Beta-glucuronidase as well as nucleotide sequence and application of gene of beta-glucuronidase

Abstract

The invention discloses beta-glucuronidase, a nucleotide sequence of a gene of the beta-glucuronidase and application of the beta-glucuronidase, the gene of the beta-glucuronidase and a protein sequence of the beta-glucuronidase are related to food-borne hazardous substances, and the beta-glucuronidase capable of efficiently and specifically activating cancerogens such as heterocyclic amine HAAs is constructed. The inhibitor designed aiming at the enzyme can reduce interference on intestinal effective microbial communities and human body GUS activity to the maximum extent, side effects are smaller, a research and development basis is provided for further high-throughput drug screening, computer-aided drug design and antibody drug development, and the inhibitor has a great application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to a beta-glucuronidase, a nucleotide sequence of its gene and its application. BACKGROUND

[0002] Heat treatment is one of the main means of food processing, which endows food with unique color, flavor and texture. However, during the heat processing of food, especially protein-rich meat products, a variety of harmful substances are easily produced, among which heterocyclic aromatic amines generated by the complex reaction of amino acids, glucose and creatine (creatinine) are a kind of polycyclic aromatic compounds with strong mutagenicity and carcinogenicity, mainly including PhIP (2-amino-1-methyl-6-phenyl imidazo [4, 5-b] pyridine), IQ (2-amino-3-methyl-3H-imidazo quinoline), AαC (2-amino-9H-pyrido [2, 3-b] indole) and MeIQx (2-amino-3, 8-dimethyl imidazo [4, 5-f] quinoline) and the like. Heterocyclic amines HAAs are listed as class 2 carcinogens, and also have potential neurotoxicity and cardiotoxicity, so it is urgent to effectively prevent and control them.

[0003]

[0004] In order to reduce the generation of heterocyclic amines HAAs in meat products, the existing technology often adopts the way of controlling processing mode and condition, microwave and plasma activated water pretreatment before heat processing or adding natural antioxidants (anthocyanins, tea polyphenols, catechins, myricetin, etc.). Recently, researchers have focused on developing composite aerogels based on metal organic frameworks for efficient adsorption and removal of HAAs in food, but before its practical application, the safety of the adsorption material itself needs to be evaluated in detail.

[0005] The human gut microbiota is complex and dynamic, co-evolved and co-developed with the host. The gut microbiota catalyzes various metabolic reactions of structurally diverse endogenous / exogenous compounds, among which reduction and hydrolysis dominate. Glucuronidation is the main detoxification pathway in the liver, in which UDP-glucuronosyltransferases catalyze the combination of hydrophobic xenobiotics and endobiotics with glucuronic acid to improve their solubility. The produced glucuronide enters the intestine, and the intestinal bacterial beta-D-glucuronidase (bGUS) removes the glucuronic acid (GlcA) to release the original molecule into the intestinal lumen. Intestinal bacteria-derived GUS enzymes are widely distributed in the human gut microbiota and play an important role in the metabolic transformation of endogenous and exogenous substances by catalyzing the hydrolysis of beta-D-glucuronides, reversing the glucuronidation reaction in the liver. As the most abundant bacteria-derived GUS enzyme in the intestine, the screening of inhibitors and the application of E. coli-derived GUS enzymes are considered to have the potential to reduce the toxicity of drugs, environmental and foodborne hazards. However, previous studies have confirmed that different intestinal bacteria-derived GUS enzymes have great differences in structural characteristics, functional properties, substrate specificity and catalytic efficiency. Therefore, it is particularly important to analyze the key strains and specific bacteria-derived GUS enzymes involved in the reactivation of HAAs. SUMMARY

[0006] The purpose of the present application is to provide a beta-glucuronidase and its gene nucleotide sequence, and the application of the above beta-glucuronidase in beta-GUS inhibitors and foodborne hazard reactivation preparations.

[0007] The beta-glucuronidase of the present application has the amino acid sequence shown in SEQ ID NO: 2.

[0008] The present application provides a gene fragment encoding beta-glucuronidase, and the nucleotide sequence thereof is shown in SEQ ID NO: 1.

[0009] The present application provides an expression vector containing the nucleotide sequence, and a host cell containing the above-mentioned expression vector.

[0010] The host cell is selected from bacteria, actinomycetes, filamentous fungi, yeast, plant cells, animal cells, wherein the bacteria are selected from Escherichia coli, the actinomycetes are selected from Streptomyces, the filamentous fungi are selected from Basidiomycetes and filamentous ascomycetes, the yeast is selected from Pichia, Saccharomyces, Schizosaccharomyces, and the animal cell is selected from insect cells. The selected cells include but are not limited to the above-mentioned host cells.

[0011] The application provides a recombinant protein with the function of beta-glucuronidase, wherein the amino acid sequence of the functional region of the recombinant protein is encoded by the nucleotide sequence shown in SEQ ID NO: 1.

[0012] The application provides application of the above beta-glucuronidase or beta-glucuronidase recombinant protein or host cell containing the beta-glucuronidase in hydrolysis and removal of glucuronic acid groups.

[0013] The application provides application of the above beta-glucuronidase or beta-glucuronidase recombinant protein or host cell containing the beta-glucuronidase in preparation of beta-glucuronidase inhibitors.

[0014] The application provides application of the above beta-glucuronidase or beta-glucuronidase recombinant protein or host cell containing the beta-glucuronidase in preparation of specific activators of foodborne hazards heterocyclic amines HAAs.

[0015] Invention principle: the application discloses a brand-new beta-GUS gene and protein sequence related to foodborne hazards for the first time, the beta-glucuronidase sequence is essentially different from known GUS enzymes (such as E. coli GUS), the beta-glucuronidase of the application can efficiently and specifically activate procarcinogens such as heterocyclic amines HAAs, so that the compound is a more ideal drug target. Metabolism of heterocyclic aromatic amines into glucuronide form can effectively detoxify, but the intestinal GUS enzyme can convert it into a toxic form, prolonging its metabolism time in the body. It is found that the intestinal GUS enzyme activity of mice fed with PhIP for a long time is significantly increased, so the bacteria related to the activity increase are locked; inhibitors that can specifically inhibit the enzyme are predicted from a natural product small molecule library, which can minimize the interference with the intestinal beneficial flora and the GUS activity of the human body, and have smaller side effects, and the inhibition effect is better than that of existing inhibitors.

[0016] Beneficial effects: compared with the prior art, the application has the following remarkable advantages: the application discloses a brand-new beta-GUS gene and protein sequence related to foodborne hazards, constructs a beta-glucuronidase gene that can efficiently and specifically activate procarcinogens such as heterocyclic amines HAAs, and the inhibitors designed for the enzyme can minimize the interference with the intestinal beneficial flora and the GUS activity of the human body, and have smaller side effects, which provides a research and development basis for further high-throughput drug screening, computer-aided drug design and antibody drug development, and has great application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 Results of fecal GUS activity detection of mice;

[0018] Figure 2The sequencing results of the mouse feces;

[0019] Figure 3 The recombinant expression vector map of β-glucuronidase;

[0020] Figure 4 The GUS gene expression amount of Lactobacillus reuteri in the feces of the mice in the control group and the experimental group;

[0021] Figure 5 The left side is the alignment result of the predicted structure of LRgus and the 3LPF structure, and the right side is the comparison of the active pockets of the two, light red is the active site of 3LPF, and dark red is the predicted active site of LRgus;

[0022] Figure 6 The agarose gel electrophoresis map of the PCR product of the β-glucuronidase gene. DETAILED DESCRIPTION

[0023] The technical solutions of the present application will be further described below in combination with examples, and the test materials used in the examples can be purchased through a conventional route.

[0024] Example 1

[0025] The exploration of the in vivo metabolism of the heterocyclic aromatic amine PhIP includes the following steps:

[0026] (1) C57 mice are selected and divided into a control group and an experimental group, 5 mice in each group. In addition, in order to better distinguish each mouse, an ear number marking method is selected to mark the mice;

[0027] (2) Prepare a PhIP solution, which is dissolved in a 1% DMSO aqueous solution and ultrasonically assisted;

[0028] (3) The experimental group mice are given 20 mg / kg PhIP by gavage every day, and the control group mice are given the same amount of solvent by gavage every day. When gavaging, the left thumb and index finger pinch the mouse's neck and back skin, the little finger fixes the tail root, so that the mouse is in a vertical body position, the head, neck and trunk are in a straight line, the double forelimbs are spread out, the mouth is slightly open, and the breathing is unobstructed. The right hand holds the gavaging device that has been sucked, inserts the needle along the right corner of the mouth, presses the tongue against the upper jaw, and makes the oral cavity and esophagus in a straight line. Slowly push the needle, when encountering resistance, rotate the needle body to stimulate swallowing, and continue to insert to the predetermined depth;

[0029] (4) Continue for 28 days and collect the mouse feces, then anesthetize the mouse with ether, place the anesthetized mouse on the clean bench, sacrifice the mouse and collect the mouse organs;

[0030] (5) Take 50-70 mg of mouse feces, add PBS at a volume ratio of 1:9, grind evenly, take the supernatant and centrifuge at 9391g, 4℃ for 20 minutes. Use the BCA kit to detect the protein content in the supernatant. Take 20 μL of supernatant and add it to 80 μL of PBS and 20 μL of 3 mMP NPG. After reacting for 30 minutes, add 250 μL of 2% NaOH to terminate the reaction. Take 200 μL of the reaction solution into a 96-well plate and detect the absorbance at 405nm. The results are as follows. Figure 1 As shown;

[0031] (6) Perform 16S rDNA sequencing on fresh feces. The sequencing results are as follows: Figure 2 As shown; Lactobacillus reuteri was identified as a key bacterium affecting PhIP metabolism, and the GUS gene was queried through NCBI;

[0032] The results showed that the concentration of GUS in the feces of mice increased after gavage with PhIP, and sequencing results showed that the concentration of Lactobacillus in the experimental group was significantly higher than that in the control group. Correlation analysis showed a high correlation with Lactobacillus reuteri, indicating that Lactobacillus reuteri is associated with increased GUS activity.

[0033] Example 2

[0034] The construction of the PET-28a(+)-GUS recombinant plasmid includes the following steps:

[0035] (1) The β-glucuronidase gene of Lactobacillus reuteri was searched in the NCBI database. No labeled β-glucuronidase was found. Different types of β-glucuronidase sequences were searched with Lactobacillus reuteri. Possible β-glucuronidase sequences were found. The nucleotide sequence encoding the protein is shown in SEQ ID NO: 1, with a length of 1887 bp; the protein sequence is shown in SEQ ID NO: 2, with a length of 628 aa.

[0036] (2) The β-glucuronidase gene was synthesized by Shanghai Sangon Biotech Co., Ltd. and ligated into the E. coli expression vector PET-28a(+) to construct the recombinant plasmid PET-28a(+)-GUS, as shown below. Figure 3As shown, the protein was correctly constructed into the PET-28a(+) E. coli expression plasmid. This plasmid was transformed into E. coli DH5α, plated on LB agar plates containing the antibiotic Kana, and incubated overnight at 37°C. Single colonies were picked and inoculated into LB liquid medium containing Kana, incubated overnight at 37°C and 200 rpm, then the plasmid was extracted, stored at -20°C, and sequenced. The sequence completely matched the target sequence.

[0037] The results showed that the PET-28a(+)-GUS recombinant plasmid was successfully constructed.

[0038] The nucleotide sequence of the gene fragment for β-glucuronidase is shown in SEQ ID NO: 1:

[0039]

[0040] The β-glucuronidase has the amino acid sequence shown in SEQ ID NO: 2:

[0041] MDADIKWLDEPETFRVNOLPAHSDHYYYGNYDEWRHNNSRFAONLDGOWOFNFAENPRKRENDFYKVDYDSSSFGTIEVPSEIELNNYAQNNYINTLIPWEGKIYRRPAYALSPDDAOEGSFSDGDDNTVGEYLKHFDLEPSLRGKOIRIRFDGVER AMYVWLNGHFIGYAEDSFTPSEFDLTPYIODEGNVLAVEVFKHSTASWIEDODMFRFSGIFRSVNLLAOPLVHVEDLHIRPIVTDNYQDGIFNVDLQLHGEKTGNVNVRVIDNDGNTLVNETHPVDSTVKVQDQFLENVHLWDNHDPYLYQLLIEIR DDEGNLVELVPYRFGFRRIEINKDHVVLLNGORLIINGVNRHEWNAKRGRAITMDDMTSDIHTFKENNINAVRTCHYPDOIPWYYLCDDNGIYMMAENNLESHATWOKMGAIEPSYNVPGSVPOWRDVVVDRARTNYETFKNHPSILFWSLGNESYA GDNIVKMNEFYKKHDDSRLVHYEGVCHTPEYRDRISDVESWMYLPPKEVEEYLKNNPDKPFMECEYMHDMGNSDGGMGSYISLLDKYPQYFGGFIWDFIDQALLVKDPVSGQEVMRYGGDFDDRHSDYEFSGDGLMFADRTPKPAMOEVRYYYGLHK

[0042] Example 3

[0043] Inducing and validating recombinant GUS protein expression, and predicting inhibitors, includes the following steps:

[0044] (1) Primers for Lactobacillus reuteri β-glucuronidase were designed, and the primer sequences are shown in SEQ ID NO:3 and SEQ ID NO:4. qPCR was performed with cDNA extracted from mouse feces, and the results are as follows: Figure 4This figure illustrates the increased abundance of GUS enzymes in *Lactobacillus reuteri*. The sequence was structurally predicted and compared with a known GUS structure (3LPF), and the activity pockets of the two were also compared. The results are as follows: Figure 5 The active pockets showed significant differences. Autodock vina was used to score the docking of PhIP-G (Compound CID: 71313317) with two other proteins, revealing differences. Furthermore, PhIP-G showed even greater differences in docking with another known GUS structure (6JZ8), indicating specific binding. The scores are shown in Table 1; a lower molecular docking score indicates stronger binding affinity.

[0045] SEQ ID NO: 3:

[0046] F:TCCGGAGTGTAAACTTATTAGCC

[0047] SEQ ID NO: 4:

[0048] R:ACCGTGAAGTTGAAGGTCAAC

[0049] Table 1. Autodock Vina docking scores for PhIP-G (Compound CID: 71313317) and two proteins.

[0050] ;

[0051] (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 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 r 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 the antibiotic Kana, spread it evenly, and incubate upside down overnight;

[0052] (3) Inoculate the bacteria into LB liquid medium containing Kana and incubate at 37°C and 200 rpm for 6 h. Then perform colony PCR using the KOD enzyme system. The results are as follows: Figure 6 The presence of a band at 2000bp indicates successful plasmid transformation. The PCR-positive bacterial culture was inoculated into LB liquid medium containing Kana and cultured at 37°C and 200 rpm until the logarithmic growth phase. IPTG was then added, and the culture was incubated overnight at 18°C ​​and 120 rpm.

[0053] (4) Centrifuge the bacterial culture at 5500 r for 10 min, 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 r for 25 min at 4 °C. React the supernatant with 3 mM pNPG.

[0054] (5) Autodock vina was used to screen small molecules of natural products, compared with known inhibitors D-Saccharic acid 1,4-lactone hydrate, and molecular docking and scoring with other known GUS enzymes. Natural products that can specifically inhibit LRgus were found as candidate inhibitors. The results are shown in Table 2. This shows that the known inhibitors are not as effective as the candidate inhibitors in inhibiting LRgus, and the candidate inhibitors specifically inhibit LRgus.

[0055] Table 2. Molecular docking scores of GUS expressed by LRgus and 3LPF for screened candidate inhibitors and known inhibitors.

[0056] ;

[0057] The results of induced expression and validation of recombinant GUS protein showed that: Figure 5 The image shows a comparison between the sequence structure of this invention and known GUS structures. The green model represents the 3LPF structure, the blue model represents the predicted LRgus structure, and the right side shows the active pocket alignment. Light red represents the 3LPF active site, and dark red represents the predicted LRgus active site. In mice, elevated expression of β-glucuronidase from *Lactobacillus reuteri* was verified. This protein differs significantly in structure from known β-glucuronidases, and its active pocket also differs, suggesting a different conformation or functional state. Furthermore, protein expression was successful.

Claims

1. A β-glucuronidase, characterized in that, The beta-glucuronidase has an amino acid sequence as shown in SEQ ID NO:

2.

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

3. The genetic segment of claim 2, wherein, The nucleotide sequence of the gene fragment is shown in SEQ ID NO:

1.

4. An expression vector containing the nucleotide sequence of claim 2 or 3.

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

6. The host cell of claim 5, wherein, The host cell is selected from bacteria, actinomycetes, filamentous fungi, yeasts, plant cells, animal cells.

7. The host cell of claim 6, wherein, The bacteria are selected from Escherichia coli, the actinomycetes are selected from Streptomyces, the filamentous fungi are selected from Basidiomycetes, filamentous Ascomycetes, the yeasts are selected from Pichia, Saccharomyces, Schizosaccharomyces, and the animal cells are selected from insect cells.

8. A recombinant protein having β-glucuronidase function, characterized in that, The amino acid sequence of the functional region of the recombinant protein is encoded by the nucleotide sequence shown in SEQ ID NO:

1.

9. Use of the beta-glucuronidase of claim 1 or the beta-glucuronidase recombinant protein of claim 8 or the host cell containing the beta-glucuronidase in hydrolysis of removing glucuronide groups.

10. Use of the beta-glucuronidase of claim 1 or the beta-glucuronidase recombinant protein of claim 8 or the host cell containing the beta-glucuronidase in preparing beta-glucuronidase inhibitors.

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