Biogenic amines (BAs) are potential amine hazards that are detected in fermented foods and alcoholic beverages. Excessive intake of BAs may lead to allergic symptoms such as difficulty in breathing, nausea, and vomiting. Degradation of BAs by multicopper oxidase (MCO) is a promising method as it has little effect on the fermentation process, food nutrition, and flavor. However, the application of MCO in food industry was restricted due to its poor catalytic properties and low productivity. In this work, food-grade expression of the Bacillus amyloliquefaciens MCO (MCOB) and its three mutants were successfully constructed in Lactococcus lactis NZ3900. The expression level of MCOB in L. lactis NZ3900 was dramatically enhanced by optimizing the cultivation conditions, and the highest expression level reached 4488.1 U/L. This was the highest expression level of food-graded MCO reported so far, to our knowledge. Interestingly, the optimal reaction pH of MCOB expressed in L. lactis NZ3900 switched to 4.5, it would be more suitable for degrading BAs in food as the pH value of most fermented foods was found to be 4.5. Moreover, MCOB expressed in L. lactis NZ3900 was quite stable (with more than 80% residual activity) in the pH range of 4.0–5.5, the catalytic rate constant (kcat) and specific activity of MCOBLS were all dramatically increased compared with that of MCOB expressed in Escherichia coli. Using histamine as the substrate, the degradation of BAs within 24 h by MCOB expressed in L. lactis NZ3900 was 69.7% higher than that expressed in E. coli. The results demonstrated the potential applications of MCOB in food industry for reduction of biogenic amines.
Biogenic amines (BAs) are a group of low-molecular-weight amine compounds that have been detected in a variety of foods such as cheese, sausage, fish, soy sauce, and wine . The common BAs are detected in food include histamine, tyramine, putrescine, cadaverine, spermine, spermidine, phenethylamine, and tryptamine . They are mainly generated through decarboxylation of amino acids by lactic acid bacteria (LAB) in the process of food fermentation [3, 4]. Low levels of BAs in animals’ body are essential for keeping normal metabolic functions. However, intake of BAs present at a high concentrations in food (total BAs > 1000 mg/kg food) may cause allergic symptoms such as nausea, headache, respiratory distress, and blood pressure increasing [5,6,7]. Moreover, histamine and tyramine could bring severe acute allergic effects that affect human health . Food and Drug Administration (FDA) gives a guidance level of histamine in the edible portion of fish and suggests rejecting any fish are found with a histamine level greater than or equal to 50 ppm . European Food Safety Authority (EFSA) limits histamine between 200 and 400 mg/kg in fish products with enzyme maturation treatment in brine [10, 11]. For food products such as meat and dairy, the same legislation is applied . Histamine and tyramine are the two major BAs in soy sauce, and their presence in Chinese brands reached a level of 478 mg/L and 257 mg/L, respectively . In fish sauce, the total BAs are found to be 49–2331 mg/kg, and the major BAs, such as histamine, tyramine, cadaverine, and putrescine, are determined at the level of 2–783 mg/kg, 14–469 mg/kg, 13–606 mg/kg, and 7–277 mg/kg, respectively . Thus, it is important to reduce the content of BAs in food to a safety level.
Technical measures, such as using of antimicrobial compounds, irradiation, or adjustment of fermentation conditions to decrease the population of LAB, were performed to reducing or eliminating BAs formation during food fermentation [15, 16]. However, food fermentation especially traditional food fermentation is a complex process, even though a successful reduction or elimination of BAs formation by techniques, it is worth noting that these methods may cause alterations of the aroma or quality of foods [17, 18]. However, enzymatic degradation is the most promising method to reduce BAs in fermented foods without adjusting or changing the fermentation process, and it has no unwanted impact on nutrition and flavor of food products [19, 20]. Enzymes that are found being capable of degrading BAs include amine oxidases, amine dehydrogenase, and multicopper oxidase [21, 22]. Amine oxidase and amine dehydrogenase catalysis, the degradation of specific BA, and their activity can be strongly inhibited by ethanol and carbonyl reagents [23, 24]. Multicopper oxidase (MCO) is a group of enzymes that oxidize a wide range of phenolic and nonphenolic aromatic compounds and concomitantly with the reduction of dioxygen into water . MCOs that can degrade BAs in fermented foods have been characterized from some lactic acid bacteria. The gene encoding multicopper oxidase has been verified in the genome of Lactobacillus curvatus G-1, and this enzyme exhibits capability in degradation of six common BAs including histamine, tyramine, and putrescine . MCO from Lactobacillus plantarum J16 can degrade tyramine, histamine, and putrescine, while the one from Pediococcus acidilactici CECT 5930 degrades tyramine only [27,28,29]. MCO from Lactobacillus fermentum Y29 showed activity against a broad range of substrates (histamine, tyramine, tryptamine, phenylethylamine, putrescine, cadaverine, and spermidine), and was found to be tolerant to 18% (w/v) NaCl . The optimal pH for catalysis of most bacterial MCOs is 3.0–4.0, and their activity at pH 4.5 (the pH of most fermented food, condiments, and alcohol beverages) is much lower than those at the optimal ones [31, 32]. Moreover, these MCOs have poor salt tolerance and their activity remains only 10% in the presence of 1 M NaCl [33, 34]. In the previous work, using site-direct mutagenesis, both the catalytic efficacy and pH stability (in the range of pH 2.5–5.0) of the MCOBs (MCO from Bacillus amyloliquefaciens) were dramatically increased. In addition, the salt tolerance of a MCOB triple mutant T317N/L386Y/S427E was increased by 61.3% compared with that of MCOB in the presence of 15% (w/v) NaCl . Heterologous expressions of BA-degrading MCOs were done in Escherichia coli, but the expression levels were relatively low. The highest expression and secretion expression levels of MCOs in E. coli were 3545.7 U/L and 238.1 U/L, respectively [36, 37]. On the other hand, recombinant MCOs produced by E. coli are not acceptable for food applications and have safety issues as E. coli produces endotoxin. These recombinant MCOs must be extensively purified before application, but still lack the food-grade and GRAS attributes [38,39,40]. At present, no food-grade expression of MCO was reported and there were no food-grade enzymes for degrading BAs available. Therefore, food-grade and high-efficiency expression of MCO is critical for its applications in degradation of BAs in fermented foods.
Lactococcus lactis gene expression system is widely used in food industry for production of a variety of enzymes [41, 42]. In this work, L. lactis NZ3900 was used to construct recombinants for food-grade expression of MCOs with Bas’ degradation activity. The expression level of MCOBLS was improved by optimization of the induced expression conditions, and enzymatic properties and capacity in degrading BAs of recombinant MCOBs were investigated. The results may provide a good reference for study and development of food-grade enzymatic degradation strategies for reduction of BAs in fermented foods.
Materials and methods
Strains and cultivation conditions
The bacterial strains and plasmids used in this work are listed in Table 1. E. coli BL21(DE3) and L. lactis NZ3900 were used as the host for expression of MCOs. E. coli BL21(DE3) recombinants were cultivated in LB broth supplemented with 50 μg/mL kanamycin and incubated overnight at 37 °C with shaking. L. lactis NZ3900 and its recombinants were cultivated in GM17 medium (M17 supplemented with 5 g/L glucose) at 30 °C for overnight.
Construction of L. lactis NZ3900 recombinants for food-grade expression of MCO
Construction of plasmids for over-expression of MCO L. lactis NZ3900 is shown in Fig. 1. The vector pNZ8149 was firstly linearized by PCR amplification using primers P1 and P2 (Table 2). Genes encode B. amyloliquefaciens MCO (MCOB) (GenBank: WP_088612302.1) and its mutants (L386Y, T317N/L386Y and T317N/L386Y/S427E) were amplified using primers B-L/B-R. Genes encode L. fermentum MCO (MCOF) and Weissella cibaria MCO (MCOW) were amplified by primers F-L/F-R and W-L/W-R, respectively. Then, the purified PCR products of MCOs and pNZ8149 were assembled by the Gibson assembly method using the ABclonal MultiF Seamless Assembly Mix kit (ABclonal Technology, Wuhan, China) .
Enzyme activity assay
MCO activity was analyzed by detection of the oxidation rate of 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid, ABTS) using a method described previously . Briefly, 50 μL of enzyme was mixed with 950 μL reaction buffer (50 mM sodium citrate–phosphate, 1 mM CuCl2, 1 mM ABTS, and pH 4.5) and incubated at 50 °C for 1 min. Oxidation of ABTS was determined by measuring the absorbance at 420 nm [ε = 36 L/(mM·cm)]. The amount of enzyme that oxidizes 1 μmol ABTS per minute was defined as one unit (U). MCO activity was calculated using Eq. (1)
ΔOD is the change of absorbance; Δt is the reaction time; ε is the molar absorption coefficient of ABTS at 420 nm; V1 is the total volume of the reaction solution; V2 is the volume of enzyme solution.
Expression of MCO
Escherichia coli BL21(DE3) recombinants for expression of MCOs were cultivated in LB broth supplemented with 50 μg/mL kanamycin and incubated overnight at 37 °C with shaking . Then, 2% (v/v) of the culture was inoculated into TB medium (supplemented with 50 μg/mL kanamycin) and cultured at 37 °C. Expression of MCO was induced with 0.1 mM IPTG when the OD600 reached 0.6, and 1 mM CuCl2 were added, induction was done at 20 °C for 20 h. Recombinant L. lactis NZ3900 strains were cultivated in GM17 medium and incubated at 30 °C for 16 h before subculturing in fresh GM17 medium. To induce the expression of MCOs, 0–45 ng/mL nisin (Sigma, St. Louis, USA) was added when OD600 reached approximately 0.5, and then, induction was done at different temperatures (24–32 °C) for 0–12 h, and CuCl2 (0.1–0.5 mM) was added when needed.
Cells of L. lactis NZ3900 that expressed MCOBLS (MCOBs expressed in L. lactis) were harvested by centrifugation, and washed and resuspended in 50 mM phosphate-buffer saline (pH 7.4). The cell suspension was disrupted by sonicating for 3 s, with 2 s intervals for 20 min. Then, the soluble fractions were obtained by centrifugation at 12,000 × g for 30 min at 4 °C. The ÄKTA Explorer system (GE Healthcare Life Sciences, Piscataway, NJ, USA) was used for protein purification. Crude enzyme was applied to a StrepTrap™ HP affinity column (GE Healthcare, Uppsala, Sweden), which had been equilibrated with binding buffer (20 mM phosphate buffer, 0.5 M NaCl, pH 7.4). The target protein (MCOLS) were eluted with elution buffer (20 mM phosphate buffer, 0.5 M NaCl, 0.5 M imidazole, pH 7.4) followed by a desalting step. Protein concentration was determined by the Bradford method using a protein assay kit (Sangon Biotech, Shanghai, China) . MCOBES (MCOBs expressed in E. coli) were purified using the same method.
Determination of biochemical properties of MCO
The effect of temperature on MCOs activity was determined at the range of 35–75 °C. For thermal stability tests, MCOs were incubated in 50 mM phosphate-buffer saline (pH 7.4) at different temperatures (35–75 °C) for 2 h. The optimal reaction pH and stability of MCOs at different pH were evaluated in 50 mM sodium citrate buffer (pH 2.5–5.5). For testing pH stability of MCOs, enzyme solutions were incubated in buffers with different pH values for 2 h at 4 °C. Kinetic parameters of the purified recombinant MCOs were determined using ABTS (0.1–1.0 mM) as the substrate. Data were fitted in the Michaelis–Menten equation to calculate Vmax and Km values .
Degradation of BA by MCO
To compare the capability of different recombinant MCOs in degradation of BA, purified MCOBE0 (MCOB expressed in E. coli) and MCOBL0 (MCOB expressed in L. lactis) (5000 U/L) were mixed with 50 mM sodium phosphate buffer (pH 4.5) containing histamine (final concentration 50 mg/L) and incubated at 37 °C for 0–48 h. For evaluation of MCO in degradation of different BAs, purified MCOBL3 (2400 U/L) was mixed with sodium phosphate buffers (50 mM, pH 4.5) containing histamine, tyramine, or putrescine (final concentration 100 mg/L) and incubated still or with shaking (100 rpm) at 37 °C for 24 h. BA degradation rate is calculated using Eq. (2)
M is the percentage of BA degradation; A and B represent the initial and remain BA content, respectively.
Determination of biogenic amines
Biogenetic amines were determined by HPLC as previously described by Li et al. [47, 48]. BA standards and samples were derivatized by dansyl chloride (5 mg/mL). The analysis was operated on an Agilent 1260 system equipped with ODS2 HYPERSIL C18 (250 mm × 4.6 mm, 5 µm) (Thermo Scientific, Bellefonte, USA) using defined column temperature (30 °C), flow rate (0.8 mL/min), and detection wavelength (254 nm). Acetonitrile and H2O were used as mobile phase A and B, and the solvent linear gradient procedure was set as: 55% A (0–7 min), 65% A (7–14 min), 70% A (14–20 min), then from 70 to 55% of solvent A to return to initial condition (within 20–22 min).
Results and discussion
Construction of recombinant strains for food-grade expression of MCOs
Recombinant strains of L. lactis NZ3900 for food-grade expression of MCOs were successfully constructed using the strategy shown in Fig. 1. Among them, MCOFL0 was not expressed (Fig. 2a), while MCOWL0 (57.3 kDa) and MCOBLS (MCOBL0 and its mutants MCOBL1, MCOBL2 and MCOBL3, 59.2 kDa) were successfully expressed in L. lactis NZ3900 (Fig. 2a, b). The expression level of the wild-type MCOBL0 was 231.1 U/L, mutant MCOBL1 (L386Y) showed the highest expression level at 336.2 U/L, MCOBL2 (T317N/L386Y), and the salt tolerant triple mutant MCOBL3 (T317N/L386Y/S427E) were expressed at 323.8 U/L and 282.8 U/L, respectively (Fig. 3). However, no active MCOWL0 was expressed in L. lactis NZ3900, though it had been successfully expressed in E. coli previously . Thus, L. lactis NZ3900 recombinants for expression of MCOBLS were selected for further studies.
Optimization of MCOBLS expression in L. lactis
To improve the expression of MCOBLS in L. lactis, induction conditions were optimized. It was found that 2% (v/v) of inoculum size and induction at 30 °C were the best for expression of MCOBLS in L. lactis NZ3900. Under these conditions, expression of MCOBL0 was increased to 803.6 U/L, and the highest expression level reached 1136.4 U/L for MCOBL1 (Fig. 4a, b). The optimal concentration of nisin for induction was slightly different for MCOBLS. As shown in Fig. 4c, 40 ng/mL nisin was the best for induced expression of MCOBL0, while 35 ng/mL was the best for expression of its mutants MCOBL1, MCOBL2, and MCOBL3. Induced with 35 ng/mL nisin, expression of all MCOBLS reached to the highest levels after inducing for 12 h, and the highest expression level was 1813.8 U/L for MCOBL1 (Fig. 4d). It has been reported that heterologous expression of the active form of recombinant MCO needs copper for proper folding, and addition of copper during expression contributed to enhanced activity . The optimum Cu2+ concentration for expression of MCOBL0 was 0.4 mM, while it was 0.15 mM for the mutants (Fig. 4e). The different effect of copper on MCOBLS may be related to their tolerance to copper caused by amino acid residue differences [49, 50]. For all MCOBLS, the best time for adding Cu2+ was 4 h after nisin induction (Fig. 4f). Under the best induction conditions, expression of MCOBL0, MCOBL1, MCOBL2, and MCOBL3 dramatically increased to 2325.8 U/L, 4488.1 U/L, 4289.8 U/L, and 2254.5 U/L, and these were 9.1, 12.4, 12.3 and 7.0 times higher than those of the non-optimized, respectively. Moreover, the highest expression level of MCOB in L. lactis was 1.3 times higher than that expressed in E. coil (3545.7 U/L) . For expression of MCOB, an enzyme from the Gram-positive bacteria Bacillus, Lactococcus is probably a better host than the Gram-negative bacteria E. coli. In addition, expression of heterologous genes in E. coli sometime is inefficient due to incorrect folding and formation of inclusion bodies [41, 51].
Enzymatic properties of MCOBLS
To compare the biochemical properties of MCOBLS with MCOBES, MCOBLS were purified using the affinity chromatography (Fig. 5). As shown in Table 3, in spite of the increasing in Km values of MCOBLS, the catalytic rate constant (kcat) and specific activity of MCOBLS were dramatically increased compared with that of MCOBES. They were increased by 1.4–2.1-fold and 1.1–2.5-fold, respectively. Except for MCOBL0, the catalytic efficiency (kcat/Km) of other MCOBLS was not significant changed, compared with MCOBES. The Km values of both MCOBLS and MCOBES were lower than that of the P. acidilactici MCO (1.7 mM) . The changes in kinetic properties of MCOBLS indicated a potential improved performance of them in degradation of BAs.
For further evaluation of the potential applications of MCOBLS, the optimal reaction temperature, pH, and their stability under these conditions were investigated. It was found that the optimum temperature of MCOBLS was 65 °C (Fig. 6a), and they were stable at the temperature range of 35–45 °C with 73.0–99.5% residual activity (Fig. 6b). The thermal stability of MCOB was better than the L. plantarum MCO, of which the optimal temperature was 60 °C. The L. plantarum MCO lost more than 30% activity in 10 min when incubated at 35–45 °C . Interestingly, the optimal reaction pH of MCOBLS was detected to be 4.5 (Fig. 6c), and this was different from that of MCOBs expressed in E. coli (pH 3.0 for MCOBES) and other multicopper oxidases [35, 49]. Moreover, MCOBLS were quite stable at the pH range of 2.5–5.5, in particularly, showed greater than 80% residual activity between pH 4.0 and 5.5 (Fig. 6d).
It has been reported previously that biochemical properties of a protein may be changed when expressed in varies strains. Physiological characteristics and intracellular environment of the host for recombinant protein (such as MCO and histidine decarboxylase) will facilitate considerable contributions . Our results showed that host strains might affect the biochemical properties of recombinant enzymes. These changes are crucial for their application as the pH values for most of fermented foods are around 4.5 [54, 55].
Degradation of BAs by MCOBLS
Previous work demonstrated that the MCO from B. amyloliquefaciens (MCOB) showed the best capability in degradation of histamine. Thus, we compared the capability of both MCOBL0 and MCOBE0 in degradation of histamine. The result showed that MCOBL0 was better in degradation of histamine than that of MCOBE0. The degradation rate of histamine by MCOBL0 (5000 U/L) within 24 h was 27.7%, and this was 69.7% higher than that by MCOBE0 (Fig. 7a). In addition, we also tested the capability of the salt tolerant triple mutant MCOBL3 (MCOB T317N/L386Y/S427E) in degradation of multiple BAs. As shown in Fig. 7b, MCOBL3 (2400 U/L) could efficiently degrade histamine and tyramine, the most abundant BAs in soy sauce , and was capable of degrading putrescine. Under the aerobic (with shaking) reaction condition, MCOBL3 had better capability in degradation of tyramine and putrescine. In comparison with the MCO from P. acidilactici, no degradation of histamine and putrescine was observed for this enzyme, although it showed less than 20% degradation of total BAs . Other studies demonstrated only 10% and 5% degradation of histamine and putrescine by MCOs from LAB [32, 57]. At present, the degradation rate of BAs using MCOs was lower than that using LAB strains , degradation of BAs by food-grade enzymes is still a promising method as the expression level of enzyme can be easily enhanced, and the catalytic efficiency of enzymes can be improved by various molecular modification strategies [58,59,60].
In this work, food-grade expression of the MCO from B. amyloliquefaciens (MCOBLS) was successfully achieved in L. lactis NZ3900. Expression of MCOBLS was optimized and reached to 4488.1 U/L, which was 13.3 times of the unoptimized one and was the highest food-grade expression level of MCO reported so far. Interestingly, the optimal reaction pH of the recombinant MCOBLS switched to 4.5 and they were found to be more stable (with more than 80% residual activity) in the pH range of 4.0–5.5 than MCOBES. To our knowledge, pH 4.5 was not the optimal pH of the reported MCO with capability in degrading BAs. In addition, MCOBLS also showed high thermal stability at 35–45 °C. The changes in enzymatic properties of MCOBLS demonstrated a better performance of them in degrading BAs than the E. coli recombinant MCOBES and other MCOs. Hence, we showed that the degradation rate of histamine by MCOBLS was 69.7% higher than by MCOBES within 24 h. The results demonstrated that MCOB expressed in L. lactis was better in degradation of BAs than those expressed in E. coli, and indicated its potential application prospect in food industry.
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This work was supported by the National Key Research and Development Program of China (2017YFC1600405) and National Natural Science Foundation of China (31771955).
Conflict of interest
The authors declare that there are no conflicts of interest.
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Ni, X., Chen, J., Du, G. et al. Food-grade expression of multicopper oxidase with improved capability in degrading biogenic amines. Syst Microbiol and Biomanuf (2021). https://doi.org/10.1007/s43393-021-00061-9
- Biogenic amines
- Multicopper oxidase
- Food-grade expression