Considering the inconvenience of Ochratoxin A (OTA) detection and degradation in the food industry, we have designed an OTA recognition-triggered structure switching-based molecular beacon assay for ultrasensitive and highly specific detection of OTA.

We also introduced sustainable semi-interpenetrating polymer networks to degrade OTA efficiently. We offered novel OTA detection ideas and degradation strategies for food safety detection, which is of great significance for wine quality.

We designed a molecular beacon assay strategy for OTA detection that a structure-switching locked hairpin (LHP) triggered rolling circle amplification (RCA) with dumbbell template. To amplify the signal, we designed a biotin-labelled LHP contained the aptamer and RCA primer as the recognition domain. Through the streptavidin-biotin interaction, the LHP was readily attached to the streptavidin-coated magnetic beads. At the same time, the dumbbell structure DNA with shorter stem was designed as RCA template. To generate the final signal, we used molecular beacons to hybridize with the RCA products.

In the absence of the target, the molecular beacons formed a hairpin structure, which rendered the fluorophore extremely closed to the quencher, and extinguished the fluorescence . When introduced, the RCA products situates itself between the two short self-complementary segments. This disrupts the hairpin structure, leading to the separation of the fluorophore and the quencher. As the distance between them increases, fluorescence is restored, which can be easily detected by instruments.

We choosed carboxypeptidase A (CPA) derived from bovine pancreas to degrade OTA. CPA is an enzyme with rich crystal structures and a clear catalytic mechanism. In order to obtain a CPA with enhanced thermal stability and improved tolerance to pH variations, we truncated 110 residues from the propeptide and signal peptide to construct mature CPA (M-CPA). However, expressing CPA in prokaryotic systems proved challenging, so we also chose amidohydrolase ADH3 from Stenotrophomonas acidaminiphila, which was reported to exhibit 57 to 35,000-fold higher activity than previously reported enzymes. Both enzymes can hydrolyze the amide bond of OTA and produce the nontoxic ochratoxin α (OTα) and L-α-phenylalanine (L-α-Phe).

In the degradation device, we added an autolysis circuit, which can cause oscillations in bacterial population density over time. The lysis circuit contains two modules. The first module consists of a lysis gene E from phage φX174, placed behind the P_tra promoter. The second module consists of a TraR gene and a TraI gene under the control of an isopropyl β-D-1-thiogalactopyranoside (IPTG) inducible promoter (P_lac). Thus, high cell densities lead to triggers quorum sensing and greater E protein expression, which then results in lysis of the bacteria. At the same time, few engineered bacteria that survive will continue to grow until the next lysis, and the cycle repeats.

SpyTag and SpyCatcher are a pair of reactive protein partners that can spontaneously react to reconstitute the intact folded CnaB2 domain under mild conditions. Hydrophilic elastin-like polypeptides (ELPs) composed of tandem pentapeptides of the form (VPGXG)(n) (where X may be any amino acid except proline) always serve as versatile model systems for biomaterials.

We used ELPs as the backbone of the monomers. Each monomer was fused with 3 SpyTags or 3 SpyCatchers. The polymerization between these two types of monomers can proceed efficiently under multiple conditions. We linked degrading enzymes (M-CPA/ADH3) into the SpyTag monomer to immobilize the enzyme and increase the stability of degrading enzymes.

Chitosan and sodium alginate were selected as encapsulation materials to provide a concentrated monomer environment and facilitate covalent bond formation between the monomers. The positive-charged amine groups of chitosan are cross-linked by non-toxic polyanion negative-charged tripolyphosphate, and its amine groups can be protonated to protect the cells at acidic environment. Additionally, chitosan can be used together with other polysaccharide polymers such as alginate to prepare microcapsules. We used a dual-layer encapsulation of chitosan and sodium alginate to prevent the escape of engineered bacteria.

Semi-interpenetrating polymer networks (sIPN) is a polymeric capsule encapsulating engineered bacteria. Under various conditions, the formation of sIPNs leads to strengthening of the mechanical property of the proteins and the versatile functionalization of the scaffold polymer by incorporating other proteins of interest. In addition, we made engineered bacteria under the control of the autonomous lysis circuit, so that engineered bacteria can undergo autonomous lysis at a high density. After lysis, the bacteria released two types of monomers after the fusion of the enzyme. When the concentration of the bacteria dropped to a certain extent, the bacteria resumed growth, thereby continuously releasing the monomers, realizing the sustainable production and assembly of the enzyme.

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