Involvement of a lysine residue in the active site of a thermostable xylanase from Thermomonospora sp.

A highly thermostable xylanase (Xyl I) produced by Thermomonospora sp. was purified to homogeneity and was classified as a family 10 xylanase based on its molecular weight (38,000 Da) and isoelectric point (4.1). K2d analysis showed that the secondary structure of Xyl I was made up of 38% alpha -hel ix and 10% P-sheet. The optimal temperature for the activity of Xyl I was 8 0 degreesC. Xyl I was highly thermostable with half-lives of 86, 30, and 15 min at 80, 90, and 100 degreesC respectively. Xyl I was stable in an expan sive pH range of 5 to 10 with more than 75% residual activity. Our present investigation using o-phthalaldehyde (OPTA) as the chemical initiator for f luorescent chemoaffinity labeling and trinitrobenzenesulphonic acid (TNBS) as chemical modifier have revealed the presence of a single lysine residue in the active site of Xyl I, The high pK value for the basic limb of the pH profile reflects the ionization of a lysine residue. The higher K-m values and similar K-cat values of the TNBS modified enzyme in comparison to nati ve enzyme and the substrate protection against OPTA and TNBS, suggested the presence of the lysine residue in the substrate-binding site, (C) 2001 Aca demic Press.