Abstract 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 Km 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.

中文翻译：

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来自碱杆菌属的新型多结构域、耐盐和 SDS 抗性碱性支链淀粉酶的基因克隆、表达和生化表征。SL3

摘要 从碱杆菌属克隆了一个新的支链淀粉酶编码基因 pulSL3。SL3 并在大肠杆菌中表达。推断的 PulSL3 包含 2026 个氨基酸残基，由一个推定的信号肽、四个碳水化合物结合模块、一个 E_set 支链淀粉酶域、一个 α-淀粉酶域和一个支链淀粉酶域组成。纯化的 rPulSL3 仅表现出支链淀粉酶活性。为了鉴定α-淀粉酶和支链淀粉酶结构域的功能，还在大肠杆菌中表达了N-和C-末端截短的突变体(pulSL3△N和pulSL3△C)。纯化的rPulSL3△N具有支链淀粉酶活性，而rPulSL3△C无α-淀粉酶和支链淀粉酶活性。rPulSL3 和 rPulSL3△N 在 pH 9.0 和 50 °C 时均表现出最大活性，并且在高达 4 M 的 NaCl 浓度下具有高活性和稳定性，并且对一些表面活性剂和洗涤剂具有抗性。与野生型相比，rPulSL3△N 具有更好的热稳定性（在 52 °C 下保持 33.6% 的活性 30 分钟）、更高的底物亲和力（Km 为 0.10 对 0.15 mg mL-1）和更高的催化效率（836.6 与 520.3 mL mg-1 s-1）。这些优异的性能使rPulSL3△N在洗涤剂、食品和环境修复等行业具有应用潜力。