Signal peptide (SP) prediction is used, but not known correct or not. The Aspergillus niger GH10 xylanase (XynB) SP is predicted to be 1-19 (Met(1)-Ser(19)) residue, differing from the Penicillium simplicissimum xylanase 1-25 residue SP. To determine the real SP, two types of XynB, Xyn Delta 19 and Xyn Delta 25, were constructed by respectively deleting the 19 (Met(1)-Ser(19)) or 25 (Met(1)-Arg(25)) residues. The Xyn Delta 25 had 10 degrees C higher T-opt and 21.6-times longer thermostability than the Xyn Delta 19 (46 vs. 36 degrees C and 47.6 vs. 2.2 m). When the kinetics were assayed, the Xyn Delta 25 had similar to 2.1-times higher V-max and higher binding-affinity for xylan than the Xyn Delta 19 (53.7 vs. 25.3 mu mol/ml/m and 2.43 vs. 2.96 mg/ml). Thus, the XynB real SP is the 1-25 and not predicted 1-19 residues. The extra six N-terminal residues (Glu(20)Pro(21)Ile(22)Glu(23)Pro(24)Arg(25)) drastically interfered with the Xyn Delta 19 thermal activity, thermostability, and catalytic efficiency. (C) 2012 Elsevier Ltd. All rights reserved.