Characterization and Preliminary Application of a Novel Lipoxygenase from Enterovibrio norvegicus

Lipoxygenases have proven to be a potential biocatalyst for various industrial applications. However, low catalytic activity, low thermostability, and narrow range of pH stability largely limit its application. Here, a lipoxygenase (LOX) gene from Enterovibrio norvegicus DSM 15893 (EnLOX) was cloned and expressed in Escherichia coli BL21 (DE3). EnLOX showed the catalytic activity of 40.34 U mg−1 at 50 °C, pH 8.0. Notably, the enzyme showed superior thermostability, and wide pH range stability. EnLOX remained above 50% of its initial activity after heat treatment below 50 °C for 6 h, and its melting point temperature reached 78.7 °C. More than 70% of its activity was maintained after incubation at pH 5.0–9.5 and 4 °C for 10 h. In addition, EnLOX exhibited high substrate specificity towards linoleic acid, and its kinetic parameters of Vmax, Km, and Kcat values were 12.42 mmol min−1 mg−1, 3.49 μmol L−1, and 16.86 s−1, respectively. LC-MS/MS analysis indicated that EnLOX can be classified as 13-LOX, due to its ability to catalyze C18 polyunsaturated fatty acid to form 13-hydroxy fatty acid. Additionally, EnLOX could improve the farinograph characteristics and rheological properties of wheat dough. These results reveal the potential applications of EnLOX in the food industry.


Introduction
Lipoxygenases (LOXs) are a family of non-heme iron dioxygenases, which catalyze the regiospecific and stereospecific insertion of molecular oxygen in polyunsaturated fatty acids (PUFAs), containing 1-Z, 4-Z-pentadiene moieties into hydroperoxides fatty acid with (Z), (E)-diene conjugations [1][2][3]. LOXs have a broad potential for application in food, chemical, and pharmaceutical industries, of which the application prospects in dough products are due to the reinforcement effect by enhancing the gluten network [4,5]. LOX is extracted mainly from soybean powder [6][7][8][9], which contains multiple enzymes, and may reduce the catalyzing effects of LOX and produce unpleasant odor after the catalytic process, thus, limiting the application of LOXs in flour products [7,10,11]. Hence, the hunt for LOXs with desired properties has gained widespread attention for its application prospects in dough products.
LOXs are widely found in animals, plants, and microorganisms, but only microbial-LOXs are of industrial significance due to its high yield, low cost, and simple preparation process [12,13]. To date, many LOXs from microorganisms have been characterized [14,15], especially LOXs from Anabaena [16], Burkholderia thailandensis [15], Pseudomonas aeruginosa [14], and Myxococcus xanthus [1]. However, these LOXs cannot meet all the requirements of high catalytic activity, good thermostability, and wide pH-stability simultaneously. Directed evolution, rational design, and semi-rational design were used to improve biochemical characteristics of LOX [17][18][19][20]. Consensus design method was used to improve the thermostability of LOX from Anabaena [21]; the specific activity of P. aeruginosa LOX was improved using the fused self-assembling amphipathic peptides [19]. Although researchers have made a lot of efforts to improve the properties of LOXs by protein engineering [17,19,21], LOX, with the comprehensive properties, has not yet been obtained. Meanwhile, these methods are dependent on the analysis of crystal structures or the establishment of a high-throughput screening library, and it is hard to obtain mutant enzymes with superior properties in a short period of time [20]. Therefore, microbial-LOXs have been reported to not be suitable for industrial application, which calls for high yield, high purity, acid and alkali resistance, good thermal stability, and high catalytic activity [7,21]. Considering the demand of LOXs with satisfactory properties, it is reasonable to gain new types of high-efficiency, thermo-stable, and wide pH-range stable LOXs by excavating the genetic reservoir in nature [20,22].
In this study, a novel LOX gene from E. norvegicus DSM 15893 was analyzed using bioinformatics technology, then cloned and expressed in Escherichia coli BL21 (DE3). The properties of the recombinant E. norvegicus LOX (EnLOX) have been characterized. Moreover, the influence of EnLOX on the farinograph characteristics and rheological properties of dough were evaluated.
to improve biochemical characteristics of LOX [17][18][19][20]. Consensus design method used to improve the thermostability of LOX from Anabaena [21]; the specific activity o aeruginosa LOX was improved using the fused self-assembling amphipathic peptides Although researchers have made a lot of efforts to improve the properties of LOX protein engineering [17,19,21], LOX, with the comprehensive properties, has not yet b obtained. Meanwhile, these methods are dependent on the analysis of crystal struct or the establishment of a high-throughput screening library, and it is hard to ob mutant enzymes with superior properties in a short period of time [20]. Theref microbial-LOXs have been reported to not be suitable for industrial application, w calls for high yield, high purity, acid and alkali resistance, good thermal stability, and h catalytic activity [7,21]. Considering the demand of LOXs with satisfactory propertie is reasonable to gain new types of high-efficiency, thermo-stable, and wide pH-ra stable LOXs by excavating the genetic reservoir in nature [20,22].
In this study, a novel LOX gene from E. norvegicus DSM 15893 was analyzed u bioinformatics technology, then cloned and expressed in Escherichia coli BL21 (DE3). properties of the recombinant E. norvegicus LOX (EnLOX) have been characteri Moreover, the influence of EnLOX on the farinograph characteristics and rheolog properties of dough were evaluated.

Phylogenetic Analysis and Structural Modeling of EnLOX
SWISS-MODEL was used to build the three dimensional (3D) model [23]. Pymol was used to analyze the 3D structure of EnLOX [24]. ProtParam tool of Expasy was used to predicted the molecular mass of EnLOX [25]. The nucleotide and amino acid sequences of EnLOX were subjected to homology sequence similarity retrieval and analysis on the NCBI, and then the percent identities among the nucleotide and amino acid sequences were calculated by MEGA 5.05.

Expression and Purification of EnLOX
E. coli BL21 (DE3)/pET28a-EnLOX combination was inoculated into the Luria Broth medium (LB) containing 100 µg mL −1 kanamycin at 37 • C and agitation rate of 180 rpm until the optical density at 600 nm reached 0.6-0.8. The final concentration of 100 µg mL −1 IPTG was added to induce the protein expression at 16 • C for 16 h. The cells were collected by centrifugation (8000× g, 5 min), and resuspended in Tris-HCl buffer (0.05 mol L −1 Tris-HCl, 0.3 mol L −1 NaCl, pH 7.5). After cells were completely broken by D-3L High Pressure Homogenizer (PhD Technology LLC, Minnesota, USA), cell debris were removed by centrifugation (4 • C, 10000× g, 30 min). The supernatant loaded onto Ni-NTA affinity column for purification. The purity and molecular mass of EnLOX were checked by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) [1,21].

Determination of EnLOX Activity and Protein Concentration
EnLOX activity was assayed in 20 mmol L −1 Tris-HCl buffer (pH 8.0) at 50 • C by monitoring the increase in absorbance at 234 nm. The final concentration of 1.73 mmol L −1 PUFAs was added to a 3 mL reaction system. One unit of activity was defined as the amount of enzyme required to synthesize 1 mmol hydroperoxide per min with an extinction coefficient of 25,000 L mol −1 cm −1 [1,20,21,26]. The protein concentration was measured by Bradford method [27,28].

Kinetic Parameters of EnLOX
Substrate specificity of EnLOX was determined with different final concentration of PUFAs from 10 to 300 mmol L −1 . All measurements were carried out at 50 • C in 20 mmol L −1 Tris-HCl buffer (pH 8.0). The kinetic parameters of K m , V max , and K cat were calculated by nonlinear regression fitting of the experimental data to Michaelis-Menten equation using GraphPad software. Michaelis-Menten equation is shown as follows [29,30]: where v 0 , V max , [S], and K m represent the initial rate, the maximum velocity of reaction, the substrate concentration, and the Michaelis constant, respectively.

Product Detection of EnLOX
Product specificity of EnLOX was determined by incubating PUFAs, EnLOX, and Tris-HCl buffer (pH 8.0) at 50 • C for 10 min. Hydroperoxides formed were reduced to their corresponding hydroxides with addition of 10 mmol L −1 cysteine. The reaction mixtures were extracted according to the method of HUANG et al. [31]. The products were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS), with an UPLC column (2.1 × 100 mm ACQUITY UPLC BEH C18 column containing 1.7 µm particles). Buffer A consisted of 0.1% formic acid in water; buffer B consisted of 0.1% formic acid in acetonitrile. The gradient was 20% Buffer B for 0.5 min, 20-98% Buffer B for 10 min, and 98% Buffer B for 4 min, with a flow rate of 0.4 mL min −1 . Mass spectrometry was performed using electrospray source in negative ion mode with MS acquisition mode, with a selected mass range of 100-1000 m/z. The lock mass option was enabled using leucine-enkephalin (m/z 556.2771) for recalibration. The ionization parameters were the following: capillary voltage was 2.5 kV, sample cone was 40 V, source temperature was 120 • C, and desolvation gas temperature was 800 • C. Data acquisition and processing were performed using Masslynx 4.1.

Effect of pH and Temperature on EnLOX Activity and Stability
The effect of pH on EnLOX activity was measured from pH 3.0 to pH 9.5 using citrate sodium citrate buffer (pH 3.0-6.0), phosphate buffer solution (pH 6.0-7.0), Tris-HCl buffer (pH 7.0-9.0), and borax-sodium hydroxide buffer (pH 9.0-9.5) at 50 • C [32,33]. The highest activity of EnLOX as 100% to calculate the relative activities of varied pH value. To determine the pH stability, we measured the residual activity after incubating the enzyme in buffers with different pH at 4 • C for 10 h [3].
The enzyme activity was measured at different temperatures (20-80 • C) to determine the optimum temperature of EnLOX. The maximum activity was set as 100%; the relative activities at different temperatures were plotted to obtain the optimum temperature of EnLOX [34,35]. Thermostability was analyzed by measuring the residual enzyme activity after incubation at different temperatures (20-70 • C) for different times (0-12 h) before a 5 min ice bath [3,21,35]. Enzyme activity without heat treatment was defined as 100% for comparisons.

Thermal Shift Assay of EnLOX
Protein Thermal Shift was performed using Sypro Orange dye from Sigma-Aldrich [36]. The thermal shift assay was conducted in Light Cycler 480 Real Time Detection System, designed for PCR, for melting point temperature analysis. An amount of 1 µL of 1 ng µL −1 of EnLOX, 2 µL of 10X Sypro orange, and 16 µL of 40 mmol L −1 Tris-HCl (pH 8.0) were added to the wells of the 96-well Cycler PCR plate. The plate was heated from 25 to 95 • C with a heating rate of 0.5 • C min −1 . The melting point temperature (T m ) tested is the results of multiple independent experiments, each in triplicate and reported as mean ± standard deviation (SD).

Dynamic Rheological Properties
Rheological properties were measured by using a Physica MCR301 rheometer with a parallel plate (40 mm diameter, 2.5 mm gap), according to the reported research with some modifications [4,41]. The wheat dough was prepared by Brabender farinograph-E, as described before. The dough was taken out immediately and stored at 25 • C for 1 h with plastic film sealing. The rim of the dough was coated with paraffin oil to prevent dryness of the sample. The dough was relaxed for another 5 min to relax the residual stress during sample loading. The tests were measured at 25 • C. The linear viscoelastic zone was tested by stress sweeps (0.01-30%) at 1.0 Hz frequency. Frequency scan tests were performed from 0.1 to 20.0 Hz at constant strain amplitude (30% strain). Data were reported as the means of three measurements.

Statistical Analysis
All the tests were performed in triplicate, and data were expressed as mean ± SD. SPSS 21.0 were used to analyze the differences between the variables. Statistical differences in correlation analysis and linear regression analysis were determined by one-way analysis of variance (ANOVA), and Duncan's test and the significance level of p < 0.05 were used [42,43].

Bioinformatic Analysis of EnLOX
The EnLOX sequence resides in an open reading frame encoding 726 amino acids, with the calculated molecular mass of 81.45 kDa. The nucleic acid sequences of original and codon-optimized EnLOX were illustrated in Supplementary Materials Figure S1. A phylogenetic tree of LOXs, which were reported and functionally identified, was constructed ( Figure 2). Analysis of amino acid sequences in Figure 2a revealed that EnLOX had the maximum identity with E. norvegicus LOX (WP_017011269). Nevertheless, no characterization experiments of LOX from E. norvegicus have been reported so far. The nucleotide sequences showed significant differences between EnLOX and LOXs from other sources (Figure 2b). The differences for LOX genes between nucleotide and amino acid levels indicated the distinction among the organisms. As aligned with amino acid sequences of the characterized linoleate 13-lipoxygenases from soybean, P. aeruginosa, Calothrix, and Rivularia ( Figure S2), the catalytic key residues of EnLOX (His404-His409-His601-Asn605-Ile726) are completely conserved. sample loading. The tests were measured at 25 °C . The linear viscoelastic zone was tested by stress sweeps (0.01-30%) at 1.0 Hz frequency. Frequency scan tests were performed from 0.1 to 20.0 Hz at constant strain amplitude (30% strain). Data were reported as the means of three measurements.

Statistical Analysis
All the tests were performed in triplicate, and data were expressed as mean ± SD. SPSS 21.0 were used to analyze the differences between the variables. Statistical differences in correlation analysis and linear regression analysis were determined by oneway analysis of variance (ANOVA), and Duncan's test and the significance level of p < 0.05 were used [42,43].

Bioinformatic Analysis of EnLOX
The EnLOX sequence resides in an open reading frame encoding 726 amino acids, with the calculated molecular mass of 81.45 kDa. The nucleic acid sequences of original and codon-optimized EnLOX were illustrated in Supplementary Materials Figure S1. A phylogenetic tree of LOXs, which were reported and functionally identified, was constructed ( Figure 2). Analysis of amino acid sequences in Figure 2a revealed that EnLOX had the maximum identity with E. norvegicus LOX (WP_017011269). Nevertheless, no characterization experiments of LOX from E. norvegicus have been reported so far. The nucleotide sequences showed significant differences between EnLOX and LOXs from other sources (Figure 2b). The differences for LOX genes between nucleotide and amino acid levels indicated the distinction among the organisms. As aligned with amino acid sequences of the characterized linoleate 13-lipoxygenases from soybean, P. aeruginosa, Calothrix, and Rivularia ( Figure S2), the catalytic key residues of EnLOX (His404-His409-His601-Asn605-Ile726) are completely conserved. In order to identify the structural differences with other LOXs, a 3D structural model of EnLOX was predicted. The 3D structure of EnLOX was predicted by Swiss-Model using P. aeruginosa LOX (PDB ID: 4rpe) structure as a template, which revealed that EnLOX was composed of 44.0% α-helix, 8.4% strands, and 47.6% random coil (Figure 3a). The proportion of α-helix in an EnLOX structure is similar to P. aeruginosa LOX (PDB ID: 4rpe) and Cyanothece LOX (PDB ID: 5med). The results also suggested that EnLOX had the In order to identify the structural differences with other LOXs, a 3D structural model of EnLOX was predicted. The 3D structure of EnLOX was predicted by Swiss-Model using P. aeruginosa LOX (PDB ID: 4rpe) structure as a template, which revealed that EnLOX was composed of 44.0% α-helix, 8.4% strands, and 47.6% random coil (Figure 3a). The proportion of α-helix in an EnLOX structure is similar to P. aeruginosa LOX (PDB ID: 4rpe) and Cyanothece LOX (PDB ID: 5med). The results also suggested that EnLOX had the lowest (21.76%) and the highest (37.47%) identity with Gaeumannomyces graminis LOX (PDB ID: 5fx8) and P. aeruginosa LOX, which indicated that it was a novel LOX. Soy flour is used in flour and dough products due to its abundant of soybean LOXs [44]. Soybean LOX-3 (PDB ID: 1rrl) shared the highest structural homology with EnLOX among all of  Figure 3b. EnLOX has only one domain instead of two, as in soybean LOX-3. The N-terminal domain in the soybean LOX-3 is β-folds, which is substituted by α-helices in EnLOX. Increasing α-helix ratio could improve the stability of LOXs [20]. The α-helix ratio of EnLOX is higher than that of most LOXs, such as human arachidonate 12-LOX (α-helix ratio: 38.3%, PDB ID: 3d3l), porcine leukocyte 12S-LOX (39.8%, 3rde), and soybean LOX-1 (31.8%, 4wfo), which may contribute to thermostability of the enzyme. lowest (21.76%) and the highest (37.47%) identity with Gaeumannomyces graminis LOX (PDB ID: 5fx8) and P. aeruginosa LOX, which indicated that it was a novel LOX. Soy flour is used in flour and dough products due to its abundant of soybean LOXs [44]. Soybean LOX-3 (PDB ID: 1rrl) shared the highest structural homology with EnLOX among all of the soybean LOXs. The comparison of the 3D structures of soybean LOX-3 and EnLOX is shown in Figure 3b. EnLOX has only one domain instead of two, as in soybean LOX-3 The N-terminal domain in the soybean LOX-3 is β-folds, which is substituted by α-helices in EnLOX. Increasing α-helix ratio could improve the stability of LOXs [20]. The α-helix ratio of EnLOX is higher than that of most LOXs, such as human arachidonate 12-LOX (α helix ratio: 38.3%, PDB ID: 3d3l), porcine leukocyte 12S-LOX (39.8%, 3rde), and soybean LOX-1 (31.8%, 4wfo), which may contribute to thermostability of the enzyme.

Expression and Purification of EnLOX
The purified EnLOX showed a single band with a molecular mass of approximately 80 kDa in SDS-PAGE (Figure 4), which was consistent with the predicted value 81. 45

Expression and Purification of EnLOX
The purified EnLOX showed a single band with a molecular mass of approximately 80 kDa in SDS-PAGE (Figure 4), which was consistent with the predicted value 81.45 kDa. The biochemical properties of EnLOX and LOXs from various sources are shown in Supplemental Data Table S1. The molecular mass of the enzyme was similar to those of B. thailandensis (75 kDa) [15] and M. xanthus (80 kDa) [1]. The specific activity of purified EnLOX was 40.34 U mg −1 , which was higher than that of most other sources of LOXs,  [15], and P. aeruginosa (28.5 U mg −1 ) [17] but lower than that of Calothrix (73.1 U mg −1 ) [26], Rivularia (68.8 U mg −1 ) [26], and A. aegerita LOX (51.34 U mg −1 ) [35]. In addition, the total yield of the purification for EnLOX was 55.3%, with a purification fold of 31.29. 11,2864 aeruginosa (28.5 U mg −1 ) [17] but lower than that of Calothrix (73.1 U m (68.8 U mg −1 ) [26], and A. aegerita LOX (51.34 U mg −1 ) [35]. In addition the purification for EnLOX was 55.3%, with a purification fold of 31.29

Effects of Temperature and pH on the Activity and Stability of EnLOX
The activity of EnLOX was measured at different temperatures, as shown in Figure 5a. The optimum temperature of most LOXs varied from 30-40 • C [1,16,35,48,49], however, En-LOX showed the highest catalytic activity at 50 • C. At present, one of the disadvantages for the industrial application of LOX to overcome is the poor thermostability [19]. Noteworthily, EnLOX exhibited high thermostability; more than 50% of the original activity remained after being stored at 20 • C-50 • C for 6 h (Figure 5c). The half-life (T 1/2 ) of EnLOX at 50 • C was about 6 h, which was much longer than that of LOXs from soybean (T 1/2 = 30 min at 45 • C) [50], M. xanthus (7.1 min, 50 • C) [1], P. aeruginosa (10 min, 50 • C) [19], and Anabaena (3.8 min, 50 • C) [21]. The thermal shift assay of EnLOX was completed to further verify the thermostability of the enzyme ( Figure S3). The melting point temperature (T m ) value of En-LOX was 78.7 • C, much higher than that of human 5-LOX (36.5 • C) [36] and human 15-LOX (50.0 • C) [51], which revealed the superior thermostability of EnLOX [52]. These results suggested EnLOX is thermotolerant and has potential value in industrial applications.  LA is linoleic acid, ALA is α-linolenic acid, GLA is γ-linolenic acid, 13-HODE is 13hydroxyoctadecaenoic acid, and 13S-HOTrE is 13(S)-hydroxyoctadeca trienoic acid.

Effects of Temperature and pH on the Activity and Stability of EnLOX
The activity of EnLOX was measured at different temperatures, as shown in Figure  5a. The optimum temperature of most LOXs varied from 30-40 °C [1,16,35,48,49], however, EnLOX showed the highest catalytic activity at 50 °C . At present, one of the disadvantages for the industrial application of LOX to overcome is the poor thermostability [19]. Noteworthily, EnLOX exhibited high thermostability; more than 50% of the original activity remained after being stored at 20 °C -50 °C for 6 h (Figure 5c). The half-life (T1/2) of EnLOX at 50 °C was about 6 h, which was much longer than that of LOXs from soybean (T1/2 = 30 min at 45 °C ) [50], M. xanthus (7.1 min, 50 °C ) [1], P. aeruginosa (10 min, 50 °C ) [19], and Anabaena (3.8 min, 50 °C ) [21]. The thermal shift assay of EnLOX was completed to further verify the thermostability of the enzyme ( Figure S3). The melting point temperature (Tm) value of EnLOX was 78.7 °C , much higher than that of human 5-LOX (36.5 °C ) [36] and human 15-LOX (50.0 °C ) [51], which revealed the superior thermostability of EnLOX [52]. These results suggested EnLOX is thermotolerant and has potential value in industrial applications. The activity of EnLOX was assayed at different pHs, as shown in Figure 4b. EnLOX showed activity in a wide range of pH 3.5-9.5 and had a maximum activity at pH 8.0. This result was consistent with the most reported optimum pH of LOXs, which are weakly alkaline [16,35,48,49]. EnLOX displayed excellent stability in assayed pH range (4.0-9.5); it could retain more than 50% relative activity after being incubated at 4 °C for 10 h, and it could retain above 70% relative activity at a broad pH range (5.0-9.5). Surprisingly, EnLOX remained above 90% relative activity at pH 7.5-8.0 after being stored at 4 °C for 10 h (Figure 5d). Excellent stability over a broad pH range may be due to the reversible denaturation of the enzyme, so that there is weak effect on the EnLOX activity on incubation at different pH [53]. Nevertheless, M. xanthus LOX is only stable in the range of pH 3.0-6.0 [1]. LOXs from soybean [46], B. thailandensis [15], and Cyanothece [54] are only stable under alkaline conditions. Therefore, EnLOX could be well-suited for industrial production, as it was stable in weak acidic and alkaline environment.

Effect of Metal Ions and Solvents on EnLOX Activity
As shown in Table 3, the activity of EnLOX varies with the addition of different metal ions and solvents. Ca 2+ , Mn 2+ , Fe 3+ , and DMSO enhanced EnLOX activity by 2.37, 2.12, 1.89, and 2.13 times, respectively. The promotion of enzyme activity by metal ions possibly related to the N-terminal PLAT domain, cofactors, and coenzyme of EnLOX [55,56]. These results were similar to M. xanthus LOX [1] and Cyanobacterium Cyanothece LOX [55]. Zn 2+ , Li + , and K + had no effects on the enzyme activity. The activating effect on EnLOX of DMSO may be due to DMSO changing some secondary bonds in the enzyme and influencing the flexibility of the activity site [38]. The enzyme activity was completely inhibited by EDTA, SDS, and β-ME. EDTA could chelate iron ion in LOX, thus EnLOX was proved to be a metal enzyme [57]. Some solvents could affect iron ion or change the conformation of LOX, thus, showing the inhibitory effect of the enzyme activity [58]. The activation function of Ca 2+ , Mn 2+ , Fe 3+ , and DMSO provide new methods for the industrial application of EnLOX to optimize the fermentation conditions. The activity of EnLOX was assayed at different pHs, as shown in Figure 4b. EnLOX showed activity in a wide range of pH 3.5-9.5 and had a maximum activity at pH 8.0. This result was consistent with the most reported optimum pH of LOXs, which are weakly alkaline [16,35,48,49]. EnLOX displayed excellent stability in assayed pH range (4.0-9.5); it could retain more than 50% relative activity after being incubated at 4 • C for 10 h, and it could retain above 70% relative activity at a broad pH range (5.0-9.5). Surprisingly, EnLOX remained above 90% relative activity at pH 7.5-8.0 after being stored at 4 • C for 10 h (Figure 5d). Excellent stability over a broad pH range may be due to the reversible denaturation of the enzyme, so that there is weak effect on the EnLOX activity on incubation at different pH [53]. Nevertheless, M. xanthus LOX is only stable in the range of pH 3.0-6.0 [1]. LOXs from soybean [46], B. thailandensis [15], and Cyanothece [54] are only stable under alkaline conditions. Therefore, EnLOX could be well-suited for industrial production, as it was stable in weak acidic and alkaline environment.

Effect of Metal Ions and Solvents on EnLOX Activity
As shown in Table 3, the activity of EnLOX varies with the addition of different metal ions and solvents. Ca 2+ , Mn 2+ , Fe 3+ , and DMSO enhanced EnLOX activity by 2.37, 2.12, 1.89, and 2.13 times, respectively. The promotion of enzyme activity by metal ions possibly related to the N-terminal PLAT domain, cofactors, and coenzyme of EnLOX [55,56]. These results were similar to M. xanthus LOX [1] and Cyanobacterium Cyanothece LOX [55]. Zn 2+ , Li + , and K + had no effects on the enzyme activity. The activating effect on EnLOX of DMSO may be due to DMSO changing some secondary bonds in the enzyme and influencing the flexibility of the activity site [38]. The enzyme activity was completely inhibited by EDTA, SDS, and β-ME. EDTA could chelate iron ion in LOX, thus EnLOX was proved to be a metal enzyme [57]. Some solvents could affect iron ion or change the conformation of LOX, thus, showing the inhibitory effect of the enzyme activity [58]. The activation function of Ca 2+ , Mn 2+ , Fe 3+ , and DMSO provide new methods for the industrial application of EnLOX to optimize the fermentation conditions. Table 3. Effect of metal ions and solvents on EnLOX activity. EnLOX activity was plotted as the relative activity (%), with the highest activity as 100%.

Metal Ion and Solvent
Relative Activity (%)

Effect of EnLOX on Farinographical Properties
The effects of EnLOX on the farinographical properties of wheat flour are shown in Table 4. Compared with the blank group, water absorption, dough development time, and stability time of flours fortified with 5-30 IU g −1 EnLOX increased, softening degree decreased. Water absorption, dough development time, and stability time of flours fortified with 20 IU g −1 EnLOX increased by 3.89%, 0.50 min, and 1.14 min, respectively; softening degree was reduced by 39 FU. Fatty acid hydroperoxides generated by EnLOX in the catalytic process can induce protein polymerization [5], and, at the same time, oxidize the sulfhydryl group (-SH) of gluten to the disulfide bond (-S-S-) to the enhanced gluten network [59], which promotes the stability of the wheat dough, manifested as dough development time and stability time prolongation, and decreases the softening degree value [11]. The indicators of flour, which added KBrO 3 , had no significant changes apart from the softening degree decreasing significantly. EnLOX exhibited favorable farinographical properties of wheat flour and could be added in flour products to improve its quality.

Effect of EnLOX on Rheological Properties
Rheological characteristics of dough treated with different concentrations of EnLOX were evaluated ( Figure 6). Dough samples added with EnLOX showed a significant increase in elastic value (G ) (Figure 6a) and viscous (G ) value ( Figure 6b); both of the two values elevated with the increase in EnLOX concentration, revealing the good elasticity and extensibility of dough [4]. The degree of polymerization in dough can be described by tan δ (G /G ) [60]. With the increase in the frequency, the tan δ value was always lower than 1.0 (Figure 6c), which suggested that elastic properties were more prominent, and the dough exhibits solid phase properties. Dough treated with 10-50 IU g −1 EnLOX exhibited higher elastic and viscous characteristics than that of the blank group, indicating that EnLOX could enhance the cross-linking of gluten [60]. EnLOX has been proven to have the reinforcement ability in wheat dough, similar to previously reported Anabaena LOX [4] and soybean LOX [46], indicating that the enzyme could be a potential enzymatic preparation [7].
Data are shown as means ± SD of three independent experiments. Means with different lowercase letters indicate significant differences between groups (p < 0.05). WA is water absorption, DDT is dough development time, ST is stability time, and SD is softening degree.

Effect of EnLOX on Rheological Properties
Rheological characteristics of dough treated with different concentrations of EnLOX were evaluated ( Figure 6). Dough samples added with EnLOX showed a significant increase in elastic value (G′) (Figure 6a) and viscous (G′′) value ( Figure 6b); both of the two values elevated with the increase in EnLOX concentration, revealing the good elasticity and extensibility of dough [4]. The degree of polymerization in dough can be described by tan δ (G"/G') [60]. With the increase in the frequency, the tan δ value was always lower than 1.0 (Figure 6c), which suggested that elastic properties were more prominent, and the dough exhibits solid phase properties. Dough treated with 10-50 IU g −1 EnLOX exhibited higher elastic and viscous characteristics than that of the blank group, indicating that EnLOX could enhance the cross-linking of gluten [60]. EnLOX has been proven to have the reinforcement ability in wheat dough, similar to previously reported Anabaena LOX [4] and soybean LOX [46], indicating that the enzyme could be a potential enzymatic preparation [7].

Conclusions
To overcome the low enzyme activity, poor thermal stability, and narrow pH range stability of existing LOX enzymes, a novel LOX from Enterobacter norvegicus was expressed and characterized in this study. EnLOX only showed 37.47% identity with P. aeruginosa LOX, and the enzyme exhibited superior properties; good thermostability at 50 • C; outstanding pH stability at pH 5.0-9.5; and high activity of LA, achieving 40.34 U mg −1 . In addition, the application of EnLOX to wheat dough further improved the farinograph and rheological properties. Our research indicated EnLOX is a potential enzyme agent for food industrial applications.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/foods11182864/s1, Figure S1: The nucleic acid sequences alignment of original and codonoptimized EnLOX. Figure S2: Multiple sequences alignment of LOXs. Figure S3: Melt curves of EnLOX in Thermal shift assay. Table S1: Biochemical properties of EnLOX and LOXs from various sources.