Laboratory of Microbial Biotechnology and Engineering Enzymes (LMBEE), Centre of Biotechnology of Sfax (CBS), University of Sfax, Road 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), P.O. Box 32, El Alia, Bab Ezzouar, 16111, Algiers, Algeria.
Extremophiles. 2019 Sep;23(5):529-547. doi: 10.1007/s00792-019-01105-8. Epub 2019 Jun 24.
The present study investigates the purification and biochemical characterization of a novel extracellular serine alkaline protease, subtilisin (called SAPN) from Melghiribacillus thermohalophilus Nari2A. The highest yield of protease (395 IU/g) with white shrimp shell by-product (40 g/L) as a unique source of nutriments in the growth medium was achieved after 52 h at 55 °C. The monomeric enzyme of about 30 kDa was purified to homogeneity by ammonium sulfate fractionation, heat treatment, followed by sequential column chromatographies. The optimum pH and temperature values for subtilisin activity were pH 10 and 75 °C, respectively, and half lives of 9 and 5 h at 80 and 90 °C, respectively. The sequence of the 25 NH-terminal residues pertaining of SAPN exhibited a high homology with those of Bacillus subtilisins. The inhibition by DFP and PMSF indicates that this enzyme belongs to the serine proteases family. SAPN was found to be effective in the deproteinization (DDP %) of blue swimming crab (Portunus segnis) and white shrimp (Metapenaeus monoceros) by-products, with a degree of 65 and 82%, respectively. The commercial and the two chitins obtained in this work showed a similar peak pattern in Fourier-Transform Infrared (FTIR) analysis, suggesting that SAPN is suitable for the bio-production of chitin from shell by-products.
本研究从嗜热盐单胞菌 Nari2A 中纯化和生化表征了一种新型的胞外丝氨酸碱性蛋白酶，枯草菌素（称为 SAPN）。在 55°C 下，以虾壳副产物（40g/L）为唯一营养源的生长培养基中，经过 52 小时的培养，获得了最高产量的蛋白酶（395IU/g）。通过硫酸铵分级沉淀、热处理和顺序柱层析，将约 30kDa 的单体酶纯化为均一性。该酶的最适 pH 和温度值分别为 pH 10 和 75°C，半衰期分别为 80°C 和 90°C 下的 9 和 5 小时。SAPN 的 25 个 N 端残基序列与枯草杆菌枯草菌素的序列具有高度同源性。DFP 和 PMSF 的抑制作用表明该酶属于丝氨酸蛋白酶家族。SAPN 对蓝蟹（Portunus segnis）和白虾（Metapenaeus monoceros）副产物的蛋白水解（DDP%）有效，分别达到 65%和 82%。本工作中获得的商业壳聚糖和两种甲壳素在傅里叶变换红外（FTIR）分析中显示出相似的峰型模式，表明 SAPN 适合从壳副产物生物生产甲壳素。
