A new pullulanase-encoding gene, pLdSL3, was cloned from Alkalibacterium sp. SL3 and expressed in Escherichia coil. Deduced PulSL3 contains 2026 amino acid residues and consists of a putative signal peptide, four carbohydrate-binding modules, an E_set pullulanase domain, an alpha-amylase domain, and a pullulanase domain. The purified rPulSL3 exhibited pullulanase activity only. To identify the functions of the alpha-amylase and pullulanase domains, the N- and C-terminus truncated mutants (pulSL3 Delta N and pulSL3 Delta C) were also expressed in E. coli. The purified rPulSL3 Delta N had pullulanase activity, while rPulSL3 Delta C had no either alpha-amylase or pullulanase activity. Both rPulSL3 and rPulSL3 Delta N exhibited maximum activities at pH 9.0 and 50 degrees C, and were highly active and stable at the concentration of NaCl up to 4 M and resistant to a few surfactants and detergents. In comparison to the wild type, rPulSL3 Delta N had better thermostability (retaining 62.9 vs. 33.6% activity at 52 degrees C for 30 min), higher substrate affinity (with K-m of 0.10 vs. 0.15 mg mL(-1)) and higher catalytic efficiency (836.6 vs. 520.3 mL mg(-1) s(-1)). These excellent properties make rPulSL3 Delta N potential for the application in the industries of detergent, food, and environmental remediation.