Laboratory of Microbial Biotechnology and Engineering Enzymes (LMBEE), Centre of Biotechnology of Sfax (CBS), University of Sfax, Road of Sidi Mansour Km 6, P.O. Box 1177, 3018, Sfax, Tunisia.
Laboratory of Cellular and Molecular Biology (LCMB), Microbiology Team, Faculty of Biological Sciences, University of Sciences and Technology of Houari Boumediene (USTHB), El Alia, P.O. Box 32, 16111, Bab Ezzouar, Algiers, Algeria.
Extremophiles. 2019 Nov;23(6):687-706. doi: 10.1007/s00792-019-01123-6. Epub 2019 Aug 12.
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 NH-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.
一种热稳定的胞外碱性蛋白酶（称为 SAPA）由 Anoxybacillus kamchatkensis M1V 产生（4600 U/mL），经纯化至均一性，并进行了生化特性分析。SAPA 是一种单体，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-酶谱和高效液相色谱（HPLC）的分子筛分析，估计其分子量为 28 kDa。其 NH2-末端氨基酸序列与芽孢杆菌蛋白酶具有高度同源性。DFP 和 PMSF 对 SAPA 的不可逆抑制证实其属于丝氨酸蛋白酶家族。SAPA 的最佳活性在 pH 11 和 70°C。sapA 基因在大肠杆菌的细胞外部分被克隆和表达。SAPA 与枯草芽孢杆菌 WT 168 的肽酶 S8 具有最高的序列同一性值（95%），但有 16 个氨基酸的差异。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 的水解程度分别比来自解淀粉芽孢杆菌 CBS 的 SAPB 和来自地衣芽孢杆菌的枯草杆菌蛋白酶 A 高 1.24 和 2.6 倍。此外，与商业酶相比，rSAPA 表现出较高的去污剂相容性和出色的去污能力： savinase™ 16L、type EX 和 alcalase™ Ultra 2.5 L。
