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 -helix and 10% P-sheet. The optimal temperature for the activity of Xyl I was 80 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 expansive pH range of 5 to 10 with more than 75% residual activity. Our present investigation using o-phthalaldehyde (OPTA) as the chemical initiator for fluorescent 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 native enzyme and the substrate protection against OPTA and TNBS, suggested the presence of the lysine residue in the substrate-binding site, (C) 2001 Academic Press.