was also detected.
[0061] Example 4:
[0062] Enzyme kinetic parameter determination:
[0063] 4.1 Establishing the standard curve:
[0064] Prepare a series of KIP or QIP standard solutions with concentrations (1–200 μM), and determine the peak area using the HPLC-MS method described in Example 2.3 in multiple reaction monitoring (MRM) mode. Figure 7 As shown, a standard curve is plotted with concentration on the x-axis and peak area on the y-axis. The equation of the KIP standard curve is y = 121628x - 3065.03 (R²). 2 = 0.9984), the equation of the QIP standard curve is y = 6064.07x - 200.39 (R = 0.9984). 2 = 0.9978).
[0065] 4.2 Determination of initial reaction rate:
[0066] Add 490 μL of ammonium acetate buffer, 5 μL of 2 μM high-activity YhhW protein (or QueD), and 5 μL of substrate at different concentrations (1–200 μM) (kaempferol or quercetin) to a 1.5 mL centrifuge tube. Incubate at 25°C (YhhW* for 10 min, QueD for 4 min). Terminate the reaction by adding 500 μL of 5% formic acid acetonitrile. After ultrafiltration, determine the amount of intermediate formed by HPLC-MS. Calculate the product concentration based on the standard curve and convert it to the initial reaction rate v (μM·min). -1 ).
[0067] 4.3 Dynamic Fitting:
[0068] A scatter plot was drawn with substrate concentration [S] on the x-axis and initial reaction rate v on the y-axis. The Hill equation was used for nonlinear fitting (Origin software, Analysis → Fitting → Nonlinear Curve Fit). Figure 8 As shown, the highly active YhhW protein catalyzes the K+ of quercetin. m The K catalyzed by kaempferol was 9.49 ± 0.75 μM. m The maximum reaction rate was 7.97 ± 1.41 μM; max The values were 1.48 ± 0.04 μM·min. -1 and 1.14±0.06 μM·min -1 .
[0069] Example 5:
[0070] Product stability and separation:
[0071] After reacting according to the method in Example 2, the stop solution was subjected to 10 kDa ultrafiltration to remove the enzyme protein. The filtrate was extracted three times with ethyl acetate, and the organic phases were combined and concentrated by rotary evaporation. The resulting solid was separated by preparative HPLC (C18 column, methanol / water gradient) to obtain the purified KIP product. Figure 9 As shown, the product can be stably stored at 4°C for 8 days, at 25°C for 50 hours, and at 37°C for 20 hours.
[0072] As shown above, the highly active YhhW protein prepared by the method of this invention can efficiently catalyze the 2,4-dioxygenation reaction of flavonol substrates (kaempferol, quercetin), generating characteristic phenolic acid intermediates and releasing CO. Its catalytic mechanism is consistent with that of known QueD enzymes. The reaction process can be clearly characterized by HPLC-MS and CO detection, providing a new tool enzyme for the enzymatic degradation of flavonols and the study of gut microbiota metabolism.
[0073] The YhhW protein prepared in this invention exhibits two distinct active states during the experimental process. Through optimized purification and screening methods, a highly active YhhW protein was successfully obtained. This highly active protein can efficiently catalyze the specific cleavage of flavonol substrates at positions 2 and 4 of the C-ring, generating characteristic phenolic acid intermediates and releasing carbon monoxide. Its catalytic efficiency is significantly superior to that of conventional low-activity YhhW protein, providing a novel and highly efficient biocatalyst for the enzymatic degradation of flavonols.
[0074] This invention provides a standardized operating procedure from gene expression, affinity chromatography gradient elution, activity screening to catalytic reaction, product separation and identification, achieving stable acquisition of highly active YhhW protein and reliable evaluation of its catalytic performance. This method not only helps elucidate the physiological function of YhhW protein in regulating flavonol metabolism in the gut microbiota, but can also be directly applied to the biotransformation of flavonols, the targeted removal of flavonols from food, and the preparation of related metabolic intermediates. It has advantages such as simple operation, high catalytic specificity, and easy product detection.
[0075] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.
Claims
1. A method for the catalytic degradation of flavonols by highly active YhhW protein, characterized in that, Includes the following steps: The gene encoding the YhhW protein was introduced into host cells, and after induction of expression, the bacterial cells were collected and broken down to obtain a crude extract containing the YhhW protein. The crude extract was subjected to nickel column affinity chromatography, and impurities were eluted sequentially with buffers containing 10 mM, 20 mM, and 50 mM imidazole. The eluted fraction with flavonol degradation activity was then collected after elution with buffer containing 300 mM imidazole to obtain highly active YhhW protein. The highly active YhhW protein was mixed with flavonol substrates in an oxygen-containing buffer system and subjected to an oxidative cleavage reaction at 25°C to 37°C, which caused the 2- and 4-position bonds of the flavonol C ring to break, generating condensed phenolic acid intermediates and releasing carbon monoxide. The reaction was terminated by adding a 5% (v / v) formic acid acetonitrile solution to the reaction system. The enzyme protein was removed by ultrafiltration, the filtrate was collected, and the condensed phenolic acid intermediate or its hydrolysis product was obtained.
2. The method for catalytic degradation of flavonols by highly active YhhW protein according to claim 1, characterized in that, When collecting the elution fractions with flavonol degradation activity, kaempferol was used as the substrate, and the substrate consumption rate was monitored at a wavelength of 368 nm. The elution fractions with a substrate absorbance decrease of more than 90% per unit time were selected as highly active YhhW proteins.
3. The method for catalytic degradation of flavonols by highly active YhhW protein according to claim 1, characterized in that, In the oxidative cleavage reaction, the molar ratio of highly active YhhW protein to substrate is 1:25 to 1:50, the buffer system is ammonium acetate buffer or PBS buffer at pH 7.5, and the reaction time is 10 to 20 minutes.
4. The method for catalytic degradation of flavonols by highly active YhhW protein according to claim 1, characterized in that, The phenolic acid intermediate is 2,4-dihydroxy-6-((4-hydroxybenzoyl)oxy)benzoic acid KIP or 2-((3,4-dihydroxybenzoyl)oxy)-4,6-dihydroxybenzoic acid (QIP), and is monitored and quantified in real time by high performance liquid chromatography-mass spectrometry-HPLC-MS in negative ion mode with characteristic ion peaks at m / z 289 or m / z 305.
5. The method for catalytic degradation of flavonols by highly active YhhW protein according to claim 1, characterized in that, The amount of carbon monoxide released was detected in real time using a portable infrared carbon monoxide analyzer, and the amount of CO generated by the known QueD enzyme catalyzing the same substrate was used as a positive control to verify that the catalytic mechanism of the highly active YhhW protein belongs to the 2,4-dioxygenase type.
6. The method for catalytic degradation of flavonols by highly active YhhW protein according to claim 1, characterized in that, The enzyme kinetic parameters of the highly active YhhW protein for flavonol substrates were determined by fitting the Michaelis-Menten equation: Michaelis constant K when quercetin is used as a substrate m The value was 9.49 ± 0.75 μM. When kaempferol is used as a substrate, K m The value was 7.97 ± 1.41 μM.
7. The method for catalytic degradation of flavonols by highly active YhhW protein according to claim 1, characterized in that, After the reaction is terminated, the enzyme protein is removed by centrifugation at 4°C and 4000 g using a 10 kDa ultrafiltration tube, and the filtrate is collected for subsequent product separation or analysis.
8. The application of the highly active YhhW protein prepared by the method according to any one of claims 1-7 in the catalytic degradation of flavonols.