Analysis of Prokaryotic Expression and Gossypol Degradation Activity of Catechol 2,3-Dioxygenase of Bacillus subtilis Strain M-15
XU Ming-Yang1, LI Jia1,*, WANG Wei1, ZHANG Cai-Xuan1,2, YANG Chen-Xi1, HAO Zhi-Min1,*
1 College of Life Science, Hebei Agricultural University/Hebei Bioinformatic Utilization and Technological Innovation Center for Agricultural Microbes, Baoding 071000, China; 2 School of Light Industry Science and Engineering, Beijing Technology and Business University, Beijing 100048, China
Abstract：Gossypol (GOS) is a polyphenolic antinutritional factor naturally present in cottonseed, which severely limits the widespread application of cottonseed by-products in the food and feed industries. Our research group screened Bacillus subtilis M-15 for its ability to degrade GOS efficiently and found that catechol 2,3-dioxygenase (C23O) may be a key enzyme in the GOS degradation process. This study aimed to verify the degradation effect of BsC23O on GOS in Bacillus subtilis M-15. The changes in the expression level of BsC23O under GOS stress were verified by qPCR. The physicochemical properties of BsC23O were predicted using bioinformatics methods. Subsequently, its expression was induced in an Escherichia coli expression system, and the effect of the expressed product on the degradation of gossypol (GOS) was tested. Additionally, the potential interaction mechanism between BsC23O and GOS was explored using structural confirmation and molecular docking. The results showed that the expression of BsC23O was up-regulated significantly under GOS stress (P<0.05). Bioinformatics analysis revealed that the protein encoded by BsC23O consisted of 285 amino acids, with a relative molecular mass of 31.56465 kD and an isoelectric point of 5.48. This protein lacked a signal peptide, had a relatively stable structure, was a hydrophilic protein, and was mainly located in the cytoplasm. Its structure contained 2 main domains: the VOC_BsCatE_like_N domain located at the N-terminus and the VOC_BsCatE_like_C domain located at the C-terminus. Additionally, the protein contained 23 potential phosphorylation sites and was primarily composed of α-helices and random coils. In the prokaryotic expression system, the activity of BsC23O reached 97.79 U/L. The expression product degraded 29.42% of GOS within 1 h. Molecular docking showed that the multiple benzene rings of GOS formed hydrophobic and conjugated interactions with residues around the pocket, promoting stable binding. This study successfully expressed highly active BsC23O and validated its pivotal role in GOS degradation. These findings provide critical theoretical support for the development of efficient GOS degradation technology and practicing enzyme engineering for safe utilization of cottonseed byproducts.
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