Laboratory of Microorganisms and Biomolecules, 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. 2013 Sep;60:165-77. doi: 10.1016/j.ijbiomac.2013.05.019. Epub 2013 May 30.
An extracellular alkaline elastase was produced from Pseudomonas aeruginosa CTM50182. It was chromatographically purified using HPLC and Mono Q Sepharose column. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis revealed that the purified enzyme (called AMPP) was a monomer with a molecular mass of 33,015.18 Da. The N-terminal 29 amino acid sequence of AMPP showed high homology with those of Pseudomonas elastases. It showed optimal activity at pH 12 and 80 °C and was stable at a pH range of 9-12 after 120 h of incubation. Its thermoactivity and thermostability were upgraded in the presence of 5 mM Co(2+). Its half-life times at 70 and 80 °C were 16 and 10 h, respectively. It was completely inhibited by ethylene glycol-bis (β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), and 1,10-phenanthroline, suggesting that it belongs to the metalloprotease family. AMPP also exhibited high catalytic efficiency, organic solvent-tolerance, and hydrolysis. The lasB gene encoding AMPP was cloned, sequenced, and expressed in Escherichia coli. The biochemical properties of the extracellular purified recombinant enzyme (rAMPP) were similar to those of native AMPP. This organic solvent-stable protease could be considered a potential candidate for application as a biocatalyst in the synthesis of enzymatic peptides.
一株铜绿假单胞菌 CTM50182 可产生细胞外碱性弹性蛋白酶。该酶经 HPLC 和 Mono Q Sepharose 柱层析纯化。基质辅助激光解吸电离飞行时间质谱（MALDI-TOF/MS）分析表明，该纯化酶（称为 AMPP）为单体，分子量为 33015.18 Da。AMPP 的 N 端 29 个氨基酸序列与假单胞菌弹性蛋白酶具有高度同源性。它在 pH 12 和 80°C 下表现出最佳活性，在孵育 120 小时后在 pH 9-12 范围内稳定。在 5 mM Co(2+) 的存在下，其热活性和热稳定性得到提高。它在 70 和 80°C 下的半衰期分别为 16 和 10 h。它完全被乙二胺四乙酸（EGTA）和 1,10-邻菲啰啉抑制，表明它属于金属蛋白酶家族。AMPP 还表现出高催化效率、有机溶剂耐受性和水解活性。编码 AMPP 的 lasB 基因被克隆、测序并在大肠杆菌中表达。细胞外纯化的重组酶（rAMPP）的生化特性与天然 AMPP 相似。这种有机溶剂稳定的蛋白酶可以被认为是作为生物催化剂在酶肽合成中的潜在候选者。
