Multi-copper oxidase mutant sourced from Psychrobacter sp. And capable of efficiently degrading biogenic amine and application of multi-copper oxidase mutant

Abstract

The invention discloses a multi-copper oxidase mutant sourced from Psychrobacter sp. And capable of efficiently degrading biogenic amine and application of the multi-copper oxidase mutant. According to the multi-copper oxidase mutant, the 183rd site of an amino acid sequence shown in SEQ ID NO.1 is mutated into histidine from phenylalanine, the 221st site of the amino acid sequence shown in SEQ ID NO.1 is mutated into aspartic acid from asparagine, the 261st site of the amino acid sequence shown in SEQ ID NO.1 is mutated into glutamic acid from glutamine, and the 292nd site of the amino acid sequence shown in SEQ ID NO.2 is mutated into arginine from lysine. Compared with wild type multi-copper oxidase, the multi-copper oxidase mutant disclosed by the invention has a good degradation effect on tryptamine, phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine and spermine, and the degradation rate on the spermidine and the spermine is increased to 100%, so that the multi-copper oxidase mutant has a good application prospect in food fermentation safety production.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to a multi-copper oxidase mutant derived from Psychrobacter sp. and capable of efficiently degrading biogenic amines, and application thereof. Background Art

[0002] Biogenic amines are a class of biotoxic organic bases primarily formed by the decarboxylation of amino acids produced by protein breakdown. Based on their molecular structure, they can be divided into three main categories: monoamines (such as histamine), diamines (such as putrescine), and polyamines (such as spermidine). These compounds are widely found in fermented foods, aquatic products, and meat. When biogenic amine concentrations exceed safety thresholds, they can cause severe headaches, gastrointestinal discomfort, and even life-threatening allergic reactions. Therefore, establishing an effective biogenic amine control system has become a critical issue in the food safety field.

[0003] Multicopper oxidases (MCOs) are a class of oxidoreductases containing four copper ions. They catalyze the reduction of oxygen to water through their copper active centers, simultaneously oxidizing a variety of substrates, including biogenic amines. MCO catalysis utilizes a unique electron transfer mechanism to oxidize biogenic amines to their corresponding aldehydes, ammonia, and water, making it a promising candidate for application. Notably, MCOs from different sources exhibit significant differences in their degradation efficiency against various biogenic amines, particularly for complex biogenic amines such as histamine and putrescine. Existing MCOs exhibit low catalytic efficiency, likely due to spatial constraints within the enzyme's substrate pocket. In recent years, researchers both domestically and internationally have made breakthroughs in the degradation of biogenic amines using MCOs. These include confirming the biogenic amine degradation efficacy of MCOs from various sources, achieving promising results in food safety applications and high-concentration biogenic amine degradation technologies. Current research on MCOs focuses primarily on their enzymatic properties, with limited structural analysis. Therefore, further research is needed to identify and optimize MCOs with broad-spectrum degradation capabilities and to enhance their degradation efficiency against specific biogenic amines through site-directed mutagenesis. Summary of the Invention

[0004] The present invention aims to provide a multi-copper oxidase mutant derived from Psychrobacter sp. that efficiently degrades biogenic amines and its use. This invention aims to address the problem of high biogenic amine content in existing aquatic products. By providing a multi-copper oxidase mutant with improved biogenic amine degradation efficiency and using a recombinant enzyme to degrade biogenic amines, the present invention aims to reduce the biogenic amine content, thereby improving the quality of traditional aquatic products.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] The present invention first provides a multi-copper oxidase mutant derived from Psychrobacter sp. and capable of efficiently degrading biogenic amines. The mutant comprises a mutated amino acid sequence shown in SEQ ID NO. 1, wherein position 183 of the phenylalanine (F) is mutated to histidine (H), position 221 of the asparagine (N) is mutated to aspartic acid (D), position 261 of the glutamine (Q) is mutated to glutamic acid (E), and position 292 of the lysine (K) is mutated to arginine (R).

[0007] The present invention also provides a method for constructing a multi-copper oxidase mutant that efficiently degrades biogenic amines: AlphaFold 2 is used to predict the three-dimensional structure of the multi-copper oxidase through a de novo prediction method, the model with the highest score is selected as the structure for subsequent research, and the Dali Server server is used to align proteins with spatial structures similar to the multi-copper oxidase, and the differences in key sites in the spatial structure between proteases with RMSD values ​​≤ 2 and the multi-copper oxidase are compared. The tertiary structure of the multi-copper oxidase is compared through structural superposition, and the four key amino acid sites of Phe183, Asn221, Gln261 and Lys292 are screened and mutated to Phe183His, Asn221Asp, Gln261Glu and Lys292Arg. Finally, the catalytic effect of the key amino acid sites on biogenic amines is verified by molecular docking.

[0008] The present invention also provides an expression vector containing the nucleic acid sequence of the multi-copper oxidase mutant. Preferably, the expression vector is a pET series expression vector.

[0009] The present invention also provides an expression vector containing the nucleic acid sequence of the carbonyl reductase mutant. Preferably, the expression vector is a pET series expression vector.

[0010] The present invention also provides a recombinant engineered bacterium, wherein the recombinant engineered bacterium is Escherichia coli containing the expression vector. Preferably, the genetically engineered recombinant bacterium is Escherichia coli BL21 (DE3).

[0011] The present invention also provides a method for constructing the recombinant engineered bacteria, which comprises connecting the sequence shown in SEQ ID NO. 3 or SEQ ID NO. 4 to a vector and transforming the vector into Escherichia coli cells.

[0012] The present invention also provides a method for producing a multi-copper oxidase mutant, which comprises inoculating the recombinant engineered bacteria into a culture medium for culturing, and collecting the multi-copper oxidase mutant.

[0013] In one embodiment of the present invention, the method is to collect bacterial cells from a cell culture solution, and disrupt the cells to obtain a crude enzyme solution containing the mutant multi-copper oxidase mutant.

[0014] The present invention also provides the use of the multi-copper oxidase mutant in degrading biogenic amines. The use includes reducing the content of biogenic amines in aquatic products. The biogenic amines include one or more of tryptamine, phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine, and spermine. The multi-copper oxidase mutant degrades biogenic amines at a temperature of 25°C and a pH of 7.0.

[0015] The present invention has the beneficial effects that, compared with the wild-type multi-copper oxidase, the multi-copper oxidase mutant provided by the present invention has stronger performance in degrading biogenic amines and a broad degradation spectrum, and can degrade tryptamine, phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine, and spermine within 48 hours. In a biogenic amine simulation system, the degradation rate of total amines reaches 100% (total amines are approximately 450 mg / L), among which the degradation rates of spermidine and spermine are increased to 100%, which helps to further improve the safety of processed aquatic products. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Agarose gel electrophoresis verification diagram of the multi-copper oxidase mutant gene of Example 1; wherein lanes 1-3 are: DNA Maker, Mcob and mutant, respectively.

[0017] Figure 2 This is an electrophoresis diagram of the protein separation and purification process of the multi-copper oxidase mutant in Example 1; Lane M: Unstained Protein Ladder; Lanes 1-2: MCOB and mutants.

[0018] Figure 3 To determine the stability of multi-copper oxidase and its mutants at different temperatures.

[0019] Figure 4 To determine the stability of multi-copper oxidase and its mutants at different pH.

[0020] Figure 5 To analyze the ability of multi-copper oxidase to degrade biogenic amines.

[0021] Figure 6 To analyze the ability of multi-copper oxidase mutants to degrade biogenic amines. DETAILED DESCRIPTION

[0022] The present invention is further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any form.

[0023] The reagents, methods and equipment used in the following examples are conventional reagents, methods and equipment in the art.

[0024] SEQ ID No. 1: Amino acid sequence of MCOB derived from Psychrobacter sp.

[0025] SEQ ID No. 2: MCOB nucleotide sequence derived from Psychrobacter sp.

[0026] SEQ ID No. 3: Amino acid sequence of MCOB mutant F183H / N221D / Q261E / K292R from Psychrobacter sp.

[0027] SEQ ID No. 4: Nucleotide sequence of MCOB mutant F183H / N221D / Q261E / K292R derived from Psychrobacter sp.

[0028] Example 1 Construction of multi-copper oxidase MCOB recombinant plasmid

[0029] The specific steps are as follows:

[0030] (1) Using DNA from Psychrobacter sp. preserved in our laboratory as a template, the multi-copper oxidase gene was amplified. The primers required for amplification are as follows:

[0031]

[0032] (2) PCR amplification was performed using DNA from Psychrobacter sp. as a template. The PCR product was verified by 1% agarose gel electrophoresis. The size of the amplified sequence was consistent with that of the target gene sequence.

[0033] The PCR amplification system was as follows: pre-denaturation at 98°C for 2 min, denaturation at 98°C for 10 s, annealing at 62°C for 30 s, extension at 72°C for 30 s, and a total of 34 cycles from denaturation to extension.

[0034] (3) The amplified fragment was ligated with the plasmid pET-28a(+) by homologous recombination to construct a recombinant vector. The recombinant vector was transformed into E. coli DH5α, cultured, and the plasmid was extracted and screened for sequencing to confirm that the recombinant vector had the correct coding frame. The target gene was approximately 1602 bp, and the sequencing result is shown in SEQ ID NO. 2.

[0035] SEQ ID NO.1: MCOB amino acid sequence of Psychrobacter sp.

[0036] SEQ ID NO.2: MCOB nucleotide sequence of Psychrobacter sp.

[0037] (4) Screening of key amino acid sites

[0038] Firstly, the tertiary structure of MCOB was predicted using Swiss-Model, and the prediction result was not adopted because the sequence consistency was less than 25%. Therefore, AlphaFold 2 was used to predict the three-dimensional structure of MCOB by ab initio prediction method, and the highest score model was selected as the structure for subsequent research. Since there are few studies on the multi-copper oxidase derived from psychrophilic bacteria at present, the Dali Server server was used to compare the protein with similar spatial structure to MCOB. The difference in key sites between the proteinase with RMSD value ≤2 and MCOB in spatial structure was compared. By comparing the tertiary structure of multi-copper oxidase, four key amino acid sites, Phe183, Asn221, Gln261 and Lys292, were screened, and they were mutated to Phe183His, Asn221Asp, Gln261Glu and Lys292Arg. Then, the catalytic effect of the key amino acid sites on biological amines was further verified by molecular docking.

[0039] (5) Design of primers for site-directed mutagenesis

[0040] The key amino acid sites of MCOB were determined by structure prediction and molecular docking computer simulation technology, and the F183H / N221D / Q261E / K292R mutant was further constructed by experiment to verify the experiment.

[0041]

[0042]

[0043] Note: Lowercase letters indicate mutation sites

[0044] (6) Site-directed mutagenesis reaction

[0045] The PCR amplification system is as follows: 98°C pre-denaturation for 2 min, 98°C denaturation for 10 s, 62°C annealing for 30 s, 72°C extension for 105 s, and denaturation to extension for a total of 34 cycles.

[0046] The recombinant plasmid of multi-copper oxidase MCOB was used as a template for PCR amplification, and the PCR product was verified by 1% agarose gel electrophoresis. The amplified product was digested by Dpn I enzyme and transformed into E. coli. The recombinant plasmid was used as a template for amplification of the target fragment, as shown in Figure 1 The size of the amplified sequence is the same as that of the target gene sequence, about 1602 bp, indicating that the amplification is successful, and the transformant is sent to the company for sequencing.

[0047] (7) Transform the correct plasmid into E. coli BL21 to obtain the seed liquid. Pick a single colony into 5-10 mL of LB medium and shake it as a seed liquid, and incubate it at 37°C, 200 r / min overnight.

[0048] (8) Activate the expression bacteria and inoculate them into the induction medium (LB / Kana concentration 50 μg / mL) at V / V = 1:100, 220 r / min, 37°C, and incubate them for about 1-4 h until the OD 600 of the culture reaches 0.6-0.8. Take 1 mL of the bacterial liquid as the uninduced control, and reduce the remaining culture to 16°C, add IPTG (final concentration 0.1 mM) and 10 mL of 5 mmol / L CuCl2, and induce expression at 37°C, 220 r / min for 4 h. Then centrifuge the resulting fermentation broth at 4°C, 6000 r / min for 10 min, collect the bacterial precipitate, and store it at -80°C or immediately sonicate the bacterial cells.

[0049] (9) Sonicate in an ice water bath, and the sonication conditions are as follows: about 400 w, work for 2 s, interval of 4 s, and sonication time of 20 min. After sonication, centrifuge at 10,000 r / min, 10 min, 4°C, filter the supernatant with a 0.45 μm filter, and use it for subsequent column chromatography (soluble expression in the supernatant).

[0050] (10) Elute the proteins bound to the nickel column with eluents containing 0, 5, 150, and 500 mM imidazole, respectively, and the target protein is eluted at 150 mM imidazole. Detect the protein concentration, enzyme activity, and SDS-PAGE protein electrophoresis of the purified protein. As shown in Figure 2 , the purified multicopper oxidase and its mutants have a protein size of about 60.12 kDa, which is consistent with the expected size.

[0051] Example 2 Enzyme activity determination of multicopper oxidase and its mutants F183H / N221D / Q261E / K292R

[0052] The enzyme activity determination method is as follows: visible light absorption method is used to determine the multicopper oxidase activity: 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) is used as the substrate, and the amount of ABTS oxidized by the enzyme is used to calculate the enzyme activity of the multicopper oxidase mutant. At room temperature, 190 μL of a buffer containing 0.5 mM ABTS is added with 10 μL of the multicopper oxidase mutant, and incubated for 10 min, and the enzyme activity is determined by measuring the oxidation of ABTS. The determination of kinetic parameters is determined by the oxidation of ABTS (0.1 -1 mM).

[0053] One international unit (IU) is defined as the amount of enzyme required to catalyze the oxidation of 1 μmol of ABTS per minute.

[0054] The enzyme activity formula is as follows:

[0055]

[0056] The molar absorption coefficient of ABTS at 420 nm is ε = 3.6 × 10 4 M -1 cm -1

[0057] Δ t :Reaction time (min)

[0058] Δ OD : Change in absorbance at 420nm

[0059] V1: total reaction volume (μL)

[0060] V2: enzyme volume (μL)

[0061] Example 3 Effects of temperature and pH on the activity of multi-copper oxidase and its mutant F183H / N221D / Q261E / K292R

[0062] (1) Determination of stability at different temperatures.

[0063] The initial enzyme activity of MCOB and its mutant F183H / N221D / Q261E / K292R was determined using phosphate buffer (10 mM, pH 5.0, containing 0.5 mM ABTS). The residual enzyme activity of MCOB and its mutant F183H / N221D / Q261E / K292R was determined after incubation at 15-45°C for 5 h. The stability of MCOB at different temperatures was determined. Figure 3 As shown, the optimal temperature of multi-copper oxidase and its mutant F183H / N221D / Q261E / K292R is 25℃.

[0064] (2) Determination of stability at different pH values.

[0065] The initial enzyme activity of MCOB and its mutant F183H / N221D / Q261E / K292R was determined using phosphate buffer (10 mM, pH 2.0-11.0, containing 0.5 mM ABTS). The residual enzyme activity of MCOB and its mutant F183H / N221D / Q261E / K292R was determined after incubation at pH 2.0-11.0 for 5 h. The stability of MCOB at different pH values ​​was determined as shown in the following table. Figure 4 As shown, the optimal pH of multi-copper oxidase and its mutant F183H / N221D / Q261E / K292R is 7.0.

[0066] Example 4

[0067] Degradation of biogenic amines by multicopper oxidase and its mutant F183H / N221D / Q261E / K292R under optimal conditions

[0068] Tryptamine, phenylethylamine, putrescine, cadaverine, histamine, octopamine, internal standard, tyramine, spermidine and spermine purchased from Aladdin Biochemical Technology Co., Ltd. were weighed and prepared into a biogenic amine mixture with a monoamine concentration of 50 mg / L.

[0069] At 25°C and pH 7.0, a final concentration of 8 U / mL enzyme solution was added to a 50 mg / L biogenic amine mixed solution and incubated for 48 hours. The reaction was terminated by heating, and the sample was derivatized and analyzed for biogenic amine content using HPLC. The results of biogenic amine degradation by MCOB wild-type enzyme and mutants are shown in Figure 2. Figure 5 and Figure 6 As shown, the peak numbers 1-10 in the figure represent tryptamine, phenylethylamine, putrescine, cadaverine, histamine, octopamine, internal standard, tyramine, spermidine and spermine, respectively.

Claims

1. A Psychrobacter sp . A multi-copper oxidase mutant with high efficiency in degrading biogenic amines derived from The amino acid sequence shown in SEQ ID NO.1 was mutated from phenylalanine at position 183 to histidine, from asparagine at position 221 to aspartic acid, from glutamine at position 261 to glutamic acid, and from lysine at position 292 to arginine.

2. The gene encoding the multi-copper oxidase mutant according to claim 1.

3. An expression vector, characterized in that The expression vector comprises the nucleotide sequence of claim 3.

4. The expression vector according to claim 4, characterized in that The expression vector is a pET series expression vector.

5. A recombinant engineered bacterium, characterized in that: The recombinant engineered bacteria is Escherichia coli containing the expression vector according to claim 4.

6. A method for constructing a multi-copper oxidase mutant capable of efficiently degrading biogenic amines, characterized in that: AlphaFold 2 was used to predict the three-dimensional structure of multi-copper oxidase by ab initio prediction. The model with the highest score was selected as the structure for subsequent research. Proteins with similar spatial structures to multi-copper oxidase were compared using Dali Server. The differences in key sites in the spatial structure between proteases with RMSD values ​​≤ 2 and multi-copper oxidase were compared. The tertiary structure of multi-copper oxidase was compared by structural superposition, and four key amino acid sites, Phe183, Asn221, Gln261 and Lys292, were screened and mutated to Phe183His, Asn221Asp, Gln261Glu and Lys292Arg. Finally, molecular docking was used to verify the catalytic effect of the key amino acid sites on biogenic amines.

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