Facultad de Farmacia y Bioquímica, Laboratorio de Biología Molecular, Universidad Nacional Mayor de San Marcos, Lima, Peru.
Dept. of Biochemical Engineering, The Advanced Centre for Biochemical Engineering, University College London, London, WC1E 6BT, U.K.
Biotechnol Prog. 2019 Jan;35(1):e2728. doi: 10.1002/btpr.2728. Epub 2018 Oct 22.
Proteases are the most important group of industrial enzymes and they can be used in several fields including biorefineries for the valorization of industrial byproducts. In this study, we purified and characterized novel extremophilic proteases produced by a Pseudomonas aeruginosa strain isolated from Mauritia flexuosa palm swamps soil samples in Peruvian Amazon. In addition, we tested their ability to hydrolyze distillers dried grains with solubles (DDGS) protein. Three alkaline and thermophilic serine proteases named EI, EII, and EIII with molecular weight of 35, 40, and 55 kDa, respectively, were purified. EI and EIII were strongly inhibited by EDTA and Pefabloc being classified as serine-metalloproteases, while EII was completely inhibited only by Pefabloc being classified as a serine protease. In addition, EI and EII exhibited highest enzymatic activity at pH 8, while EIII at pH 11 maintaining almost 100% of it at pH 12. All the enzymes demonstrated optimum activity at 60°C. Enzymatic activity of EI was strongly stimulated in presence of Mn (6.9-fold), EII was stimulated by Mn (3.7-fold), while EIII was slightly stimulated by Zn , Ca , and Mg . DDGS protein hydrolysis using purified Pseudomonas aeruginosa M211 proteases demonstrated that, based on glycine released, EIII presented the highest proteolytic activity toward DDGS. This enzyme enabled the release 63% of the total glycine content in wheat DDGS protein, 2.2-fold higher that when using the commercial Pronase®. Overall, our results indicate that this novel extremopreoteases have a great potential to be applied in DDGS hydrolysis. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2728, 2019.
蛋白酶是最重要的工业酶类群之一，它们可应用于多个领域，包括生物炼制厂对工业副产物的增值利用。在本研究中，我们从秘鲁亚马逊地区 Mauritia flexuosa 棕榈沼泽土壤样本中分离出一株铜绿假单胞菌，对其产生的新型极端耐热蛋白酶进行了纯化和特性分析。此外，我们还测试了它们水解酒糟蛋白的能力。三种碱性和嗜热丝氨酸蛋白酶，分别命名为 EI、EII 和 EIII，分子量分别为 35、40 和 55 kDa。EI 和 EIII 被 EDTA 和 Pefabloc 强烈抑制，被归类为丝氨酸金属蛋白酶，而 EII 仅被 Pefabloc 完全抑制，被归类为丝氨酸蛋白酶。此外，EI 和 EII 在 pH 8 时表现出最高的酶活性，而 EIII 在 pH 11 时保持近 100%的活性，在 pH 12 时也有一定的活性。所有酶在 60°C 时表现出最佳的酶活性。Mn 强烈刺激 EI 的酶活性（提高 6.9 倍），Mn 刺激 EII 的酶活性（提高 3.7 倍），而 Zn、Ca 和 Mg 略微刺激 EIII 的酶活性。用纯化的铜绿假单胞菌 M211 蛋白酶水解酒糟发现，根据释放的甘氨酸计算，EIII 对酒糟具有最高的蛋白水解活性。与使用商业 Pronase®相比，该酶可使小麦酒糟蛋白中总甘氨酸含量的 63%释放出来，提高了 2.2 倍。总体而言，我们的研究结果表明，这些新型极端蛋白酶在酒糟水解中具有很大的应用潜力。© 2018 美国化学工程师协会生物科技进展，35:e2728，2019 年。
