Purification and biochemical characterization of a novel thermostable protease from the oyster mushroom Pleurotus sajor-caju strain CTM10057 with industrial interest
Purification and biochemical characterization of a novel thermostable protease from the oyster mushroom Pleurotus sajor-caju strain CTM10057 with industrial interest
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DOI：
10.1186/s12896-019-0536-4
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发表时间：
2019-07-01
影响因子：
3.5
通讯作者：
Jaouadi, Bassem
中科院分区：
文献类型：
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作者：
Benmrad, Maroua Omrane;Mechri, Sondes;Jaouadi, Bassem
蛋白酶是在蛋白质和多肽水平上催化肽链裂解反应的水解酶，具有不同程度的特异性。这一群体引起了业界的注意。超过三分之一的蛋白酶是丝氨酸蛋白酶。通常，丝氨酸蛋白酶在中性到碱性ph值下具有活性。丝氨酸蛋白酶被研究用于工业用途，特别是洗涤剂。它们是世界市场上最具商业价值的酶群。总的来说，真菌产生细胞外蛋白酶，很容易通过过滤从菌丝体中分离出来。结果鉴定出一种新的担子菌CTM10057，该真菌高产一种新型蛋白酶（10500 u /mL），为平菇（Pleurotus saor -caju）。经热处理（80℃20min）纯化至均匀，硫酸铵沉淀(35-55%)-透析，UNO Q-6 FPLC离子交换层析，最后HPLC-ZORBAX PSM 300 HPSEC凝胶过滤层析，并进行生化表征。通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）、Native-PAGE、酪蛋白酶谱分析和HPLC粒度排除，估计其分子量为65kDa。nh2末端氨基酸序列的前26个氨基酸残基与蘑菇蛋白酶具有较高的同源性。苯基甲基磺酰氟（PMSF）和二碘多丙基氟磷酸盐（DFP）对SPPS有较强的抑制作用，表明SPPS是丝氨酸蛋白酶家族的成员。最适pH值为9.5℃，最适温度为70℃。有趣的是，与SPTC、Flavourzyme (R) 500L和Thermolysin type X蛋白酶相比，SPPS具有最高的水解水平和催化效率。更值得注意的是，与商业蛋白酶Thermolysin X型和Flavourzyme (R) 500L相比，SPPS对有机溶剂具有较高的耐受性，并且具有相当的洗涤剂稳定性。结论SPPS具有优良的性能，具有广泛的工业应用前景。
BackgroundProteases are hydrolytic enzymes that catalyze peptide linkage cleavage reactions at the level of proteins and peptides with different degrees of specificity. This group draws the attention of industry. More than one protease in three is a serine protease. Classically, they are active at neutral to alkaline pH. The serine proteases are researched for industrial uses, especially detergents. They are the most commercially available enzyme group in the world market. Overall, fungi produced extracellular proteases, easily separated from mycelium by filtration.ResultsA new basidiomycete fungus CTM10057, a hyperproducer of a novel protease (10,500U/mL), was identified as Pleurotus sajor-caju (oyster mushroom). The enzyme, called SPPS, was purified to homogeneity by heat-treatment (80 degrees C for 20min) followed by ammonium sulfate precipitation (35-55%)-dialysis, then UNO Q-6 FPLC ion-exchange chromatography and finally HPLC-ZORBAX PSM 300 HPSEC gel filtration chromatography, and submitted to biochemical characterization assays. The molecular mass was estimated to be 65kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Native-PAGE, casein-zymography, and size exclusion by HPLC. A high homology with mushroom proteases was displayed by the first 26 amino-acid residues of the NH2-terminal aminoacid sequence. Phenylmethanesulfonyl fluoride (PMSF) and diiodopropyl fluorophosphates (DFP) strongly inhibit SPPS, revealing that it is a member of the serine-proteases family. The pH and temperature optima were 9.5 and 70 degrees C, respectively. Interestingly, SPPS possesses the most elevated hydrolysis level and catalytic efficiency in comparison with SPTC, Flavourzyme (R) 500L, and Thermolysin type X proteases. More remarkably, a high tolerance towards organic solvent tolerance was exhibited by SPPS, together with considerable detergent stability compared to the commercial proteases Thermolysin type X and Flavourzyme (R) 500L, respectively.ConclusionsThis proves the excellent proprieties characterizing SPPS, making it a potential candidate for industrial applications especially detergent formulations.