Abstract

A thermostable extracellular alkaline protease (called SAPA) was produced (4600 U/mL) by Anoxybacillus kamchatkensis M1V, purified to homogeneity, and biochemically characterized. SAPA is a monomer with a molecular mass of 28 kDa estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Native-PAGE, casein-zymography, and size exclusion using high performance liquid chromatography (HPLC). The sequence of its NH₂-terminal amino-acid residues showed high homology with those of Bacillus proteases. The SAPA irreversible inhibition by diiodopropyl fluorophosphates (DFP) and phenylmethanesulfonyl fluoride (PMSF) confirmed its belonging to the serine proteases family. Optimal activity of SAPA was at pH 11 and 70 °C. The sapA gene was cloned and expressed in the extracellular fraction of E. coli. The highest sequence identity value (95%) of SAPA was obtained with peptidase S8 from Bacillus subtilis WT 168, but with 16 amino-acids of difference. The biochemical characteristics of the purified recombinant extracellular enzyme (called rSAPA) were analogous to those of native SAPA. Interestingly, rSAPA exhibit a degree of hydrolysis that were 1.24 and 2.6 than SAPB from Bacillus pumilus CBS and subtilisin A from Bacillus licheniformis, respectively. Furthermore, rSAPA showed a high detergent compatibility and an outstanding stain removal capacity compared to commercial enzymes: savinase™ 16L, type EX and alcalase™ Ultra 2.5 L.

Graphic Abstract

中文翻译：

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从嗜热嗜热衣原体M1V中鉴定出一种新型蛋白酶，并将其用作洗衣洗涤剂添加剂。

摘要

堪萨克氏厌氧杆菌M1V生产了一种热稳定的细胞外碱性蛋白酶（称为SAPA）（4600 U / mL），纯化至均一并进行了生化表征。SAPA是通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE），Native-PAGE，酪蛋白酶谱分析和使用高效液相色谱（HPLC）进行尺寸排阻估计的分子量为28 kDa的单体。其NH ₂末端氨基酸残基的序列与芽孢杆菌蛋白酶显示高度同源性。二碘丙基氟代磷酸酯（DFP）和苯基甲磺酰氟（PMSF）对SAPA的不可逆抑制作用证实了它属于丝氨酸蛋白酶家族。SAPA的最佳活性是在pH 11和70°C下。在S APA该基因被克隆并在大肠杆菌的细胞外部分表达。SAPA的最高序列同一性值（95％）是由枯草芽孢杆菌WT 168的肽酶S8获得的，但有16个氨基酸差异。纯化的重组细胞外酶（称为rSAPA）的生化特性与天然SAPA相似。有趣的是，rSAPA的水解度分别比短小芽孢杆菌CBS的SAPB和地衣芽孢杆菌的枯草杆菌蛋白酶A分别高1.24和2.6 。此外，与商业酶savinase™16L，EX型和alcalase™Ultra 2.5 L相比，rSAPA显示出高洗涤剂相容性和出色的去污能力。

图形摘要