CarE can degrade gossypol, Hence, an evaluation of its structure is necessary. Degrading gossypol is a process which aims to mitigate the toxicity of FG by converting it into less toxic or non-toxic substances. One way to reduce the toxicity of gossypol and enhance its biological properties is to convert it into azepine derivatives, such as Schiff bases or hydrazones. The Schiff base or hydrazone gossypol can undergo Schiff base formation [58,59], ozonation [15], oxidation [60], and methylation [61] to form gossypol derivatives.CarEs is a serine hydrolase that reacts with many compounds with different structures that contain ester bonds [62,63,64,65,66]. As mentioned above, the toxicity of gossypol can be attributed to six phenolic hydroxyl groups and two aldehyde groups, and CarEs can catalyze the hydrolysis of esters or amide compounds into corresponding alcohols and carboxylic acids [67]. CarEs are widely considered attractive and advantageous biocatalysts due to their characteristics, such as their ability to accept a wide range of substrates and their high stereospecificity, high tolerance to organic solvents, and lack of cofactors required for the reaction [68]. Conserved region prediction analysis was performed with the H. armigera CarE CCE001a protein (https://www.genome.jp/tools/motif//, 18 April 2022). The protein showed a co-esterase family (PF00135) domain and a dehydrogenase family (PF07859) domain (Figure S3). The enzymatic reaction itself is a complex process involving graded metabolites. Hence, we initially hypothesized that the action of CarEs on gossypol may involve the hydrolase activity of the enzyme and the reaction of unknown intermediate metabolites. Gossypol is metabolized in different pathways in animals such as pigs [69] and hens [70]; however, these metabolites have not been completely elucidated as gossypol and its derivatives are excreted in low concentrations in animal feces [71,72]. Gossypol is bioconverted to gossypol ketone, gossypol acid, and demethylated gossypol acid [71]. The main scavenging mechanisms of gossypol include glucoaldehyde acidification and bile excretion [72]. In our reaction, CarE showed the characteristics of a hydrolase and some unknown enzymatic functions. The phenolic hydroxyl and aldehyde groups of gossypol were exposed, and the stability was relatively poor. Therefore, we explored the possibility of another degradation mechanism of gossypol. Metabolites were identified from the reaction system using UPLC-QTOF/MS and Masslynx 4.1 software (Waters Corporation, Milford, MA, USA) analysis and divided into standard, control, and test group. Gossypol formed a deprotonated molecule [M-H]− at m/z 518.1857 with a theoretical mass of 517.1910 in the negative ion scan mode. A comparison of the total ion current of the three groups (Figure 6) shows that the peak observed in the test group (which included the recombinant CarE in the reaction) at a retention interval of 19 min corresponds to gossypol, and its lower signal intensity quantitatively indicates that the gossypol content decreased under the influence of the recombinant CarE. In the endogenous enzyme group, the gossypol level was lower than that of the standard product. Although no recombinant CarE was added, the endogenous enzymes of P. pastoris may bind with gossypol to form bound gossypol, which reduced the TG content. After recombinant CarE was added to the test group (Figure 7), a new peak was observed that was not seen in the results from the standard and control groups. The peak had a retention time of 0.75 min and was measured at m/z 268.53. The chemical formula was C19H24O. We designated it as compound M0. The theoretical mass was 269.19, and the product was confirmed to be hemigossypol [73]. Gossypol itself is a polyphenol binaphthyl, which endows the binaphthyl bond with instability due to photosensitivity [74]. Another peak in the test group was observed at a retention interval of 4.51 min with m/z as 487.01 This compound, designated M1, had a chemical formula of C31H36O5 and a theoretical mass of 489.26. It was also the product of recombinant CarE activity, during which the exposed groups of gossypol were removed to detoxify gossypol. The degradation of gossypol by enzymes is less frequently examined. Previous studies have focused on the oxidation of gossypol by H. armigera P450 enzymes [57]. Laccase cyclization-hydroxyl aldehyde condensation [61] is a reaction of gossypol that renders it less toxic. This finding also coincides with the fact that enzymes have a wide range of substrates. Another detoxification product, M2, appeared at a retention time of 4.97 min, m/z of 600.03. This substance, with a chemical formula of C34H36N2O8 and theoretical mass of 601.25, has not been observed in other studies. Compound M2 was formed owing to the effect of CarE on the toxic aldehyde group of gossypol. This reaction formed an intermediate carboxylic acid in place of the aldehyde. Further, hydrolysis of the carboxylic acid derivative realized the complete elimination of the aldehyde group. Another detoxification product corresponding to the peak at 5.28 min was designated M3 with an m/z of 713.05. The structural formula of M2 was C47H59N3O3, with a theoretical mass of 714.46, and it is considered to be an Aza derivative of gossypol. We analyzed the recombinant CarE and found that the alanine and leucine levels were the highest. Furthermore, the aldehyde group on gossypol could react with the α-NH2 of amino acids to form Schiff bases, thereby forming azides. This finding is consistent with those of previous reports, which showed that binding or removing toxic aldehyde groups from gossypol can effectively reduce its toxicity. The metabolic pathway for the in vitro degradation of gossypol is shown in Figure 8. The exact mass, elemental composition, and molecular formula of the gossypol degradation products are listed in Table 2. In general, the metabolism of gossypol by the enzymatic reaction of H. armigera CCE001a is a complex process involving hydrolysis, dehydrogenation, and covalent binding to amine products. Hence, defining the metabolism of gossypol at all levels is challenging.