Mechri Sondes, Ben Elhoul Berrouina Mouna, Omrane Benmrad Maroua, Zaraî Jaouadi Nadia, Rekik Hatem, Moujehed Emna, Chebbi Alif, Sayadi Sami, Chamkha Mohamed, Bejar Samir, Jaouadi Bassem
Laboratory of Microbial Biotechnology and Engineering Enzymes, Centre of Biotechnology of Sfax, University of Sfax, Road of Sidi Mansour Km 6, P.O. Box 1177, Sfax 3018, Tunisia.
Laboratory of Environmental Bioprocesses, LMI COSYS-Med, Centre of Biotechnology of Sfax, University of Sfax, Road of Sidi Mansour Km 6, P.O. Box 1177, Sfax 3018, Tunisia.
Int J Biol Macromol. 2017 Jan;94(Pt A):221-232. doi: 10.1016/j.ijbiomac.2016.09.112. Epub 2016 Oct 5.
The present study investigates the purification and physico-chemical characterization of an extracellular protease from the Aeribacillus pallidus strain VP3 previously isolated from a geothermal oil-field (Sfax, Tunisia). The maximum protease activity recorded after 22h of incubation at 45°C was 3000U/ml. Pure enzyme, designated as SPVP, was obtained after ammonium sulfate fractionation (40-60%)-dialysis followed by heat-treatment (70°C for 30min) and UNO Q-6 FPLC anion-exchange chromatography. The purified enzyme is a monomer of molecular mass about 29kDa. The sequence of the 25 NH-terminal residues of SPVP showed a high homology with those of Bacillus proteases. The almost complete inhibition by PMSF and DIFP confirmed that SPVP is a member of serine protease family. Its optima of pH and temperature were pH 10 and 60°C, respectively. Its half-life times at 70 and 80°C were 8 and 4h, respectively. Its catalytic efficiency was higher than those of SAPCG, Alcalase Ultra 2.5L, and Thermolysin type X. SPVP exhibited excellent stability to detergents and wash performance analysis revealed that it could remove blood-stains effectively and high resistance against organic solvents. These properties make SPVP a potential candidate for applications in detergent formulations and non-aqueous peptide biocatalysis.
本研究对先前从突尼斯斯法克斯的一个地热油田分离出的苍白芽孢杆菌VP3菌株的一种细胞外蛋白酶进行了纯化及理化特性分析。在45°C孵育22小时后记录到的最大蛋白酶活性为3000U/ml。通过硫酸铵分级分离（40 - 60%）-透析，随后进行热处理（70°C处理30分钟）和UNO Q - 6 FPLC阴离子交换色谱法，获得了纯酶，命名为SPVP。纯化后的酶是一种分子量约为29kDa的单体。SPVP的25个N端残基序列与芽孢杆菌蛋白酶的序列具有高度同源性。PMSF和DIFP几乎完全抑制作用证实SPVP是丝氨酸蛋白酶家族的一员。其最适pH和温度分别为pH 10和60°C。它在70°C和80°C下的半衰期分别为8小时和4小时。其催化效率高于SAPCG、碱性蛋白酶Ultra 2.5L和X型嗜热菌蛋白酶。SPVP对洗涤剂表现出优异的稳定性，洗涤性能分析表明它能有效去除血迹且对有机溶剂具有高抗性。这些特性使SPVP成为洗涤剂配方和非水相肽生物催化应用的潜在候选者。
