title: Kinetics and substrate selectivity of a Triticum aestivum xylanase inhibitor (TAXI) resistant D11F/R122D variant of Bacillus subtilis XynA xylanase.
abstract: This study examined the kinetics and substrate selectivity of a GH11 Bacillus subtilis XynA xylanase (BsX) sensitive to inhibition by TAXI and an engineered variant, which is much less inhibited by TAXI (BsX(mut)). The main purpose of the work was to elucidate any influence of the structural point mutations on the kinetics and substrate selectivity of the enzyme. Three-dimensional structures of both xylanases were superimposed to elucidate the structural basis for differences in their hydrolytic properties. The two xylanases were incubated individually with water-extractable arabinoxylan (WEAX), water-unextractable arabinoxylan (WUAX), birchwood xylan, and wheat bran. Both the BsX and the BsX(mut) catalyzed the release of xylo-oligosaccharides with higher degree of polymerization from WUAX than from WEAX. At equimolar addition levels the activity of the BsX(mut) was lower than that of the BsX with respect to both the initial rate and the product yields obtained after prolonged reaction on the xylan substrates. The calculated substrate selectivity factors indicated that the BsX and the BsX(mut) both had higher catalytic rate on WUAX than on WEAX. Addition of a 100:1 (TAXI:xylanase) molar ratio of inhibitor confirmed the significantly decreased inhibition of BsX(mut) by TAXI. Addition of TAXI also influenced the xylanases' selectivity factor differently.
authors.affiliation: Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Søltofts Plads, Building 229, DK-2800 Kgs. Lyngby, Denmark.
authors.forename: Louise E
authors.initials: LE
authors.lastname: Rasmussen
authors.forename: Jens F
authors.initials: JF
authors.lastname: Sørensen
authors.forename: Anne S
authors.initials: AS
authors.lastname: Meyer
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D004043:Dietary Fiber / Q000378:metabolism; D043364:Endo-1,4-beta Xylanases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D013379:Substrate Specificity; D014990:Xylans / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004043:Dietary Fiber; D010940:Plant Proteins; C422246:TAXI I protein, Triticum aestivum; D014990:Xylans; C085118:arabinoxylan; D043364:Endo-1,4-beta Xylanases