Characterizing the pH-dependent stability and catalytic mechanism of the family 11 xylanase from the alkalophilic Bacillus agaradhaerens

• Published in: Carbohydrate research Vol. 338; no. 5; pp. 415 - 421
• Main Authors: Poon, David K.Y., Webster, Philip, Withers, Stephen G., McIntosh, Lawrence P.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier Ltd 14.02.2003
• Subjects: Bacillus - enzymology; Bacillus xylanase; Extremophile; Glycosidase; Hot Temperature; Hydrogen-Ion Concentration; pH-Dependent mechanism; Protein Denaturation; Protein stability; Xylan Endo-1,3-beta-Xylosidase; Xylosidases - metabolism; Bacillus xylanase; Glycosidase; Protein stability; Extremophile; pH-Dependent mechanism
• ISSN: 0008-6215; 1873-426X
• DOI: 10.1016/S0008-6215(02)00486-X

The xylanase, BadX, from the alkalophilic Bacillus agaradhaerens was cloned, expressed and studied in comparison to a related family 11 xylanase, BcX, from B. circulans. Despite the alkaline versus neutral conditions under which these bacteria grow, BadX and BcX both exhibit optimal activity near pH 5.6 using the substrate o-nitrophenyl β-xylobioside. Analysis of the bell-shaped activity profile of BadX yielded apparent p K a values of 4.2 and 7.1, assignable to its nucleophile Glu94 and general acid Glu184, respectively. In addition to having an ∼10-fold higher k cat/ K m value with this substrate at pH 6 and 40 °C, BadX has significantly higher thermal stability than BcX under neutral and alkaline conditions. This enhanced stability, rather than a shift in its pH-optimum, may allow BadX to hydrolyze xylan under conditions of elevated temperature and pH. Graphic