Here we report heterologous expression, enzymatic characterization and structure homology modeling of a subtilisin-like alkaline serine protease (ASP) from Bacillus halodurans C-125. Encoding gene was successfully obtained by PCR and cloned into pMA0911 shuttle vector under the control of strong HpaII promoter and expressed extracellularly. ASP enzyme was successfully expressed in B. subtilis WB800 cell line lacking eight extracellular proteases and produced extracellularly in the culture medium. Km, Vmax and specific activity parameters of the recombinantly produced ASP were identified as 0.2899 mg/ml, 76.12 U/ml and 9500 U/mg, respectively. The purified enzyme revealed remarkable proteolytic activity at highly alkaline conditions with a pH optimum 12.0 and notable thermostability with temperature optimum at 60 °C. Furthermore, substrate-free enzyme revealed remarkable pH stability at pH 12.0 and maintained 93% of its initial activity when incubated at 37 °C for 24 h and 60% of its initial activity upon incubation at 60 °C for 1 h. Theoretically calculated molecular mass of ASP protein was confirmed through SDS-PAGE and western blot analysis (Mw: 28.3 kDa). The secondary and tertiary structures of ASP protein were also identified through homology modeling and further examined in detail. ASP harbors a typical S8/S53 peptidase domain comprising 17 β-sheets and 9 α-helixes within its secondary structure. The structure dynamics analysis of modeled 3D structure further revealed that transient inactivating propeptide chain is the most dynamic region of ASP enzyme with 8.52 Å2 β-Factor value. Additional residue-dependent fluctuation plot analysis also confirmed the elevated structure dynamics patterning of ASP N-terminus which could be the potential prerequisite for the autonomous propeptide removal of alkaline serine peptidases. Yet the functional domain of ASP becomes quite stable after autonomous exclusion of its propeptide. Although the sequence homology between ASP and commercial detergent additive B. lentus protease (PDB ID:1GCI) was moderate (65.4% sequence similarity), their overlaid 3D structures revealed much higher similarity (98.14%) within 0.80 Å RMSD. In conclusions, with remarkable pH stability, notable thermostability and particularly high specific activity at extreme alkaline conditions, the unveiled ASP protein stands out as a novel protease candidate for various industrial sectors such as textile, detergent, leather, feed, waste, pharmaceutical and others.

中文翻译：

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嗜盐芽孢杆菌 C-125 高碱性枯草杆菌蛋白酶样丝氨酸蛋白酶的同源建模和异源表达

在这里，我们报告了来自嗜盐芽孢杆菌 C-125 的枯草杆菌蛋白酶样碱性丝氨酸蛋白酶 (ASP) 的异源表达、酶学表征和结构同源性建模。通过PCR成功获得编码基因并克隆到在强HpaII启动子控制下的pMA0911穿梭载体中并在细胞外表达。ASP 酶在缺乏八种细胞外蛋白酶的枯草芽孢杆菌 WB800 细胞系中成功表达，并在培养基中细胞外产生。重组产生的 ASP 的 Km、Vmax 和比活性参数分别为 0.2899 mg/ml、76.12 U/ml 和 9500 U/mg。纯化的酶在高度碱性条件下显示出显着的蛋白水解活性，最适 pH 值为 12.0，并且具有显着的热稳定性，最适温度为 60 °C。此外，无底物酶在 pH 12.0 时表现出显着的 pH 稳定性，并在 37°C 下孵育 24 小时时保持其初始活性的 93%，在 60°C 下孵育 1 小时时保持其初始活性的 60%。通过 SDS-PAGE 和蛋白质印迹分析（Mw：28.3 kDa）确认了理论上计算的 ASP 蛋白的分子量。ASP 蛋白的二级和三级结构也通过同源建模进行了鉴定并进一步详细检查。ASP 包含一个典型的 S8/S53 肽酶结构域，在其二级结构中包含 17 个 β-折叠和 9 个 α-螺旋。建模的 3D 结构的结构动力学分析进一步表明，瞬态灭活前肽链是 ASP 酶最具活力的区域，具有 8.52 Å2 β-因子值。额外的残基依赖性波动图分析也证实了 ASP N 端的结构动力学模式升高，这可能是碱性丝氨酸肽酶的自主前肽去除的潜在先决条件。然而，在自主排除其前肽后，ASP 的功能域变得相当稳定。尽管 ASP 和商业洗涤剂添加剂 B. lentus 蛋白酶 (PDB ID:1GCI) 之间的序列同源性适中（65.4% 序列相似性），但它们重叠的 3D 结构在 0.80 Å RMSD 内显示出更高的相似性（98.14%）。总之，由于具有显着的 pH 稳定性、显着的热稳定性和在极端碱性条件下特别高的比活性，新推出的 ASP 蛋白是纺织、洗涤剂、皮革、