Abstract

In this work, we report on the halophilic bacterium Halobacillus blutaparonensis strain M9, which was isolated from a preserved Brazilian restinga, as a producer of alkaline serine peptidases with potential application as additives for biotechnological purposes. Gelatin-zymography assay revealed the presence of the major peptidase of 70 kDa and two minor ones (50 and 40 kDa) secreted by the living bacterial cells. When a specific chromogenic substrate for chymotrypsin-type serine peptidases was employed by means of an in-solution assay, the bacterial peptidases were completely inhibited by phenylmethylsulphonyl fluoride (PMSF) and slightly inhibited by benzamidine, N-tosyl-L-lysine chloromethyl ketone (TLCK) and 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF), which are classical serine peptidase inhibitors. The proteolytic activity remained extremely stable within a broad pH range (from 5.0 to 9.0) and also displayed moderate thermostability. In addition, the effects of osmolytes/polyols, divalent cations, denaturing agents, and detergents on the enzymatic activity and stability of these serine peptidases were tested. The proteolytic activity increased in the presence of different osmolytes/polyols (e.g., glycerol, glutamate, and mannitol). The enzymatic activity increased to 110% when incubated in the presence of 10 mM Ca²⁺, while Mg²⁺ and Mn²⁺ (both at 10 mM) decreased the proteolytic activity to 95 and 85%, respectively. Interestingly, urea at 1% induced a significant increase (around 160%) in the proteolytic activity. The peptidases showed high stability when incubated with various detergent agents and commercial detergent powders. Collectively, all these properties indicate H. blutaparonensis as a producer of extracellular serine peptidases with possible industrial applications, such as in detergent formulations.

中文翻译：

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Blutaparonensis Halobacillus blutaparonensis菌株M9作为具有生物技术用途特性的细胞外丝氨酸肽酶的来源

摘要

在这项工作中，我们报告了嗜盐细菌blutaparonensis M9菌株，该菌株从保存的巴西resta植物中分离，作为碱性丝氨酸肽酶的生产者，具有潜在的生物技术用途添加剂。明胶酶谱分析显示存在70 kDa的主要肽酶和活细菌细胞分泌的两个次要的肽酶（50和40 kDa）。当通过溶液中测定法使用胰凝乳蛋白酶型丝氨酸肽酶的特定生色底物时，细菌肽酶被苯甲基磺酰氟（PMSF）完全抑制，而被苯甲，N轻微抑制-tosyl-L-赖氨酸氯甲基酮（TLCK）和4-（2-氨基乙基）苯磺酰氟盐酸盐（AEBSF），它们是经典的丝氨酸肽酶抑制剂。蛋白水解活性在很宽的pH范围（5.0至9.0）内保持极其稳定，并显示出适度的热稳定性。另外，测试了渗透液/多元醇，二价阳离子，变性剂和去污剂对这些丝氨酸肽酶的酶活性和稳定性的影响。在不同的渗透液/多元醇（例如甘油，谷氨酸和甘露醇）存在下，蛋白水解活性增加。在10 mM Ca 2 ⁺，Mg ²⁺和Mn ²⁺的存在下孵育时，酶活性增加到110％（均为10 mM）将蛋白水解活性分别降低至95和85％。有趣的是，尿素含量为1％会引起蛋白水解活性的显着提高（约160％）。当与各种去污剂和市售去污剂粉末一起孵育时，肽酶显示出高稳定性。总的来说，所有这些性质表明，棉铃虫是细胞外丝氨酸肽酶的生产者，具有可能的工业应用，例如在洗涤剂配方中。