Gene cloning, expression and biochemical characterization of a new multi-domain, halotolerant and SDS-resistant alkaline pullulanase from Alkalibacterium sp. SL3
A new pullulanase-encoding gene, pulSL3 , was cloned from Alkalibacterium sp. SL3 and expressed in Escherichia coli . Deduced PulSL3 contains 2026 amino acid residues and consists of a putative signal peptide, four carbohydrate-binding modules, an E_set pullulanase domain, an α-amylase domain, and a pullulanase domain. The purified rPulSL3 exhibited pullulanase activity only. To identify the functions of the α-amylase and pullulanase domains, the N- and C-terminus truncated mutants ( pulSL3△N and pulSL3△C ) were also expressed in E. coli. The purified rPulSL3△N had pullulanase activity, while rPulSL3△C had no either α-amylase or pullulanase activity. Both rPulSL3 and rPulSL3△N exhibited maximum activities at pH 9.0 and 50 °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△N had better thermostability (retaining 62.9 vs. 33.6% activity at 52 °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△N potential for the application in the industries of detergent, food, and environmental remediation.
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