Recently, the research team of Fengjiao Xin, an innovation team for food enzyme engineering, identified a novel gut-bacterial Acetyl Xylan Esterase by screening the Bacteroides thetaiotaomicron genome and determined the structure of this enzyme. The relevant research results were published as a supplemental cover story in the journal J. Agric. Food Chem (IF: 5.279) with the title “Rational Design for Broadened Substrate Specificity and Enhanced Activity of a Novel Acetyl Xylan Esterase from Bacteroides thetaiotaomicron”.

Gut bacteria-derived enzymes play important roles in the metabolism of complex polysaccharides such as dietary fiber. Bacteroides thetaiotaomicron, a dominant bacterial symbiont of the human intestine and the most effective degrader of polysaccharides, is a model for studying polysaccharide metabolism in the human gut and for understanding the symbiotic host−bacterial relationships. In intestinal tract, acetyl xylan esterases (Axes) decompose acetylated xylan to produce short chain fatty acid -- acetic acid, which is beneficial to the host in weight control, insulin sensitivity, inflammation, and carcinogenesis. In this study, we identified and characterized a 29 kDa novel acetyl xylan esterase, BTAxe1, from Bacteroides thetaiotaomicron VPI5482. Then, we solved the structure of BTAxe1 and performed the rational design. Mutants N65S and N65A increased the activities toward short-chain (pNPA, pNPB) to near four-fold, and gained the activities toward longer-chain substrate (pNPO). Molecular docking analysis showed that the mutant N65S had a larger substrate binding pocket than the wild type. Hydrolysis studies using natural substrates showed that either N65S or N65A showed higher activity of that of wild-type, yielding 131.31 and 136.09 mM of acetic acid from xylan. This is the first study on the rational design of gut bacteria-derived Axes with broadened substrate specificity and enhanced activity, which can be referenced by other acetyl esterases or gut-derived enzymes.

Our postgraduate student Luyao Wang and Xue Han as co-first authors, Associate Researcher Lichao Sun and Researcher Fengjiao Xin as co-corresponding authors. This work was supported by the National Key Research and Development Plan “modern food processing and food storage and transportation technology and equipment” (2017YFD0400204), National Natural Science Foundation of China (31700701 and 31801475), and Central Public-interest Scientific Institution Basal Research Fund (S2020JBKY-13).

Figure 1. Molecular docking analysis of wild-type BTAxe1 and mutant N65S with pNPA, pNPB, and pNPO. Wild-type BTAxe1 with pNPA (A), pNPB (B), and pNPO (C), shown in green, yellow, and gray, respectively. Interactions between pNPA (D), pNPB (E), and pNPO (F) and amino acids, shown in orange. Mutant N65S with pNPA (A), pNPB (B), and pNPO (C), shown in green, yellow, and gray, respectively. Interactions between pNPA (J), pNPB (K), and pNPO (L) and amino acids, shown in orange.

Link to the paper: https://pubs.acs.org/doi/10.1021/acs.jafc.1c00750