A beta-glucuronidase and uses thereof

Abstract

The present application belongs to the technical field of genetic engineering, and particularly relates to a beta-glucuronidase and application thereof. The present application discloses a novel beta-glucuronidase derived from intestinal flora of a colorectal cancer patient with a smoking history. Through metagenomic sequencing and chemical proteomics technology, a beta-glucuronidase capable of catalyzing the deglucuronidation reaction of tobacco carcinogen metabolite NNAL-Gluc and re-releasing toxic NNAL is identified and screened, so as to weaken the detoxification ability of the body and promote the occurrence and development of colorectal cancer. According to the screening of the inhibitor of the enzyme, the influence of tobacco hazards on intestinal health can be maximally reduced. The present application is suitable for mechanism research, active ingredient screening, intestinal flora intervention and related functional food development of tobacco-related colorectal cancer.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a β-glucuronidase and its applications. Background Technology

[0002] Smoking is a significant risk factor for colorectal cancer (CRC). A meta-analysis showed that former smokers had a 17% increased risk of CRC compared to non-smokers, and this risk increased linearly with increasing smoking intensity, duration, and years of smoking. The effect of smoking on CRC risk showed a dose-response relationship: for every additional 10 cigarettes smoked per day, the risk of CRC increased by 7.8%. Smoking significantly alters the composition and function of the gut microbiota. Studies have shown that harmful substances in cigarette smoke promote CRC development by regulating gut microbiota and related metabolites.

[0003] In addition to nicotine and tar, tobacco contains potent carcinogens such as nitrosamines. Nitrosamines in tobacco have been classified as Group 1 carcinogens by the International Agency for Research on Cancer (IARC) of the World Health Organization. The nitrosamine NNK (4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone) in tobacco smoke undergoes carbonyl reduction in the body to form NNAL (4-(methylnitrosamine)-1-(3-pyridyl)-1-butanol). The harmful nitrosamine NNAL can enter the human intestines through the digestive tract, systemic blood circulation, or liver metabolism, thereby harming intestinal health.

[0004] To reduce the carcinogenic risk of NNAL, a harmful nitrosamine in tobacco, and its precursor NNK, existing technologies mainly employ interventions such as reducing the formation of tobacco-specific nitrosamines (TSNAs), promoting detoxification metabolism, and inhibiting carcinogenic activation. For example, researchers have focused on enhancing the activity of phase II metabolic enzymes, including using natural active substances such as sulforaphane and isothiocyanates to promote UDP-glucuronyltransferase (UGTs)-mediated glucuronidation, accelerating the detoxification conversion of NNAL to NNAL-Glucose (4-(methylnitrosamine)-1-(3-pyridyl)-1-butanol glucuronide). Furthermore, some studies have attempted to reduce the formation of NNK / NNAL DNA adducts and the induction of tumorigenesis by inhibiting cytochrome P450 (CYP)-mediated metabolic activation. However, there is a neglected link in the above detoxification pathway: NNAL-Gluc is hydrolyzed back to NNAL by β-glucuronidase (GUS: β-glucuronidase, EC 3.2.1.31) in the intestine, thus producing intestinal toxicity. Currently, there are no effective interventions for the gut microbiota-mediated NNAL-Gluc reactivation process, especially lacking strategies to inhibit gut-derived specific GUS enzymes.

[0005] The human gut microbiota is a complex microbial community that plays a crucial role in maintaining intestinal homeostasis. This community expresses numerous enzymes that metabolize endogenous and exogenous substances through hydrolysis, reduction, and other reactions. Among these, the function of GUS enzymes is particularly critical. By catalyzing the hydrolysis of glucuronide conjugates, GUS enzymes reverse the body's "glucuronidation" detoxification process (i.e., attaching glucuronic acid molecules to compounds to facilitate their excretion in bile or urine), causing the original compound or its active / toxic metabolites to be released back into the gut. This process not only increases systemic drug absorption but may also lead to direct damage to the gut from toxic substances, thus significantly affecting the host's endogenous metabolic homeostasis and exogenous substance disposal. GUS enzymes significantly regulate metabolite levels by catalyzing the hydrolysis of glucuronide conjugates to release free drugs or endogenous metabolites.

[0006] Studies have shown that the abundance of certain harmful bacteria in the gut of smokers is significantly increased, and this dysbiosis may promote the occurrence and development of colorectal cancer through multiple mechanisms. Furthermore, the GUS enzyme produced by the gut microbiota is related to the metabolism of certain harmful substances in cigarettes. Harmful substances in cigarettes, such as nitrosamines, accumulate in the gut, and changes in the level of GUS enzymes in the gut microbiota can affect the metabolism of harmful substances in cigarettes in the human gut.

[0007] Therefore, there is an urgent need to provide a specific GUS enzyme that can metabolize NNAL-Gluc, a nitrosamine compound in tobacco, to lay the foundation for subsequent screening of specific GUS enzyme inhibitors. Summary of the Invention

[0008] The purpose of this invention is to provide a β-glucuronidase.

[0009] To achieve the above objectives, the present invention provides the following technical solution: The present invention provides a β-glucuronidase, the amino acid sequence of which is shown in SEQ ID NO.1.

[0010] The present invention also provides a gene fragment encoding the aforementioned β-glucuronidase.

[0011] Preferably, the nucleotide sequence of the gene fragment is as shown in SEQ ID NO.2.

[0012] The present invention also provides an expression vector containing the gene fragments described above.

[0013] The present invention also provides a host cell containing the expression vector described above.

[0014] Preferably, the host cell is Escherichia coli.

[0015] The present invention also provides the application of the above-described β-glucuronidase in the preparation of a reagent for in vitro catalytic hydrolysis of NNAL-Gluc.

[0016] The present invention also provides the application of the β-glucuronidase as a target in screening candidate drugs for inhibiting NNAL reactivation in the intestine.

[0017] The present invention also provides the application of the above-described β-glucuronidase as a target in the preparation of drugs or functional foods for reducing NNAL toxicity in the intestines of smokers or preventing smoking-related intestinal damage.

[0018] Preferably, the functional food is a probiotic product, dietary supplement, or health food containing active ingredients that specifically inhibit the activity of the enzymes described above.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a novel β-glucuronidase gene and protein sequence related to tobacco-specific nitrosamine NNAL. A GUS enzyme gene that can efficiently and specifically activate procarcinogens such as tobacco-specific nitrosamine NNAL was constructed. Inhibitors designed against this enzyme can reduce the impact of endogenous GUS enzyme reactivation of toxic substances in the human body, resulting in milder toxic side effects. This invention lays a research foundation for subsequent high-throughput drug screening, computer-aided drug design, and antibody drug development, and has broad application prospects.

[0020] This invention utilizes metagenomic sequencing and chemical proteomics technologies to screen and identify a β-glucuronidase capable of catalyzing the deglucuronization of NNAL-Gluc, a metabolite of tobacco carcinogens. This enzyme promotes the conversion of NNAL-Gluc to toxic free NNAL, enhancing local intestinal toxicity exposure, weakening the body's metabolic detoxification capacity for tobacco-related harmful substances, and participating in the regulation of colorectal cancer development. This invention further constructs and obtains this β-glucuronidase, and based on its structural and functional characteristics, screens for specific inhibitors, thereby effectively reducing the adverse effects of tobacco-derived harmful substances on intestinal health. This invention is applicable to mechanistic studies of tobacco-related colorectal cancer, screening of active ingredients, intervention in gut microbiota, and the development of related functional foods. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is the result of fecal GUS enzyme activity testing in colorectal cancer patients with a history of smoking; where P represents the patient and H represents the normal individual. Figure 2 Results of metagenomic sequencing analysis of patient feces; Figure 3 Western blotting image of GUS enzymes as a probe for chemical proteomics; Figure 4 This is a map of the recombinant expression vector for the GUS enzyme; Figure 5 Agarose gel electrophoresis image of the PCR product of the GUS enzyme gene; Figure 6 The results show the docking of GUS enzyme with the nitrosamine compound NNAL-Gluc. Detailed Implementation

[0023] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0024] Unless otherwise specified, the production processes, experimental methods, or testing methods involved in the embodiments of this invention are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, which are very clear and distinct in the relevant application areas. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0025] The various instruments, equipment, raw materials or reagents used in the embodiments of this invention are not subject to any special restrictions on their source. They are all conventional products that can be purchased through regular commercial channels and can be prepared according to conventional methods known to those skilled in the art.

[0026] Example 1 The fecal GUS enzyme activity test for colorectal cancer patients with a history of smoking includes the following steps: (1) After review and approval by the ethics committee, feces were collected from colorectal cancer patients with a history of smoking and normal individuals; (2) Take 50-70 mg of feces from patients (P1-P6) and normal individuals (H1-H5), add PBS at a volume ratio of 1:9, grind evenly, and centrifuge the supernatant at 4 ℃ and 5000 rpm for 20 min. Detect the protein content in the supernatant using a protein assay kit. Add 20 μL of supernatant to 60 μL of PBS and 20 μL of 3 mM p-nitrophenyl-β-D-glucuronide (pNP-Gluc). After reacting for 30 min, add 100 μL of 10% Na2CO3 to terminate the reaction. Measure the absorbance at 405 nm in 200 μL of the reaction solution in a 96-well plate. The results are as follows: Figure 1 As shown; (3) Metagenomic sequencing was performed on fresh feces. The sequencing results are as follows: Figure 2 As shown; Depend on Figure 1 , 2 The results showed that the level of GUS enzyme in the feces of colorectal cancer patients with a history of smoking was elevated. Analysis of metagenomic sequencing results revealed that the unique GUS gene count varied greatly among different individuals, suggesting that the specific GUS enzyme in the feces of colorectal cancer patients with a history of smoking may originate from certain specific bacterial genera.

[0027] Example 2 Chemical proteomics fishing for specific GUS enzymes includes the following steps: (1) Processing fecal samples: Processing 5-10 g fecal samples: Dissolve the sample in 25 mL buffer (25 mM HEPES, 25 mM NaCl, pH 6.5), vortex to mix, centrifuge at 4 ℃ and 5000 rpm for 5 min, collect the supernatant, dissolve the precipitate again in 25 mL solution and centrifuge under the same conditions, collect the supernatant, combine the two supernatants, and centrifuge twice more under the above conditions to further remove insoluble fibers, use a 30 kDa ultrafiltration tube to remove extracellular host proteins, replace the buffer, and retain the intestinal flora; (2) Total protein extraction: The supernatant was sonicated twice on an ultrasonic homogenizer, with each pulse lasting 0.5 s and the treatment time being 1.5 min. After each sonication, the lysis buffer was inverted to mix well. The lysis buffer was centrifuged at 4 ℃ and 5000 rpm for 20 min, and the supernatant was collected. The supernatant was washed by filtering with an ultrafiltration tube with a molecular weight cutoff of 30 kDa. Each time, 1.5 mL of supernatant was added to the ultrafiltration tube, centrifuged at 4 ℃ and 5000 rpm until 0.5 mL remained, the solution was added to 1.5 mL, and centrifuged again. The buffer was replaced three times, and protein quantification was performed using a protein assay kit, and the protein was analyzed using GUS (from E. coli). EcoUsing CUS enzyme as a reference standard, the total protein concentration was diluted to 1 mg / mL, aliquoted into 500 μL / tube, flash-frozen in liquid nitrogen, and stored at -80 °C. (3) Targeted capture of GUS enzyme using chemical proteomics: Take 50 μL of total protein (1 mg / mL) and 22.4 μL of GUS-specific probe (0.5 mg / mL), incubate in 200 μL solution for 2 h, and wash 3 times with a 10 kDa ultrafiltration tube to remove unbound probe. Pretreatment of magnetic beads: Equilibrate at room temperature, vortex the magnetic beads, transfer 160 μL of the magnetic bead suspension to a centrifuge tube, place it on a magnetic rack, let it stand for 2 min, remove the supernatant, remove the magnetic rack, add 1 ml of PBS, and vortex to mix. Place it on the magnetic rack again, remove the supernatant, remove the magnetic rack, transfer all the protein to a centrifuge tube containing the magnetic beads, add PBS buffer, cover with sealing film, and incubate at low temperature overnight. After enrichment, wash the magnetic beads and target GUS with PBS buffer. Place the centrifuge tube on a magnetic rack and let it stand for 2 min, remove the supernatant. Repeat washing 3-4 times to remove non-specific adsorption. Samples were prepared and Western blot analysis was performed. The results are as follows: Figure 3 As shown; The results showed that the expression level of GUS enzyme in the intestine of patients was significantly higher than that of healthy people, and the molecular weight distribution was significantly different from that of healthy people.

[0028] (4) Preprocessing for proteomics: Trypsin digestion of proteins: Add PBS buffer containing 6 M urea to the sample, add PBS buffer containing 200 mM dithiothreitol (DTT), and incubate in a metal bath at 37 ℃ for 60 min; add PBS buffer containing 400 mM iodoacetamide (IAA) to the sample, and continue incubation in a metal bath at 37 ℃ for 30 min, discard the supernatant, and keep the magnetic bead sample; wash with 1 ml PBS, discard the supernatant, and keep the magnetic bead sample; add 200 μL PBS to each sample group, and add 5 μL trypsin at the same time, and incubate overnight at 37 ℃ and 1000 rpm.

[0029] Peptide desalting: Wash the desalting column twice with 300 μL acetonitrile, centrifuge at 10,000 rpm for 1 min at 4 ℃, wash the desalting column twice with 300 μL 0.1% trifluoroacetic acid, centrifuge at 10,000 rpm for 1 min at 4 ℃, add the sample, centrifuge at 10,000 rpm for 1 min at 4 ℃, discard the eluent, wash 5-6 times with 300 μL 0.1% trifluoroacetic acid, centrifuge at 10,000 rpm for 1 min at 4 ℃, discard the eluent, wash twice with 300 μL 0.1% trifluoroacetic acid and 50% acetonitrile, centrifuge at 10,000 rpm for 1 min at 4 ℃, collect the eluent, which is 600 μL of desalted sample. Concentrate and dry the 600 μL sample under vacuum at 30 ℃ and 1500 rpm, and redissolve it with 20 μL 0.1% formic acid aqueous solution before performing protein mass spectrometry analysis.

[0030] Proteomics data processing: The database used was the human GUS enzyme gene sequence from the National Center for Biotechnology Information (NCBI). The database search and screening criteria were: unique peptides ≥ 2, LFQ intensity not equal to 0. By comparing the unique peptides of 5 normal individuals and 6 patients, one GUS enzyme was screened: WP_152074744.1 β-glucuronidase [Collinsella aerofaciens] CaGUS ).

[0031] Example 3 The construction of the PET-28a(+)-GUS recombinant plasmid includes the following steps: (1) Search for the GUS enzyme gene in the NCBI database; (2) The entire GUS gene was synthesized by Shanghai Sangon Biotech Co., Ltd., and cloned into the E. coli expression vector pET-28a(+) to construct the recombinant plasmid pET-28a(+)-GUS, as shown below. Figure 4 As shown, the gene has been correctly cloned into the pET-28a(+) expression vector. This plasmid was transformed into *E. coli* BL21(DE3) and plated on LB agar plates containing the antibiotic Kan, then incubated overnight at 37 °C. A single colony was picked and inoculated into 5 mL of LB liquid medium containing Kan, incubated overnight at 37 °C and 200 rpm, the plasmid was extracted, stored at -20 °C, and sequenced.

[0032] The results showed that the PET-28a(+)-GUS recombinant plasmid was successfully constructed.

[0033] The GUS enzyme has the amino acid sequence shown in SEQ ID NO. 1: MLYPQQTASRFVESLDGIWDFKLDDGSAFESKWFDAPLADAMTMPVPASYNDLKEGIDFRDFCGWAFYQRAIAVPEFVRSQRVMLRFSAVTHHAKVYLNGKLVCEHVCGFLPFEVQINDYLEPGDNLLTVAVDNVIDYTTLPVGGKANMMSGLLGGMGDATEDAPKKKVNNPNFDFFNYAGITRPVYIYTTPAEHIDDVELVADVQGETTDAPSATVSYKVAATGDAACKVEMFDVQGVKVAEGEGLEGELKLDQVTLWQPGAAYLYRVKVTFGEDVYELPYGVRTVRVKGTKFLINGRPFYFKGYGKHEDTFPTGRGENMPMNVKDLSLMKWQGANSFRTSHYPYSEEMMRLCDAEGIVVIDETTAVGVNLQFGGGANFGGEKITTFDPEHGVQTQEAHKQVIRDLVARDKNHACVVMWSIANEPDSSAEGAYDYFKPLYDLARELDPQKRPCTLVSVQGTTAVTDCSAKLSDAICLNRYYGWYFGGPNLVAPFKAMRAELDEWAKLGKPIIFTEFGADTVAGMHDTTPVMYTEEYQVDYYKANLAVMDDYPCVVGEQVWNFADFATSQSLLRVQGNKKGLFTRDRKPKLAAHFFRERWHEVPDFDYKA The nucleotide sequence of the GUS enzyme described is shown in SEQ ID NO. 2: Example 4 The induction and validation of recombinant GUS enzyme expression includes the following steps: (1) Structural prediction of the sequence was performed using AutoDock vina. The sequences of NNAL-Gluc (Compound CID: 183009) and the known GUS structure derived from E. coli K-12 were compared. EcoGUS (PDB: 3LPF), known source Mediterraneibacter gnavus GUS structure ( MgGUS Molecular docking and docking affinity scoring were performed using NNAL-Gluc (PDB: 6JZ8). The results showed a significant difference in docking results between NNAL-Gluc and MgGUS. Molecular docking as... Figure 6 As shown in Table 1, the scoring results indicate that CaGUS is more likely to undergo a catalytic reaction with NNAL-Gluc.

[0034] Table 1. AutoDock vina docking affinity scores for NNAL-Gluc (Compound CID: 183009) and two other proteins.

[0035] (2) Take BL21 competent cells, add plasmid at a ratio of 10:1, and incubate on ice for 25 min. Then heat shock at 42 ℃ for 90 s, and then place on ice for 3-4 min. Add LB liquid medium to the tube, incubate at 37 ℃ and 200 rpm for 1 h, centrifuge at 4800 rpm for 2 min, aspirate the supernatant, leave a little and mix it with the precipitate, aspirate all of it and add it to LB plates containing antibiotic Kan, spread it evenly, and incubate upside down overnight; (3) Inoculate the bacteria into LB liquid medium containing Kan and incubate at 37 ℃ and 200 rpm for 6 h. Then perform colony PCR using the KOD enzyme system. The results are as follows: Figure 5 As shown, the presence of a band at 2000 bp indicates successful plasmid transformation. The PCR-positive bacterial culture was inoculated into LB liquid medium containing Kan and cultured at 37 ℃ and 200 rpm until the logarithmic growth phase. Isopropyl-β-D-thiogalactoside (IPTG) was then added, and the culture was incubated overnight at 18 ℃ and 120 rpm. (4) Centrifuge the bacterial culture at 5000 rpm for 10 min at room temperature, discard the supernatant, resuspend in 0.1 M PBS, and place on ice. Place on a low temperature ice-water bath and sonicate to disrupt the culture, then centrifuge at 12000 rpm for 25 min at 4 ℃.

[0036] (5) Protein purification: The Ni-NTA column was washed twice with ultrapure water, followed by two equilibration treatments with 20 mM imidazole solution. The soluble protein solution was added to the equilibrated Ni-NTA column, thoroughly mixed with the column material, and incubated on ice for 10 min to allow for complete protein-cohesion. After collecting the flow-through, the column was washed with 20 mM imidazole solution, followed by elution with 300 mM imidazole solution, and the eluent was collected. Finally, the purified enzyme stock solution was concentrated by centrifugation at 4 ℃ and 4000 rpm using a refrigerated centrifuge and an ultrafiltration tube with a molecular weight cutoff of 30 kDa. 80 μL of the enzyme stock solution was reacted with 20 μL of 3 mM pNP-Gluc, and the reaction was terminated with 100 μL of 1 M Na2CO3, resulting in a yellow solution.

[0037] Result: The protein was successfully expressed and was active.

[0038] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A β-glucuronidase, characterized in that, The amino acid sequence of the β-glucuronidase is shown in SEQ ID NO.

1.

2. A gene fragment encoding the β-glucuronidase of claim 1.

3. The gene fragment according to claim 2, characterized in that, The nucleotide sequence of the gene fragment is shown in SEQ ID NO.

2.

4. An expression vector containing the gene fragment of claim 2 or 3.

5. A host cell containing the expression vector of claim 4.

6. The host cell according to claim 5, characterized in that, The host cell is Escherichia coli.

7. The use of the β-glucuronidase of claim 1 in the preparation of a reagent for in vitro catalytic hydrolysis of NNAL-Gluc.

8. The use of the β-glucuronidase of claim 1 as a target in screening candidate drugs for inhibiting NNAL reactivation in the intestine.

9. The use of the β-glucuronidase of claim 1 as a target in the preparation of drugs or functional foods for reducing NNAL toxicity in the intestines of smokers or preventing smoking-related intestinal damage.

10. The application according to claim 9, characterized in that, The functional food is a probiotic product, dietary supplement, or health food containing an active ingredient that specifically inhibits the activity of the enzyme described in claim 1.

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