Detoxification of Fumonisins by Three Novel Transaminases with Diverse Enzymatic Characteristics Coupled with Carboxylesterase

Fumonisin (FB) is one of the most common mycotoxins contaminating feed and food, causing severe public health threat to human and animals worldwide. Until now, only several transaminases were found to reduce FB toxicity, thus, more fumonisin detoxification transaminases with excellent catalytic properties required urgent exploration for complex application conditions. Herein, through gene mining and enzymatic characterization, three novel fumonisin detoxification transaminases—FumTSTA, FumUPTA, FumPHTA—were identified, sharing only 61–74% sequence identity with reported fumonisin detoxification transaminases. Moreover, the recombinant proteins shared diverse pH reaction ranges, good pH stability and thermostability, and the recombinant protein yields were also improved by condition optimum. Furthermore, the final products were analyzed by liquid chromatography-mass spectrometry. This study provides ideal candidates for fumonisin detoxification and meets diverse required demands in food and feed industries.


Introduction
As one of the most prevalent foodborne fungal toxins, fumonisins (FBs) are mostly generated by fungi (Fusarium verticillioides and Fusarium oxysporum) and widely distributed in animal feed and human food [1][2][3]. FBs have an extremely stable chemical structure consisting of two methyl groups (-CH 3 ), one amino group (-NH 2 ), one to four hydroxyl groups (-OH), and two tricarboxylate groups (TCA) in different positions as well as linear polyketide derived backbones [4]. Since they have a similar molecular structure to sphingosine and sphinganine, fumonisins exert significant toxicity by interfering sphingolipid metabolism and inducing oxidative stress [4][5][6].
To date, more than 15 fumonisin congeners have been found, and fumonisins B1 (FB1) is the most toxic and abundant fumonisin [7,8]. Based on many toxicological studies, FB1 has been demonstrated to induce a wide range of diseases in animals, such as cerebral leukodystrophy in horses, pulmonary edema syndrome in pigs, nephrotoxicity in rats, and apoptosis in many other cell types [9][10][11]. Moreover, epidemiological investigations have indicated that fumonisins are related to esophageal cancer, primary liver cancer, neural tube defects, and cardiovascular disease in highly exposed human populations [12,13]. Due to its significant risk to human health and animal welfare, many international organizations have already released the temporary maximum amount of fumonisins in food, feed, and feed raw materials [8,14]. Both social and scientific concerns have also focused on controlling and resolving fumonisins contamination [15].
Fumonisins are highly soluble in water and very thermostable, thus it is challenging to destroy and remove them from food and feed [7,15]. The current detoxification strategies

Strains, Plasmids, Chemicals, and Enzyme
The plasmid pET-28a from Invitrogen (Carlsbad, CA, USA) was adopted as the expression vector in Escherichia coli DH5α and BL21 (DE3) were utilized to clone and expression of hosts, separately. The recombinant strains of E. coli were cultivated in LB medium (0.5% w/v yeast extract, 1% w/v tryptone, and 1% w/v NaCl). Phusion DNA polymerase, restriction endonuclease, and T4 DNA ligase were purchased from ThermoFisher Scientific (Shanghai, China). The plasmid extraction kit, PCR product purification kit, and isopropyl- β-D-thioga-lactopyranoside (IPTG) were purchased from Solarbio (Beijing, China). Nickel columns and nickel resin used for affinity chromatography were purchased from GE Healthcare (Uppsala, Sweden). Substrate FB1 (purity of 98%) was obtained from Pribolab Ltd. (Qingdao, China, http://wwwpribolab.com/, accessed on 4 March 2022) and dissolved in acetonitrile-water. All additional reagents and chemicals are of analytical grade.

Protein Expression and Purification of Transaminases
The gene sequences of transaminase FumUPTA, FumTSTA, and FumPHTA selected from the screening pool were codon optimized, gene synthesized, and cloned into the expression vector pET-28a(+) (GENEWIZ, Suzhou, China). The recombinant plasmids transformed into E. coli BL21(DE3) competent cells. Thereafter, the recombinant E. coli BL21(DE3) strains were incubated in LB medium containing 100 µg/mL ampicillin at 37 • C. Then, the target proteins were induced by IPTG at a final concentration of 1 mM for 20 h at 25 • C. Moreover, carboxylesterase FumDSB used for degrading the TCA groups of FB1 was induced at its optimal induced condition (30 • C, 0.6 mM IPTG, 20 h) [29].
To obtain the recombinant proteins of carboxylesterase FumDSB, transaminase FumUPTA, FumTSTA, and FumPHTA, the recombinant proteins were purified by nickel affinity chromatography (GE Health-care, Uppsala, Sweden). The molecular mass of the recombinant proteins was assayed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The concentration of purified protein was determined by the Bradford method, using a protein assay kits (Bio-Rad, Hercules, CA, USA).
For further identification of the specific proteins, the purified proteins were detected by western blotting analysis based on their terminal His-tags [9]. After separation by polyacrylamide gel electrophoresis, the proteins were transferred to NC membranes (Beyotime Biotech). The membranes were sealed at room temperature for 1 h and hatched with primary antibody: 6-HIS (Anorun, Beijing, China) overnight at 4 • C. Subsequently, the goat anti-mouse IgG was labeled as secondary antibody using HRP (1:2000, Zhennuo Biotech, Shanghai, China). The staining protein was shown by scanning the membrane with Odyssey Infrared Laser Imaging System (LI-COR, Lincoln, NE, USA).
In addition, to optimize the expression level of recombinant protein, the protein expression yield of transaminases with compared at different incubation times IPTG concentrations, and induction temperatures.

Fumonisin Degradation by Transaminases
In this study, HFB1 was firstly obtained from the reaction mixture of FB1 and esterase FumDSB, 100 µL of FB1 solution (50 µg/mL) and 900 µL of purified enzyme FumDSB (1.164 mg/mL) were reacted at 37 • C for 2 h. The producing HFB1 were further identified by LC-MS, then they were used as the substrate for transaminases directly without any extraction and purification steps. For transaminase, each assay solution (100 µL) contained 95 µL of purified transaminase (1.38 mg/mL) in PBS buffer (8 g/L NaCl, 3.58 g/L Na 2 HPO 4 ·12H 2 O, 0.2 g/L KCl, 0.27 g/L KH 2 PO 4 ), 5 µL of HFB1 solution, and pyruvate solution, mixed to a final concentration of HFB1 of 1 µg/mL. The reactions were incubated at 37 • C for 12 h and then inactivated at 100 • C for 10 min.
The intermediate degraded products (HFB1) by transaminases were detected by HPLC. The chromatography was performed on an Agilent 1200 LC system (Agilent Technologies, Santa Clara, CA, USA). The samples were derivatized by adding 500 µL of o-phthaldialdehyde (OPA) derivative solution and 400 µL of aqueous acetonitrile, after gentle mixing, then immediately filtered through a 0.22 µm nylon filter and detected by LC-LFD within 5 min. The excitation wavelength (Ex) of the fluorescence detector was set at 335 nm, and the emission wavelength was set at 440 nm; the mobile phase (methanol: 0.1 mol/L NaH 2 PO 4 = 77:23, v/v, adjust pH to 3.8 with phosphoric acid) was maintained at a flow rate of 1 mL/min. The temperature of the column was adjusted to 25 • C and the volume of injection was adjusted to 20 µL. The degradation rate of HFB1 = (original content of HFB1 − remaining content of HFB1)/original content of HFB1 × 100%. All experiments were duplicated thrice.

Analysis of Final Degradation Products by LC-MS
To identify the detoxification final product of FB1 by carboxylesterase and transaminases, the analysis of degradation products was performed on an LC-QTOF-MS system (Agilent Technologies, Santa Clara, CA, USA). The chromatographic column was ZORBAX Extend-C18 reversed-phase column (150 × 2.1 mm, Agilent particle size of 1.8 µM). The sample volume was 2 µL, and the mobile phase was divided into phase A and phase B (phase A: 0.1% formic acid in water, phase B: acetonitrile of chromatographic grade). The mobile phase gradient conditions were as follows: 95% A for 35 min, maintain 5% A for 10 min in the middle and then 95% A for 15 min. The flow rate was controlled at 0.3 mL/min, 40 • C. Typical parameters of the ESI source were as follows: ESI positive mode, spray voltage 3200 v, nitrogen as auxiliary gas pressure 34.48 kPa, nitrogen sheath pressure 206.89 kPa, capillary temperature, and ion source temperature 350 • C. Data Analysis software was applied for further data acquisition and processing.

Biochemical Characterization of Transaminases
The biochemical characterization of transaminases was identified by HPLC analysis as mentioned in Section 2.4. The degradation rate of HFB1 = (original content of HFB1−remaining content of HFB1)/original content of HFB1 × 100%. To investigate optimum temperature, the enzyme activity was detected at a temperature range of 30-70 • C. In order to determine thermostability, purified enzymes were incubated at 50 • C and 60 • C for different times without substrate, and the enzyme activities were measured under appropriate reaction conditions, respectively. The effects of pH on enzyme activity were determined at different pH values, the used buffers contained 0.1 mol/L citric acid-Na 2 HPO 4 (pH 3.0-8.0), 0.1 mol/L Tris-HCl (pH 8.0-9.0), and 0.1 mol/L glycine-NaOH (pH 9.0-10.0). The pH stability of enzyme was assessed by incubating at various pH levels (3.0-10.0) for 1 h at 37 • C. The untreated enzyme was used as a control.

Gene Mining of FB1 Detoxifying Transaminases
Food and feed contaminated with mycotoxins worldwide pose a severe threat to the health of human and animal, resulting in significant economic losses annually [2]. Biotransformation by enzyme is one of the most attractive and potential alternatives to minimize the detrimental effects of mycotoxins [29,30]. Previously, transaminases were reported to effectively remove group C2-amino and convert FB into environmentally safe non-toxic or low-toxic products without destroying the nutritional content of food [23]. To date, only two transaminases FumIB (GenBank No. ADO15008.1) and FumIS (GenBank No. 6HBS_A) were reported to detoxificate FB1 [26,28]. Thus, more novel and effective fumonisin detoxification transaminases should be explored and characterized.
For the present study, FB1 detoxification transaminase candidates were validated by several pathways, as shown in Figure 1. Amino acid sequence analysis showed that FumIB and FumIS are very diverse, the sequence identity between them is only 65%. In order to obtain more potential FB1 detoxifying transaminases, FumIB and FumIS were used as model by BLASTp analysis, respectively. The results found that FumIB and FumIS both displayed low identity (70.77% and 75.59%) to the submitted sequences from Genbank database, which suggests the novelty of FB1 detoxifying transaminases. Then, the top five sequences of each transaminase with the highest identity were downloaded to construct an alternative gene pool. After removing the redundant sequences with identity >95%, three candidate proteins named FumTSTA, FumPHTA and FumUPTA were obtained (WP_089262923.1, PZQ64330.1, WP_068876442.1), which shared 64.80%, 63.72%, and 74.19% identity with FumIS, and 64.95%, 61.86%, and 66.28% identities with FumIB (Table 1). Moreover, the identity between each other was relatively low 25%, which indicates that three transaminases might be novel FB1 detoxifying transaminases.
health of human and animal, resulting in significant economic losses annually [2]. Biotransformation by enzyme is one of the most attractive and potential alternatives to minimize the detrimental effects of mycotoxins [29,30]. Previously, transaminases were reported to effectively remove group C2-amino and convert FB into environmentally safe non-toxic or low-toxic products without destroying the nutritional content of food [23]. To date, only two transaminases FumIB (GenBank No. ADO15008.1) and FumIS (Gen-Bank No. 6HBS_A) were reported to detoxificate FB1 [26,28]. Thus, more novel and effective fumonisin detoxification transaminases should be explored and characterized.
For the present study, FB1 detoxification transaminase candidates were validated by several pathways, as shown in Figure 1. Amino acid sequence analysis showed that FumIB and FumIS are very diverse, the sequence identity between them is only 65%. In order to obtain more potential FB1 detoxifying transaminases, FumIB and FumIS were used as model by BLASTp analysis, respectively. The results found that FumIB and FumIS both displayed low identity (70.77% and 75.59%) to the submitted sequences from Genbank database, which suggests the novelty of FB1 detoxifying transaminases. Then, the top five sequences of each transaminase with the highest identity were downloaded to construct an alternative gene pool. After removing the redundant sequences with identity >95%, three candidate proteins named FumTSTA, FumPHTA and FumUPTA were obtained (WP_089262923.1, PZQ64330.1, WP_068876442.1), which shared 64.80%, 63.72%, and 74.19% identity with FumIS, and 64.95%, 61.86%, and 66.28% identities with FumIB (Table  1). Moreover, the identity between each other was relatively low 25%, which indicates that three transaminases might be novel FB1 detoxifying transaminases. Previous studies indicated that transaminases contain five families based on sequence similarity and protein structure [31]. Three transaminases FumTSTA, FumPHTA and FumUPTA were used to construct a rootless phylogenetic tree with reference transaminases from five classes (Figure 2), which showed that FumTSTA, FumPHTA, and Fu-mUPTA, and two previously reported fumonisin detoxification transaminases (FumIB and FumIS) all belong to class III transaminases ( Figure 2). Moreover, catalytic sites of Previous studies indicated that transaminases contain five families based on sequence similarity and protein structure [31]. Three transaminases FumTSTA, FumPHTA and FumUPTA were used to construct a rootless phylogenetic tree with reference transaminases from five classes (Figure 2), which showed that FumTSTA, FumPHTA, and FumUPTA, and two previously reported fumonisin detoxification transaminases (FumIB and FumIS) all belong to class III transaminases ( Figure 2). Moreover, catalytic sites of class III transaminases were also found in sequence alignment. The catalytically active residue Lys located in the loop that links the β9 and β10 chains ( Figure 3) [32].   Moreover, the three-dimensional structures of the five transaminases were modeled by Alphafold2 and simultaneously compared by Pymol (Figure 4), and they shared the similar tertiary protein structure ( Figure 4). Moreover, the RMSD value is used to estimate the protein structure similarity and reveal the discrepancies in the structure of the two proteins. The smaller RMSD value is, the more similar the two protein structures are [33]. The RMSD values between three transaminases and two reported FumIB and FumIS are all below 0.37 (Table 1). These results suggested that the internal organization of these enzymes has a high degree of unity, indicating that its binding domain and channel mightly maintain structural consistency, allowing it to be employed as a new enzyme to mine new genes. Since three transaminases FumTSTA, FumPHTA and FumUPTA shared high similarity in protein structure and identical catalytic sites, they were chosen for the following expression and characterization experiment.  Moreover, the three-dimensional structures of the five transaminases were modeled by Alphafold2 and simultaneously compared by Pymol (Figure 4), and they shared the similar tertiary protein structure ( Figure 4). Moreover, the RMSD value is used to estimate the protein structure similarity and reveal the discrepancies in the structure of the two proteins. The smaller RMSD value is, the more similar the two protein structures are [33].
The RMSD values between three transaminases and two reported FumIB and FumIS are all below 0.37 (Table 1). These results suggested that the internal organization of these enzymes has a high degree of unity, indicating that its binding domain and channel mightly maintain structural consistency, allowing it to be employed as a new enzyme to mine new genes. Since three transaminases FumTSTA, FumPHTA and FumUPTA shared high similarity in protein structure and identical catalytic sites, they were chosen for the following expression and characterization experiment.

Protein Expression and Purification
The gene sequences of FumTSTA, FumPHTA, and FumUPTA were artificially synthesized and cloned into the expression vector pET-28a(+)and successfully transformed into E. coli BL21 (DE3). The recombinant proteins were further induced by IPTG addition, and the intracellular supernatant extracted by ultrasonic wall breaking were used to detect HFB degradation activity. After purification by Ni-affinity chromatography, purified FumTSTA, FumPHTA, and FumUPTA migrated as a single band on SDS-PAGE with a molecular weight of approximately 55 kDa ( Figure 5A), which is consistent with the cal-

Protein Expression and Purification
The gene sequences of FumTSTA, FumPHTA, and FumUPTA were artificially synthesized and cloned into the expression vector pET-28a(+)and successfully transformed into E. coli BL21 (DE3). The recombinant proteins were further induced by IPTG addition, and the intracellular supernatant extracted by ultrasonic wall breaking were used to detect HFB degradation activity. After purification by Ni-affinity chromatography, purified FumTSTA, FumPHTA, and FumUPTA migrated as a single band on SDS-PAGE with a molecular weight of approximately 55 kDa ( Figure 5A), which is consistent with the calculated molecular weight of recombinant protein. Western blot was also used to confirm the expression of the target gene in E. coli, which shows the consistent molecular weight of target recombinant protein ( Figure 5B).

Protein Expression and Purification
The gene sequences of FumTSTA, FumPHTA, and FumUPTA were artificially thesized and cloned into the expression vector pET-28a(+)and successfully transfor into E. coli BL21 (DE3). The recombinant proteins were further induced by IPTG add and the intracellular supernatant extracted by ultrasonic wall breaking were used t tect HFB degradation activity. After purification by Ni-affinity chromatography, pur FumTSTA, FumPHTA, and FumUPTA migrated as a single band on SDS-PAGE w molecular weight of approximately 55 kDa ( Figure 5A), which is consistent with the culated molecular weight of recombinant protein. Western blot was also used to con the expression of the target gene in E. coli, which shows the consistent molecular w of target recombinant protein ( Figure 5B).  To evaluate the fermentation level, the effects of induction temperature, IPTG concentration, and incubation time on protein expression were relatively analyzed. As shown in Figure 6, the optimal induction conditions of three transaminases are diverse. The optimal temperature of FumTSTA, FumPHTA and FumUPTA is 20 • C, 25 • C, and 30 • C. The optimal IPTG concentrations of FumTSTA and FumPHTA are both 0.8 mM, and FumUPTA showed the highest yield at 0.5 mM. The optimal induction time is 20 h, and the protein concentration did not increase over time. Under the optimal induction condition, FumT-STA showed the highest yield of 94 mg/L, and the final protein yields of FumPHTA and FumUPTA were 39 mg/L and 34 mg/L, respectively. Previously, FumIB and FumIS were both expressed in E. coli [26,28]. However, FumIS tended to form inclusion bodies in E. coli, which severely hinders the soluble expression and practical application. The protein solubility of FumIS were further enhanced by exchanging host strain and co-expression of cold-adapted chaperonins [34]. The final concentration of recombinant proteins FumIS reached to 1.45 mg/L recombinant protein [28]. Thus, compared with FumIS, FumTSTA, FumPHTA and FumUPTA showed better protein solubility and higher protein yield, which suggests the advantage of FumTSTA, FumPHTA and FumUPTA. E. coli, which severely hinders the soluble expression and practical application. The protein solubility of FumIS were further enhanced by exchanging host strain and co-expression of cold-adapted chaperonins [34]. The final concentration of recombinant proteins FumIS reached to 1.45 mg/L recombinant protein [28]. Thus, compared with FumIS, FumTSTA, FumPHTA and FumUPTA showed better protein solubility and higher protein yield, which suggests the advantage of FumTSTA, FumPHTA and FumUPTA.

LC-MS Analysis
The degradation vitalities of three transaminases against HFB1 were measured by HPLC. As shown in Figure 7, transaminases FumTSTA, FumPHTA, and FumUPTA can completely degrade HFB1, respectively, which suggests that all three transaminases have HFB1 detoxification activity. To further identify the degradation products of HFB1 by three transaminases, substrate HFB1 and degradation products were determined by LC-MS/MS, separately. As shown in Figure 8, the substrate HFB1 molecule of the protonated cation [M+H] + was identified by QTOF-MS at m/z 406.3534 ( Figure 8C), and the fragment

LC-MS Analysis
The degradation vitalities of three transaminases against HFB1 were measured by HPLC. As shown in Figure 7, transaminases FumTSTA, FumPHTA, and FumUPTA can completely degrade HFB1, respectively, which suggests that all three transaminases have HFB1 detoxification activity. To further identify the degradation products of HFB1 by three transaminases, substrate HFB1 and degradation products were determined by LC-MS/MS, separately. As shown in Figure 8, the substrate HFB1 molecule of the protonated cation [M+H] + was identified by QTOF-MS at m/z 406.3534 ( Figure 8C), and the fragment ions such as m/z 316.3046, 334.3154, 352.3266, 370.3374 were also determined by secondary mass spectrometry ( Figure 8D). These findings were consistent with the previously reported characteristics of HFB1 fragments [14].
The detoxification products of FumUPTA were in m/z 405 and m/z 448 (Figures 9 and 10). In secondary mass spectrometry analysis of molecular ion m/z 405, fragment ions m/z 96.0814, 114.0927, 132.1035 were also detected ( Figure 9D). The molecular formula of m/z 405 is inferred to be C 22 H 44 NO 6 by the analysis software. Therefore, this molecular is presumably produced by the oxidation of the amino on HFB1. The secondary mass spectra of molecular ion m/z 448 were analyzed, the typical fragment ions of m/z 334.3155, 352.3268, 370.3374 and 388.3483 of 2-acetamide-HFB1 were also identified ( Figure 10D). The molecular formula is inferred to be C 24 H 49 O 6 by the analysis software. These results demonstrated that FumUPTA collaborate with pyruvate to acetylate or oxidize the amino functional group of HFB1, resulting in 2-acetamide-HFB1 and 2-keto-HFB1. Previous studies have shown that there are two kinds of products for transaminases FumIB [35]. The product of 2-keto-HFB1 is non-toxic. It is particularly non-toxic to GES-1 cells [23]. Different from FumUPTA, only the products m/z 448 were detected in the degraded products of FumTSTA and FumPHTA. It might imply that the catalytic mechanisms of these transaminases are different, although they all belong to Class III transaminases. ions such as m/z 316.3046, 334.3154, 352.3266, 370.3374 were also determined by secondary mass spectrometry ( Figure 8D). These findings were consistent with the previously reported characteristics of HFB1 fragments [14].   ions such as m/z 316.3046, 334.3154, 352.3266, 370.3374 were also determined by secondary mass spectrometry ( Figure 8D). These findings were consistent with the previously reported characteristics of HFB1 fragments [14].  The detoxification products of FumUPTA were in m/z 405 and m/z 448 (Figures 9  and 10). In secondary mass spectrometry analysis of molecular ion m/z 405, fragment ions m/z 96.0814, 114.0927, 132.1035 were also detected ( Figure 9D). The molecular formula of m/z 405 is inferred to be C22H44NO6 by the analysis software. Therefore, this molecular is presumably produced by the oxidation of the amino on HFB1. The secondary mass spectra of molecular ion m/z 448 were analyzed, the typical fragment ions of m/z 334.3155, 352.3268, 370.3374 and 388.3483 of 2-acetamide-HFB1 were also identified ( Figure 10D). The molecular formula is inferred to be C24H49O6 by the analysis software. These results demonstrated that FumUPTA collaborate with pyruvate to acetylate or oxidize the amino functional group of HFB1, resulting in 2-acetamide-HFB1 and 2-keto-HFB1. Previous studies have shown that there are two kinds of products for transaminases FumIB [35]. The product of 2-keto-HFB1 is non-toxic. It is particularly non-toxic to GES-1 cells [23]. Different from FumUPTA, only the products m/z 448 were detected in the degraded products of FumTSTA and FumPHTA. It might imply that the catalytic mechanisms of these transaminases are different, although they all belong to Class III transaminases.

Degradation Activity and Biochemical Characteristics
The biochemical characteristics of three transaminases were comparatively analyzed. The optimum temperature of these transaminases is 35 • C and 40 • C ( Figure 11A-C), which is not only close to the physiological temperature in the GIT of pigs and cattle but in all homeothermic vertebrates and enables them to perform their functions well in feed or animals [29]. Moreover, three transaminases displayed excellent activity at high temperatures: FumTSTA showed 70% relative activity at 50 • C, FumPHTA maintained more than 40% relative activity in the temperature range from 50 • C to 60 • C ( Figure 11A,B), FumUPTA performed more than 40% relative activity in the range of 50-70 • C ( Figure 11C). The broad reaction temperature range largely broadens the application aspects of fumonisin detoxification transaminases.
Thermostable enzymes are also extremely critical for industrial field due to their specific processes and low cost [36]. FumTSTA, FumPHTA, and FumUPTA were able to maintain 73%, 72%, and 91% relative activity after incubated at 60 • C for 5 min ( Figure 12A-C), 27%, 28% and 18% relative activity after 1 h treatments at 60 • C ( Figure 12A-C). Transaminases FumTSTA and FumPHTA showed similar thermostability. FumUPTA displayed better thermostability than previous two transaminases after incubated at 60 • C for 5 min, but worse thermostability after incubated at 60 • C for 1 h. Moreover, based on the thermostability of FumTSTA, FumPHTA, and FumUPTA might not endure the pelleting heating process, but using an adequate coating of these enzymes to avoid their thermal inactivation is also possible and possible way. The stability could be further improved by molecular modification.   played better thermostability than previous two transaminases after incubated at 60 °C for 5 min, but worse thermostability after incubated at 60 °C for 1 h. Moreover, based on the thermostability of FumTSTA, FumPHTA, and FumUPTA might not endure the pelleting heating process, but using an adequate coating of these enzymes to avoid their thermal inactivation is also possible and possible way. The stability could be further improved by molecular modification. As shown in Figure 11, three transaminases displayed different optimal pH range. FumTSTA was most active at pH 9.0 and retained activity in acidic and neutral conditions ( Figure 11D). Compared with FumTSTA, FumPHTA had a lower optimal reaction pH of 8.0 and showed high relative activity (>59.89%) at a neutral pH range of 6.0-8.0 ( Figure  11E). Previous studies showed that FumIS is also alkaline with pH optimum of 8.5, but its  pH active range is relatively narrow, from pH 7.0 to pH 9.0 [28]. Notably, different from three transaminases, FumUPTA displayed its optimal reaction pH of 4.0, and it exhibited more than 50.0% relative activity from pH 3.0 to 5.0 ( Figure 11F). Additionally, FumTSTA exhibited good pH stability in a wide pH range from pH 3.0 to pH 10.0 ( Figure 12D). However, the pH stability of FumPHTA is not as good as that of FumTSTA, which only retains 40% residual activity at pH 3.0 and 4.0 ( Figure 12E). In contrast, FumUPTA is active in acidic condition and also showed excellent pH stability under acidic conditions (pH 3.0-6.0), which retains 60% residual activity from pH 3.0-6.0 for 1 h ( Figure 12F). Fumonisin has widely existed in many application scenarios, including different food and feed materials. The pH condition of actual application scenarios for fumonisin detox enzymes are divergent. For example, the physiological pHs of the animal intestinal environment are acidic typically between 5.0 and 6.5 [29], while the gastric pHs of certain monogastric animals, such as pigs, are around 2.0 [37]. Moreover, the corn steep water for amylase production is acidic around pH 3-4 [38]. Furthermore, fumonisins have been extensively detected in manufactured aquatic feed, while the pH of fish intestinal tract in aquaculture is neutral [39,40]. Thus, fumonisins detoxification enzymes with different pH are required for removing FB in practical applications. One enzyme usually displays a narrow pH optimum. Herein, the divergent pH optimum of three transaminases significantly enlarges the application scenes of fumonisin degradation transaminases and provide more enzyme candidates, which can be used individually or combinatorially.

Conclusions
In this study, three novel transaminases were identified to effectively detoxify hydrolyzed fumonisin B1 (HFB1) by gene mining and enzymatic characterization. They displayed diverse pH reaction ranges, good pH stability and thermostability, and high protein yield, which provides ideal candidates for fumonisin detoxification and meets different required demands in food and feed industries. As shown in Figure 11, three transaminases displayed different optimal pH range. FumTSTA was most active at pH 9.0 and retained activity in acidic and neutral conditions ( Figure 11D). Compared with FumTSTA, FumPHTA had a lower optimal reaction pH of 8.0 and showed high relative activity (>59.89%) at a neutral pH range of 6.0-8.0 ( Figure 11E). Previous studies showed that FumIS is also alkaline with pH optimum of 8.5, but its pH active range is relatively narrow, from pH 7.0 to pH 9.0 [28]. Notably, different from three transaminases, FumUPTA displayed its optimal reaction pH of 4.0, and it exhibited more than 50.0% relative activity from pH 3.0 to 5.0 ( Figure 11F). Additionally, FumTSTA exhibited good pH stability in a wide pH range from pH 3.0 to pH 10.0 ( Figure 12D). However, the pH stability of FumPHTA is not as good as that of FumTSTA, which only retains 40% residual activity at pH 3.0 and 4.0 ( Figure 12E). In contrast, FumUPTA is active in acidic condition and also showed excellent pH stability under acidic conditions (pH 3.0-6.0), which retains 60% residual activity from pH 3.0-6.0 for 1 h ( Figure 12F).
Fumonisin has widely existed in many application scenarios, including different food and feed materials. The pH condition of actual application scenarios for fumonisin detox enzymes are divergent. For example, the physiological pHs of the animal intestinal environment are acidic typically between 5.0 and 6.5 [29], while the gastric pHs of certain monogastric animals, such as pigs, are around 2.0 [37]. Moreover, the corn steep water for amylase production is acidic around pH 3-4 [38]. Furthermore, fumonisins have been extensively detected in manufactured aquatic feed, while the pH of fish intestinal tract in aquaculture is neutral [39,40]. Thus, fumonisins detoxification enzymes with different pH are required for removing FB in practical applications. One enzyme usually displays a narrow pH optimum. Herein, the divergent pH optimum of three transaminases significantly enlarges the application scenes of fumonisin degradation transaminases and provide more enzyme candidates, which can be used individually or combinatorially.

Conclusions
In this study, three novel transaminases were identified to effectively detoxify hydrolyzed fumonisin B1 (HFB1) by gene mining and enzymatic characterization. They displayed diverse pH reaction ranges, good pH stability and thermostability, and high protein yield, which provides ideal candidates for fumonisin detoxification and meets different required demands in food and feed industries.