Department of Medical Services and Techniques, Şebinkarahisar Social Sciences Vocational School, 28400, Şebinkarahisar, Giresun, Turkey.
Department of Molecular Biology and Genetics, Faculty of Science, Karadeniz Technical University, 61080, Trabzon, Turkey.
Biotechnol Lett. 2021 Feb;43(2):479-494. doi: 10.1007/s10529-020-03025-6. Epub 2020 Oct 12.
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 Å 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.
在这里，我们报告了一种来自巴氏芽孢杆菌 C-125 的枯草杆菌样碱性丝氨酸蛋白酶（ASP）的异源表达、酶学特性和结构同源建模。通过 PCR 成功获得编码基因，并在强 HpaII 启动子的控制下克隆到 pMA0911 穿梭载体中，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，并在培养基中分泌到细胞外。Km、Vmax 和重组 ASP 的比活性参数分别鉴定为 0.2899mg/ml、76.12U/ml 和 9500U/mg。纯化后的酶在 pH 值为 12.0 的强碱性条件下显示出显著的蛋白水解活性和显著的热稳定性，最适温度为 60°C。此外，无底物的酶在 pH 值为 12.0 时显示出显著的 pH 稳定性，在 37°C 孵育 24 小时后保持初始活性的 93%，在 60°C 孵育 1 小时后保持初始活性的 60%。通过 SDS-PAGE 和 Western blot 分析，理论上计算出的 ASP 蛋白分子量得到了验证（Mw：28.3kDa）。通过同源建模进一步鉴定了 ASP 蛋白的二级和三级结构，并进行了详细研究。ASP 含有一个典型的 S8/S53 肽酶结构域，由 17 个β-折叠和 9 个α-螺旋组成。模型 3D 结构的结构动力学分析进一步表明，瞬态失活的前肽链是 ASP 酶最具动态性的区域，β-因子值为 8.52Å。额外的依赖于残基的波动图分析也证实了 ASP N 端结构动力学模式的提高，这可能是碱性丝氨酸肽酶自主去除前肽的潜在前提。然而，ASP 的功能域在自主去除其前肽后变得非常稳定。尽管 ASP 与商业洗涤剂添加剂 B. lentus 蛋白酶（PDB ID：1GCI）之间的序列同源性中等（65.4%序列相似性），但它们重叠的 3D 结构在 0.80Å RMSD 内显示出更高的相似性（98.14%）。总之，ASP 蛋白具有显著的 pH 稳定性、显著的热稳定性和在极端碱性条件下特别高的比活性，是纺织、洗涤剂、皮革、饲料、废物、制药等各个工业领域新型蛋白酶候选物。
