A neutral, heat-sensitive serine protease (NHSSP) originating from the feather-degrading fungus Onygena corvina (O. corvina) was described and defined as an alkaline serine protease of the subtilisin type S8 family, exhibiting an enzymatic activity at neutral pH. Generally, broad specificity proteases, such as proteinase K or trypsin, have found numerous applications in research and biotechnology. We report the cloning and expression in the yeast PichiaPink™ system, as well as purification, and characterization of the NHSSP. Recombinant, His6-tagged NHSSP was efficiently expressed from an optimized, synthetic gene and purified using a simple protocol based on ammonium sulfate fractionation and hydrophobic interaction chromatography. The enzyme shows atypical C-terminal processing, the coded preproprotein undergoes signal peptide removal and maturation through the clipping of a propeptide section and 10 amino acids (aa) from the C-terminus, including the His6-tag. The deletion variant has been constructed, devoid of the C-terminal ORF segment, thus eliminating the need for C-terminal processing. Both NHSSP variants exhibit very similar enzymatic characteristics. The purified enzymes were characterized to determine the optimal proteolytic conditions. We revealed that the mature NHSSP is reproducibly active over a wide pH range from neutral to mild acidic (pH of 5.0 to 8.5), with an optimum at pH 6.8, and at temperatures of 15 to 50 °C with an optimum at 38–42 °C. Interestingly, we demonstrated that the protease can be fully deactivated by a moderate increase in temperature of about 15 °C from the optimum to over 50 °C. The protease was partially sensitive to serine protease inhibitors, and not inhibited by chelating or reducing agents and detergents. SDS induced autolysis of NHSSP, which points to a high stimulation of its proteolytic activity. The NHSSP was produced as a recombinant protein with high efficiency. Compared to proteinase K, the most common serine protease used, NHSSP shows an approx. twofold higher specific activity. Protein sequencing can be a valuable technical application for the protease. The protein coverage is significantly higher in comparison to trypsin and reaches about 84–100% for β-lactoglobulin (BLG), antibody (mAb) light and heavy chains. Furthermore, the option to perform digestions at neutral to slightly acidic pH-values down to pH 5.0 avoids modification of peptides, e.g. due to deamidation.

中文翻译：

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真菌Onygena corvina的蛋白酶K热敏蛋白酶的另一种生物技术：克隆，工程改造，表达，表征和蛋白质测序的特殊应用。

描述了一种中性的热敏感丝氨酸蛋白酶（NHSSP），其源自羽毛降解真菌Onygena corvina（O. corvina），并被定义为枯草杆菌蛋白酶S8家族的碱性丝氨酸蛋白酶，在中性pH值下具有酶促活性。通常，广泛特异性的蛋白酶，例如蛋白酶K或胰蛋白酶，已在研究和生物技术中找到了许多应用。我们报告了酵母PichiaPink™系统的克隆和表达，以及NHSSP的纯化和鉴定。重组的，带有His6标签的NHSSP可从优化的合成基因中高效表达，并使用基于硫酸铵分级分离和疏水相互作用色谱的简单方案进行纯化。该酶显示出非典型的C末端加工，编码的前原蛋白通过剪切前肽段和C端的10个氨基酸（aa）（包括His6-tag）进行信号肽的去除和成熟。已经构建了缺失变体，没有C端ORF片段，因此消除了C端加工的需要。两种NHSSP变体均显示出非常相似的酶促特性。对纯化的酶进行表征以确定最佳的蛋白水解条件。我们发现，成熟的NHSSP在从中性到中度酸性（pH值为5.0至8.5）的宽pH范围内均具有可再现的活性，最适pH值为6.8，最适温度为15至50°C，最适温度为38-42 ℃。有趣的是，我们证明了大约15°C的温度从最佳温度适度增加到超过50°C可以使蛋白酶完全失活。该蛋白酶对丝氨酸蛋白酶抑制剂部分敏感，不受螯合或还原剂和去污剂的抑制。SDS诱导了NHSSP的自溶，这表明其蛋白水解活性受到了极大的刺激。NHSSP是作为高效重组蛋白生产的。与蛋白酶K（使用的最常见的丝氨酸蛋白酶）相比，NHSSP的酶切强度约为K。比活性高两倍。蛋白质测序可以是蛋白酶的有价值的技术应用。与胰蛋白酶相比，蛋白质的覆盖率要高得多，β-乳球蛋白（BLG），抗体（mAb）轻链和重链的蛋白质覆盖率约为84-100％。此外，可以选择在中性至弱酸性的pH值（低至pH 5.0）下进行消化，从而避免了肽的修饰（例如由于脱酰胺作用）。