Abstract

Extracellular protease SptA from the halophilic archaeon Natrinema sp. J7 was successfully expressed in Bacillus subtilis SCK6 using touchdown two-step PCR and plasmid multimer. Culture optimization was conducted to increase SptA protease activity 132-fold. It was shown that enzyme activity achieved the maximum activity at pH 8.0 and 55°C with casein as the substrate. SptA protease kept high stability and activity against many organic solvents and even maintained 60% activity in the presence of 8% NaCl. Meanwhile, SptA was redefined as a highly Ca²⁺-dependent subtilisin-like serine protease. Interestingly, SptA protease processed an autodigestion from 45 to 35 kDa in presence of Ca²⁺. Homology modeling illustrated the three binding sites of Ca²⁺ and the catalytic triad His231-Asp190-Ser384. In addition, SptA protease exhibited high compatibility with many surfactants and commercial detergents, showing 100% stability in the presence of commercial detergent Comfort. Washing performance analysis demonstrated remarkably high de-staining of the enzyme at 40°C for 20 min with a lower supplementation (500 U/mL). Accordingly, SptA protease could be considered as a promising bio-additive in the detergent industry.

中文翻译：

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表面活性稳定的钙依赖性蛋白酶的表达和表征：一种潜在的洗衣粉添加剂

摘要

来自嗜盐古菌Natrinema sp. 的细胞外蛋白酶 SptA 。J7 成功地在枯草芽孢杆菌SCK6 中使用触地两步 PCR 和质粒多聚体表达。进行培养优化以将 SptA 蛋白酶活性提高 132 倍。结果表明，酶活性在 pH 8.0 和 55°C 下以酪蛋白为底物达到最大活性。SptA 蛋白酶对许多有机溶剂保持高稳定性和活性，甚至在 8% NaCl 存在下仍保持 60% 的活性。同时，SptA 被重新定义为高度依赖Ca ^{2+ 的}枯草杆菌蛋白酶样丝氨酸蛋白酶。有趣的是，SptA 蛋白酶在 Ca ²⁺存在下处理了 45 至 35 kDa 的自消化. 同源模型说明了 Ca ²⁺和催化三联体 His231-Asp190-Ser384的三个结合位点。此外，SptA 蛋白酶与许多表面活性剂和商业洗涤剂表现出高度的相容性，在商业洗涤剂 Comfort 存在下表现出 100% 的稳定性。洗涤性能分析表明，在 40°C 下，酶在 20 分钟内具有非常高的脱色效果，并且添加量较低（500 U/mL）。因此，SptA 蛋白酶可以被认为是洗涤剂行业中一种很有前途的生物添加剂。