Laboratory of Microorganisms and Biomolecules (LMB), Centre of Biotechnology of Sfax (CBS), University of Sfax, Road of Sidi Mansour Km 6, P.O. Box 1177, 3018 Sfax, Tunisia.
Bioresour Technol. 2012 Feb;105:142-51. doi: 10.1016/j.biortech.2011.11.115. Epub 2011 Dec 3.
The sapB gene encoding for Bacillus pumilus CBS protease (SAPB) and the triple mutated sapB-L31I/T33S/N99Y gene were cloned and overexpressed in the protease-deficient Bacillus subtilis DB430 using an Escherichia coli-Bacillus shuttle vector pBSMuL2. The 34,625.13 and 34,675.11-Da enzymes were purified from the culture supernatant of B. subtilis expressing the wild-type and mutated genes, respectively. The purified proteases showed the same N-terminal sequences and biochemical properties of those expressed in E. coli. Further investigations demonstrated that, compared to wild-type and other proteases, SAPB-L31I/T33S/N99Y had the highest catalytic efficiency and the best degree of hydrolysis. The mutant enzyme was also noted to exhibit a number of newly explored properties that are highly valued in the marketplace, namely considerable stability to detergents, higher resistance towards organic solvents, and potent dehairing ability. Overall, the findings indicated that SAPB-L31I/T33S/N99Y is a promising candidate for future use in a wide range of industrial and commercial applications.
该 sapB 基因编码短小芽孢杆菌 CBS 蛋白酶（SAPB）和三突变 sapB-L31I/T33S/N99Y 基因被克隆和过表达在蛋白酶缺陷型枯草芽孢杆菌 DB430 使用大肠杆菌枯草芽孢杆菌穿梭载体 pBSMuL2。34625.13 和 34675.11-Da 酶从表达野生型和突变基因的枯草芽孢杆菌的培养上清液中纯化。纯化的蛋白酶显示出与在大肠杆菌中表达的蛋白酶相同的 N 端序列和生化性质。进一步的研究表明，与野生型和其他蛋白酶相比，SAPB-L31I/T33S/N99Y 具有最高的催化效率和最佳的水解程度。该突变酶还表现出一些在市场上高度重视的新探索的特性，即对洗涤剂具有相当的稳定性、对有机溶剂的更高抗性和强大的脱毛能力。总的来说，这些发现表明 SAPB-L31I/T33S/N99Y 是未来在广泛的工业和商业应用中使用的有前途的候选者。
