Application of multi-copper oxidase mutant in biogenic amine degradation

Abstract

The invention discloses application of a multi-copper oxidase mutant in biogenic amine degradation, and belongs to the technical field of food biology. The amino acid sequence of the multi-copper oxidase mutant is shown as SEQ ID NO.1. The application is to degrade biogenic amines in aquatic products, and the biogenic amines comprise tryptamine, phenylethylamine, putrescine, cadaverine, histamine, octopamine, tyramine, spermidine and spermine. The multi-copper oxidase mutant disclosed by the invention not only can effectively reduce the content of biogenic amines in aquatic products and reduce the generation of reaction byproducts, but also can maintain the freshness and nutritional value of the aquatic products, and has remarkable application value in the fields of aquatic product processing and food safety.

Description

Technical Field

[0001] The present invention relates to the field of food biotechnology, and in particular to application of a multi-copper oxidase mutant in degrading biogenic amines. 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] Multi-copper 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. The MCO catalytic process oxidizes biogenic amines to their corresponding aldehydes, ammonia, and water through a unique electron transfer mechanism, making it highly valuable for biogenic applications. Notably, MCOs from different sources exhibit significant differences in their degradation efficiency against different types of 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. 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 solve the problem of generally high content of biogenic amines in existing aquatic products and provides a highly active and stable multi-copper oxidase mutant for use in degrading biogenic amines.

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

[0006] A multi-copper oxidase mutant is used in the degradation of biogenic amines. The amino acid sequence of the multi-copper oxidase mutant is shown in SEQ ID NO.1.

[0007] Furthermore, the application is to degrade biogenic amines in aquatic products.

[0008] Furthermore, the aquatic products are fish, crustaceans, shellfish, cephalopods, algae, mollusks, etc.

[0009] Furthermore, the biogenic amines include tryptamine, phenylethylamine, putrescine, cadaverine, histamine, octopamine, tyramine, spermidine, and spermine.

[0010] Furthermore, the multi-copper oxidase mutant is used in the form of enzyme powder or enzyme liquid to degrade biogenic amines in the reaction system.

[0011] Furthermore, the multi-copper oxidase mutant is added to the reaction system at a concentration of 4-12 U / mL.

[0012] The present invention explores multi-copper oxidases from the genome of Psychrobacter sp. and obtains multi-copper oxidase mutants through site-directed mutagenesis based on rational design. Experimental results confirm that the multi-copper oxidase mutants can degrade nine common biogenic amines, including tryptamine, phenylethylamine, putrescine, cadaverine, and histamine, in a complex biogenic amine system. The Psychrobacter sp.-derived multi-copper oxidase mutants not only effectively reduce biogenic amine content and the formation of reaction byproducts, but also maintain the freshness and nutritional value of aquatic products, possessing significant application value in aquatic product processing and food safety.

[0013] Mutant multi-copper oxidase enzymes, in the form of enzyme powder or enzyme solution, are used to degrade biogenic amines in reaction systems. Preparation of the multi-copper oxidase powder involves construction and in vitro expression of the mutant, followed by protein purification and freeze-drying. The enzyme powder exhibits high activity and efficient biogenic amine degradation. The enzyme powder exhibits excellent storage and operational stability, along with strong biogenic amine degradation activity, and has the potential for widespread application in aquatic product processing and preservation. The enzyme is safe, environmentally friendly, operates under mild conditions, and has strong industrial applicability. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The enzyme activity of the multi-copper oxidase mutant of the present invention at different temperatures.

[0015] Figure 2 The enzymatic activity of the multi-copper oxidase mutant of the present invention at different pH values. DETAILED DESCRIPTION

[0016] 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.

[0017] The reagents, methods and equipment used in the following examples are conventional reagents, methods and equipment in the art.

[0018] Example 1 Construction of multi-copper oxidase mutants

[0019] The present invention first provides a multi-copper oxidase mutant F183H / N221D / Q261E / K292R derived from Psychrobacter sp. that efficiently degrades biogenic amines. The amino acid sequence of the wild enzyme (SEQ ID NO. 2) is modified by mutating position 183 from phenylalanine (F) to histidine (H), position 221 from asparagine (N) to aspartic acid (D), position 261 from glutamine (Q) to glutamic acid (E), and position 292 from lysine (K) to arginine (R). The amino acid sequence of the multi-copper oxidase mutant F183H / N221D / Q261E / K292R is shown in SEQ ID NO. 1, and the mutation primers are as follows:

[0020]

[0021]

[0022] Note: Lowercase letters represent mutation sites

[0023] The PCR reaction system is as follows:

[0024]

[0025] Note: PCR amplification was performed in steps using four pairs of primers.

[0026] The PCR amplification procedure is as follows:

[0027]

[0028] The amplified product was digested with DpnⅠ enzyme, transformed into E. coli DH5α, and sequenced to verify the correctness of the target fragment.

[0029] Example 2 Preparation of Multi-copper Oxidase Mutant Enzyme Powder

[0030] (1) The PCR amplification product in Example 1 was digested with DpnⅠ enzyme and transformed into E. coli DH5α. The plasmid was extracted and transformed into E. coli BL21 after sequencing. A single clone was picked and added to 5 mL of LB medium as a seed solution. After overnight culture at 37°C and 220 rpm / min, 1% was inoculated into induction medium (LB / Kana concentration 50 μg / mL) and cultured at 220 rpm and 37°C until OD 600 The pH value was 0.6-0.8. After the culture cooled to 16°C, IPTG was added to a final concentration of 0.1 mM and 5 mM CuCl2 was added. Expression was induced at 37°C and 220 rpm for 4 hours. The resulting fermentation broth was then centrifuged at 4°C and 10,000 rpm for 10 minutes. The bacterial pellet was collected and immediately sonicated.

[0031] (2) Ultrasonic disruption in an ice-water bath using an ultrasonic cell disruptor. Ultrasonication conditions: approximately 400W, 2s on, 4s off, 20min disruption. After disruption, centrifuge at 10,000rpm for 10min at 4°C. Keep the sediment and supernatant for later use as a control. The supernatant is used for subsequent column chromatography (soluble expression is in the supernatant). Filter the supernatant through a 0.45μm filter.

[0032] (3) Gel column pretreatment: First, wash the Ni column with 5 column volumes of double-distilled water, and equilibrate with eluent 1 (Talon 0: without imidazole) for 3-5 column volumes (3 mL for nickel column). Then, load the sample at a flow rate of 0.5 mL / min, and hang the column twice to allow the protein to fully bind to the gel. Collect the flow-through (SDS-PAGE is used to detect whether the flow-through contains the target protein. If it does, it indicates that the gel volume used needs to be increased).

[0033] (4) Eluent 1 (Talon 0): 200 mM NaCl; 20 mM Tris-HCl, filtered through a 0.45 μm filter membrane

[0034] The following imidazole solutions of different concentrations were eluted in sequence, and the gel was run to verify at which concentration the target band was eluted.

[0035] Eluent 2 (Talon 5): 5 mM imidazole; 200 mM NaCl; 20 mM Tris-HCl, filtered through a 0.45 μm filter membrane.

[0036] Eluent 3 (Talon 150): 150 mM imidazole; 200 mM NaCl; 20 mM Tris-HCl, filtered through a 0.45 μm filter membrane.

[0037] Eluent 4 (Talon 500): 500 mM imidazole; 200 mM NaCl; 20 mM Tris-HCl, filtered through a 0.45 μm filter membrane.

[0038] Use eluent 1 (Talon 0) to wash for 3-5 column volumes to remove impurities such as cell debris that cannot be removed by high-speed centrifugation. Use eluent 2 (Talon 5) to wash for 3-5 column volumes to remove miscellaneous proteins that are non-specifically bound to the gel, and collect the eluate in an EP tube. Use eluent 3 (Talon150) to wash for 6-8 column volumes to dissociate the target protein that is specifically bound to the nickel particles. Use eluent 4 (Talon 500) to wash for 3-5 column volumes to dissociate all proteins bound to the gel. The purified protein was tested for protein concentration, enzyme activity, and SDS-PAGE protein electrophoresis. The results showed that the purified protein was a single band, which was in line with the expected results. Collect the enzyme solution of the target protein, freeze-dry it in vacuum to make enzyme powder, and store it at 4°C for further experiments.

[0039] Example 3 Determination of enzyme activity of multi-copper oxidase mutant under different conditions

[0040] Reconstitution of enzyme powder: The enzyme activity of multi-copper oxidase lyophilized enzyme powder is 8-13 U / mg, and 1 mg of lyophilized enzyme powder is dissolved in 1 mL of 0.01 M PBS to obtain multi-copper oxidase mutant enzyme solution.

[0041] The method for determining the enzyme activity of multi-copper oxidase mutant is as follows:

[0042] Visible light absorption method is used to determine the activity of multi-copper oxidase mutant: 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) is used as a substrate, and the enzyme activity of multi-copper oxidase mutant is calculated by detecting the amount of enzyme oxidizing ABTS. 190 μL of buffer containing 0.5 mM ABTS is added to 10 μL of reconstituted multi-copper oxidase mutant enzyme solution at room temperature 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).

[0043] One international unit (IU) is defined as the amount of enzyme required to catalyze the oxidation of 1 μmol of ABTS per minute.

[0044] The formula for enzyme activity is as follows:

[0045]

[0046] The molar absorption coefficient of ABTS at 420 nm is ε = 3.6 x 104 M -1 cm -1

[0047] Δ t : reaction time (min)

[0048] Δ OD : change in absorbance value at 420 nm

[0049] V1: total reaction volume (μL)

[0050] V2: enzyme amount (μL)

[0051] (1) Enzyme activity of multi-copper oxidase mutant at different temperatures

[0052] The enzyme activity of multi-copper oxidase is determined using phosphate buffer (10 mM, pH 5.0, containing 0.5 mM ABTS). 190 μL of buffer (preheated at 15-45°C) is added to 10 μL of reconstituted multi-copper oxidase mutant enzyme solution, incubated for 10 min, and the enzyme activity of multi-copper oxidase at different temperatures is determined.

[0053] The results are as follows Figure 1As shown, the optimal temperature of the multi-copper oxidase mutant was 25°C and the enzyme activity was 28.74 U / mL.

[0054] (2) Enzyme activity of multi-copper oxidase mutants at different pH

[0055] The activity of the multi-copper oxidase mutants was determined using phosphate buffer (10 mM, pH 2.0-11.0, containing 0.5 mM ABTS). 10 μL of the reconstituted multi-copper oxidase mutant enzyme solution was added to 190 μL of buffer at room temperature and incubated for 10 minutes. The activity of the multi-copper oxidase at different pH values ​​was measured.

[0056] The results are as follows Figure 2 As shown, the optimal pH of the multi-copper oxidase mutant was 6.0, and the enzyme activity was 39.06 U / mL.

[0057] Example 4 Degradation rate of biogenic amines by multi-copper oxidase mutants under different conditions

[0058] The multi-copper oxidase mutant enzyme solution obtained by reconstitution of lyophilized multi-copper oxidase powder was mixed with a mixture of biogenic amines (tryptamine, phenylethylamine, putrescine, cadaverine, histamine, octopamine, tyramine, spermidine, and spermine, all at 50 mg / L) and incubated under specific conditions to degrade the biogenic amines. After completion of the reaction, 1 mL of the biogenic amine standard and the reaction sample were derivatized with dansyl chloride. The derivatization reaction system consisted of the following: 1 mL of saturated NaHCO₃, 250 μL of 100 mg / L internal standard solution, 100 μL of 1 mol / L NaOH, and 1 mL of dansyl chloride (10 mg / mL in acetone) were added to 1 mL of sample. The mixture was then incubated in a 60°C water bath for 15 minutes, vortexing twice. The reaction was terminated by the addition of 0.5 mL of ammonia solution, followed by mixing and incubation in a 60°C water bath for 15 minutes, vortexing twice. The mixture was extracted with 3 mL of ether. The upper organic phase was aspirated, dried under nitrogen, and dissolved in 1 mL of acetonitrile. Phenylethylamine content was analyzed using a high-performance liquid chromatography (HPLC) system (Waters e2695-2998, Waters, USA) equipped with a reversed-phase C18 column (Waters Sunfire, 5 μm, 4.6 × 250 mm). Acetonitrile and ultrapure water were used as mobile phases A and B, respectively, with an injection volume of 20 μL, a column temperature of 35°C, and UV detection at 254 nm. Analysis was performed using the following gradient elution program: 55% A (0-7 min), 65% A (7-14 min), 70% A (14-20 min), 90% A (20-30 min), 100% A (30-35 min), and 55% A (35-38 min).

[0059] (1) Biogenic amine degradation rate of multi-copper oxidase mutants at different temperatures

[0060] The multi-copper oxidase mutant was added to the system under different temperatures, enzyme concentration 8 U / mL, pH 5.0, and reacted for 48 h, and the content of biogenic amines was detected. The biogenic amine degradation results are shown in Table 1.

[0061] Table 1 Biogenic amine degradation rate of multi-copper oxidase mutant at different temperatures

[0062] temperature Tryptamine Phenylethylamine Putrescine Cadaverine histamine Octopamine Tyramine Spermidine Spermine 10℃ 38.94％ 46.84％ 71.27％ 40.56％ 27.31％ 75.41％ 99.18％ 74.66％ 51.80％ 15℃ 92.54％ 85.36％ 96.84％ 83.51％ 86.45％ 91.95％ 98.97％ 97.88％ 93.81％ 20℃ 91.93％ 93.99％ 96.90％ 91.49％ 90.42％ 98.01％ 99.37％ 98.82％ 99.23％ 25℃ 98.41％ 99.57％ 99.98％ 96.28％ 99.72％ 98.63％ 99.64％ 99.13％ 98.76％ 30℃ 95.89％ 99.36％ 96.25％ 91.11％ 99.27％ 95.88％ 94.35％ 97.25％ 94.25％ 35℃ 91.69％ 98.21％ 96.77％ 96.15％ 55.26％ 95.01％ 97.88％ 34.16％ 11.83％ 40℃ 3.54％ 2.85％ 24.63％ 4.75％ 0.70％ 0.27％ 4.33％ 1.45％ 0.91％ 45℃ 2.17％ 1.66％ 2.57％ 3.43％ 1.20％ 1.27％ 4.84％ 1.91％ 0.76％

[0063] From Table 1, it can be seen that the enzyme has the highest degradation efficiency for biogenic amines at 15-30℃, and can efficiently degrade 9 amines. The degradation efficiency of the enzyme for biogenic amines is poor at low temperature (10℃) and high temperature (40-45℃), and there is no amine degradation activity at high temperature.

[0064] (2) Biogenic amine degradation rate of multi-copper oxidase mutant at different pH

[0065] The multi-copper oxidase mutant was added to the system under different pH, enzyme concentration 8 U / mL, and reacted for 48 h at 37℃, and the content of biogenic amines was detected. The biogenic amine degradation results are shown in Table 2.

[0066] Table 2 Biogenic amine degradation rate of multi-copper oxidase mutant at different pH

[0067] pH Tryptamine Phenylethylamine Putrescine Cadaverine histamine Octopamine Tyramine Spermidine Spermine 2 2.17％ 0.47％ 3.57％ 0.64％ 0.54％ 3.48％ 7.05％ 8.57％ 2.73％ 3 9.61％ 28.91％ 27.91％ 5.54％ 1.20％ 3.87％ 22.04％ 5.65％ 1.80％ 4 33.94％ 69.88％ 52.52％ 9.94％ 9.78％ 13.90％ 53.81％ 14.97％ 4.99％ 5 91.89％ 98.25％ 98.18％ 96.02％ 53.70％ 94.91％ 97.96％ 33.93％ 11.47％ 6 95.13％ 97.73％ 99.11％ 94.91％ 56.91％ 98.50％ 99.71％ 97.86％ 92.14％ 7 93.44％ 98.23％ 99.42％ 95.95％ 81.08％ 98.81％ 99.70％ 98.53％ 99.39％ 8 95.59％ 98.74％ 98.84％ 97.44％ 75.21％ 90.91％ 99.52％ 93.36％ 96.61％ 9 9.46％ 14.94％ 98.61％ 95.70％ 23.01％ 93.31％ 99.52％ 94.48％ 98.55％ 10 2.11％ 15.78％ 98.67％ 70.58％ 7.01％ 61.48％ 52.24％ 47.80％ 35.85％

[0068] From Table 2, it can be seen that the enzyme can efficiently degrade 9 biogenic amines under the condition of pH 6.0-8.0, loses amine degradation activity under acidic conditions (pH 2.0-3.0), and inhibits amine degradation activity under alkaline conditions (pH 9.0-10.0).

[0069] (3) Biogenic amine degradation rate of multi-copper oxidase mutant at different enzyme concentrations

[0070] The multi-copper oxidase mutant was added to the system under different enzyme concentrations, reacted for 48 h at 37℃ and pH 5.0, and the content of biogenic amines was detected. The biogenic amine degradation results are shown in Table 3.

[0071] Table 3 Biogenic amine degradation rate of multi-copper oxidase mutant at different enzyme concentrations

[0072]

[0073] From Table 3, it can be seen that the amine degradation rate increases with the increase of enzyme concentration, and the amine degradation rate reaches the highest value at 12 U / mL.

[0074] Example 5 Degradation of biogenic amines in argentine squid surimi by multi-copper oxidase mutant

[0075] The multi-copper oxidase mutant enzyme solution was added to the Argentinian squid surimi at a concentration of 8 U / mL. The reaction was incubated at 25°C for 48 hours, and then the surimi was extracted to detect the biogenic amine content. The biogenic amine degradation results are shown in Table 4.

[0076] The biogenic amine content was determined as follows: 10 g of squid surimi was added to 20 mL of 5% trichloroacetic acid solution, stirred thoroughly, and then extracted with vortexing for 30 minutes. The mixture was centrifuged at 5000 rpm (8000 rpm) for 10 minutes, extracted twice with 20 mL of 5% trichloroacetic acid solution, and the supernatants were combined and diluted to the 50 mL mark with 5% trichloroacetic acid. 10 mL of the extract was placed in a 50 mL stoppered centrifuge tube, 0.5 g of sodium chloride was added, and the mixture was vortexed until the sodium chloride was completely dissolved. 10 mL of n-hexane was added and vortexed for 5 minutes. After standing for separation, the upper organic phase was discarded, and the lower sample solution was degreased again by adding 10 mL of n-hexane. 5 mL of the degreased sample solution was transferred to a 10 mL stoppered centrifuge tube and the pH was adjusted to approximately 12.0 with a few drops of 5 mol / L sodium hydroxide solution. Add 5 mL of a 1+1 mixture of n-butanol and chloroform, vortex for 5 minutes, and centrifuge at 5000 rpm for 5 minutes. Remove the sample and allow it to stand for stratification. Transfer the upper aqueous phase to another 10 mL stoppered centrifuge tube and extract once more. Combine the extracts and dilute to volume with n-butanol and chloroform (1+1). Add 200 μL of 1 mol / L hydrochloric acid to 5 mL of the extract, mix thoroughly, and blow dry in a 40°C water bath under nitrogen. Add 1 mL of 0.1 mol / L hydrochloric acid and vortex to completely dissolve the residue before derivatization.

[0077] Table 4 Biogenic amine degradation results

[0078] Content of surimiamine in Argentine squid Octopamine Cadaverine Tyramine Spermidine Spermine Before degradation (mg / L) 6.99 2.56 2.6 11.79 7.48 After degradation (mg / L) 0 0 0 0 0 Degradation rate (%) 100％ 100％ 100％ 100％ 100％

[0079] Example 6 Degradation of biogenic amines in fermented fish sauce by multi-copper oxidase mutants

[0080] An 8 U / mL multi-copper oxidase mutant enzyme solution was added to the fermented fish sauce. The reaction was incubated at 25°C for 48 h, and the supernatant was removed after lipid removal for detection of biogenic amine content. The results of biogenic amine degradation are shown in Table 5.

[0081] Table 5 Biogenic amine degradation results

[0082] Fish Sauce Amine Content Phenylethylamine Putrescine Cadaverine histamine Tyramine Spermine Before degradation (mg / L) 11.21 39.40 76.89 79.11 77.84 25.65 After degradation (mg / L) 0.00 0.00 20.74 13.95 0.00 0.00 Degradation rate (%) 100％ 100％ 76.62％ 82.37％ 100％ 100％

[0083] The degradation of biogenic amines by the multi-copper oxidase mutant of the present invention is disclosed above using fermented fish sauce and Argentine squid surimi as examples. The applicable environmental system is the aquatic product field; the environmental system includes but is not limited to fish, crustaceans, shellfish, cephalopods, algae, mollusks, aquatic plants, and processed aquatic products.

Claims

1. Use of a multi-copper oxidase mutant in the degradation of biogenic amines, wherein the amino acid sequence of the multi-copper oxidase mutant is shown in SEQ ID NO.

1.

2. The use of a multi-copper oxidase mutant in the degradation of biogenic amines according to claim 1, characterized in that: The application is to degrade biogenic amines in aquatic products.

3. The use of a multi-copper oxidase mutant in the degradation of biogenic amines according to claim 3, characterized in that: The aquatic products are fish, crustaceans, shellfish, cephalopods, algae or molluscs.

4. The use of a multi-copper oxidase mutant in the degradation of biogenic amines according to claim 1, characterized in that: The biogenic amines include tryptamine, phenylethylamine, putrescine, cadaverine, histamine, octopamine, tyramine, spermidine and spermine.

5. The use of a multi-copper oxidase mutant in the degradation of biogenic amines according to claim 1, characterized in that: The multi-copper oxidase mutant is used in the form of enzyme powder or enzyme liquid to degrade biogenic amines in a reaction system.

6. Use of a multi-copper oxidase mutant in the degradation of biogenic amines according to claim 1, characterized in that: The amount of the multi-copper oxidase mutant added to the reaction system is 4-12 U / mL.

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