Rational Design for Broadened Substrate Specificity and Enhanced Activity of a Novel Acetyl Xylan Esterase from Bacteroides thetaiotaomicron

Abstract

Gut bacteria-derived enzymes play important roles in the metabolism of dietary fiber through enabling the hydrolysis of polysaccharides. 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.

Characterisation of a Novel Acetyl Xylan Esterase (BaAXE) Screened from the Gut Microbiota of the Common Black Slug (Arion ater)

“…The treatment of LCB with accessory enzymes has been shown to increase hydrolysis efficiency and improve the activity of backbone depolymerizing enzymes [23]. For example, the synergetic action of acetyl xylan esterase from Neocallimastix patriciarum and xylanase (XynA) released higher amounts of reducing sugars compared with hydrolysis with xylanase alone [24]. Acetyl xylan esterases (AXEs; EC 3.1.1.72) are accessory enzymes able to hydrolyse ester linkages, liberating acetic acid from acetylated hemicellulose [25].…”