Ochratoxin A (OTA) is a fungal toxin that widely contaminates agricultural products. It has strong nephrotoxicity, hepatotoxicity, neurotoxicity, and carcinogenicity, posing a serious threat to human health and agricultural production. Traditional physical and chemical detoxification methods have limitations such as low efficiency, high cost, and the possibility of secondary pollution, while enzymatic detoxification has become the most promising OTA detoxification strategy due to its high specificity, environmental friendliness, and safety. ADH3 originates from Stenotrophomonas acidimaniphila and is the most efficient OTA hydrolase reported to date. It can catalyze the hydrolysis of OTA into non-toxic ochratoxin a (OT α) and L - β - phenylalanine (Phe).
This study is based on the efficient catalytic properties of ADH3 enzyme, and a novel OTA hydrolase LlAHD was obtained through bioinformatics mining. Molecular modifications were made to ADH3 and LlAHD using rational design methods, resulting in mutant ADH3-S88E, LlAH-I326A with significantly improved catalytic efficiency, as well as inactivated mutant ADH3-D344N for mechanism research. These works not only provide efficient enzyme elements for the biological detoxification of OTA, but also provide important insights into the catalytic mechanism of amide hydrolases.
Based on the sequence and structural features of ADH3, we conducted homologous sequence mining in the NCBI database. By using the BLASTP tool, the ADH3 amino acid sequence was used as the query sequence, and an E-value threshold of<1e-50 was set to screen for the amide hydrolase LlADEH derived from Lysobacter luteus. Sequence alignment showed a high similarity of 73% between LlADEH and ADH3, and the key metal coordinating amino acids (H84, H86, H252, H272, K211, and D345) were completely conserved.
Design specific primers to amplify the lladh gene (removing the predicted N-terminal 19 amino acid signal peptide), and clone it into the pET46/Ek LIC vector using EcoK I and Ligation Independent Cloning (LIC) methods to construct the recombinant plasmid pET46/LlAH. Transform the recombinant plasmid into E. coli BL21 (DE3) competent cells and induce expression using 0.4 mM IPTG at 16 ° C for 16-18 hours.
After ultrasonic fragmentation of the bacterial cells, the supernatant was purified by Ni ² ⁺ affinity chromatography column and eluted with gradient buffer containing 0-500 mM imidazole. Collect purified protein and concentrate it to above 10 mg/mL using an ultrafiltration tube. The purity is verified to be>95% by SDS-PAGE. By gel filtration chromatography (Superose ™ 6 increase 10/300 GL column) and dynamic light scattering analysis confirmed that LlAHD is a homooctamer with a molecular weight of approximately .351 kDa.
HPLC was used to determine the activity of LlAHH enzyme. The reaction system contained 20 mM Tris HCl (pH 8.0), 50 μ g/mL OTA, and 5 μ g enzyme. The reaction was carried out at 40 ° C for 20 minutes. The results showed that the optimal reaction conditions for LlAHD were 45 ° C and pH 8.5, with the best thermal stability at 20 ° C. Compared with ADH3, the hydrolytic activity of LlADEH towards OTA has been doubled.
Through sequence alignment and structural analysis, it was found that D344 in ADH3 is a highly conserved residue responsible for coordinating substrate dimerization in binuclear metal amide hydrolases. We speculate that this residue is crucial for the catalytic mechanism,and by mutating it, we can obtain inactive mutants for binding studies.
Using the Overlap PCR method and pET46/ADH3 as a template, D344N-F/R was amplified using primers. PCR program: 95 ° C for 3 minutes; 30 cycles of (95°C 15 s, 60°C 15 s, 72°C 1 min)； 72 ° C for 5 minutes. The PCR product was digested with DpnI and transformed into E. coli DH5 α. Positive clones were screened and sequenced for validation.
Mutant plasmids were transformed into E. coli BL21 (DE3) for expression and purification, resulting in the acquisition of ADH3-D344N protein. Enzyme activity assay showed that the mutant completely lost its OTA hydrolysis ability, confirming that D344 plays a critical role in the catalytic process. This mutant was successfully used for the structural analysis of OTA complexes, revealing the exact binding mode between OTA and enzymes.
Based on the structure of ADH3-D344N/OTA complex (resolution 2.7 Å), we found that the S88 residue side chain points towards the OT α part of OTA, but its interaction is weak. Through computer-aided design, we mutated S88 into negatively charged glutamic acid (S88E) with the aim of forming additional hydrogen bonding interactions with the hydroxyl group of the OT α moiety to stabilize the substrate binding conformation.
The S88E mutant was constructed using site directed mutagenesis, and a series of mutants including S88D, S88R, S88H, and S88K were also constructed as controls. After sequencing validation, all mutants were expressed and purified to obtain high-purity protein for enzyme activity determination.
The enzyme activity assay results showed that the OTA hydrolysis activity of the S88E mutant was 3.7 times higher than that of the wild-type ADH3, 2.4 times higher in S88K, and 1.3 times higher in S88H. This indicates that introducing charged residues to enhance hydrogen bonding interactions with substrates can effectively improve enzyme catalytic efficiency. Molecular dynamics simulations further confirmed the formation of a stable hydrogen bonding network between S88E and OTA.
By stacking the LlADEH structure onto the ADH3-D344N/OTA complex, it was found that the I326 residue side chain points towards the Phe portion of OTA, but substrate binding may be limited due to steric hindrance effects. We speculate that mutating this site to alanine (I326A) with a smaller side chain may provide a broader space for OTA binding.
Construct I326A mutant using the Overlap PCR method, and amplify the lladh gene using specific primers for I326A-F/R. After sequencing verification, the mutant plasmid was transformed into an expression host and purified for protein expression.
The enzyme activity assay results showed that the I326A mutant showed a 75% increase in catalytic activity towards OTA compared to the wild-type LlAH. This confirms our hypothesis that reducing the side chain volume can alleviate steric hindrance, optimize the spatial configuration of substrate binding pockets, and thus improve catalytic efficiency. The successful acquisition of this mutant demonstrates the effectiveness of rational design in enzyme modification.
To achieve a leap from one enzyme one effect to one enzyme multi effect, we continued to mine in the database and found an enzyme that can simultaneously degrade three toxins - Fpro enzyme from Bacillus megaterium HNGD-A6.
According to literature and team enzyme activity testing, Fpro enzyme has been proven to efficiently degrade AFB1, OTA, and ZEN, three fungal toxins, with relatively low degradation rates. We found in subsequent experiments that the enzyme also has the ability to degrade Patulin, achieving a preliminary functional leap of "one enzyme, four effects".
Through electron microscopy analysis, we obtained the empty structure of Fpro, as shown in the schematic diagram below.
The team has conducted rational design mutations for Fpro, but the mutant enzyme activity test results have the following issues:
This project aims to address the global challenge of co contamination of multiple mycotoxins in agricultural products and is committed to developing efficient and safe enzymatic detoxification solutions. The research team has successfully achieved a strategic leap from "one enzyme, one effect" to "one enzyme, multiple effects", and significantly improved the catalytic performance of core enzyme components through systematic rational design and modification, laying a solid foundation for industrial applications.
At the beginning of the project, we used the highly efficient OTA hydrolase ADH3 obtained earlier as a prototype and successfully extracted its homologous enzyme, the amide hydrolase LlADEH derived from Lysobacter luteus, from the NCBI database through bioinformatics analysis. Sequence alignment shows that the similarity between the two is as high as 73%, and the key metal coordinating amino acids are completely conserved. Through heterologous expression and purification, it was confirmed that LlAHD is a homologous octamer with excellent enzymatic properties. Under optimal conditions (45 ° C, pH 8.5), its hydrolytic activity against OTA was doubled compared to ADH3, making it a more promising candidate enzyme.
To gain a deeper understanding of the catalytic mechanism, we successfully resolved the complex structures of ADH3 and its substrates Phe and OTA (with a resolution of 2.5-2.7 Å). Based on structural analysis, we created an inactive mutant ADH3-D344N. Although this mutant lost enzyme activity, it became a key tool for analyzing the precise binding mode of OTA, revealing that the substrate binding pocket is composed of key amino acids such as S88, H163, L218, H253, and a dual core metal center.
We have successfully obtained efficient OTA hydrolase LlADEH and its mutant I326A, as well as S88E and D344N mutants of ADH3, through a strategy combining bioinformatics mining and rational design.
These achievements fully demonstrate the strong potential and effectiveness of structure guided rational design in enzyme engineering.
To achieve the goal of simultaneously degrading multiple toxins, we further explored Fpro enzymes derived from Bacillus megaterium. Preliminary verification shows that the enzyme can simultaneously degrade AFB1, OTA, ZEN, and patulin, demonstrating excellent "one enzyme four effect" ability, but the degradation efficiency needs to be improved. We have obtained preliminary electron microscopy images of its empty structure, providing a starting point for subsequent mechanism research and modification. Although there have been technical bottlenecks such as decreased activity in the current mutant targeting Fpro, this has clarified the next research focus.
The enzyme element and rational design strategy developed in this study provide important technical means and theoretical basis for solving the problem of OTA and even more toxin contamination in agricultural products, which is of great significance for ensuring food safety and human health. Looking ahead to the future, research will focus on the following core directions to promote the commercialization of achievements: (1) overcoming the enzymatic hydrolysis efficiency and application bottlenecks of Fpro enzyme; (2) Constructing an efficient yeast expression system; (3) Systematic evaluation of industrial adaptability and safety.
In the future, it is necessary to accelerate the scientific research pace in the field of fungal toxin pollution prevention and control, strengthen interdisciplinary cooperation, and promote the development and implementation of more efficient and safe prevention and control technologies. At the same time, it is also necessary to raise public awareness of the hazards of fungal toxin pollution through science popularization and technical training, enhance the risk prevention awareness of the entire industry chain, jointly build a quality and safety guarantee system for agricultural products, and effectively safeguard the "safety on the tongue" of the people.