Sharma Chhavi, Timorshina Svetlana, Osmolovskiy Alexander, Misri Jyoti, Singh Rajni
Amity Institute of Microbial Technology, Amity University, Noida, India.
Department of Microbiology, Faculty of Biology, Lomonosov Moscow State University, Moscow, Russia.
Front Microbiol. 2022 Apr 25;13:882902. doi: 10.3389/fmicb.2022.882902. eCollection 2022.
Microbial keratinases exhibit a momentous role in converting keratin biowastes into exceedingly valuable protein supplements. This study reports a novel, highly stable keratinase from RSA27 for the production of pure peptides rich in essential amino acids from chicken feathers. Purified keratinase showed a specific activity of 38.73 U/mg, 2.58-fold purification, and molecular weight of 36 kDa. Kinetic studies using a chicken feather as substrate report and values of 5.69 mg/ml and 142.40 μg/ml/min, respectively, suggesting significant enzyme-substrate affinity/biocatalysis. Identification and structural-functional analysis of keratinase discovered the presence of distinct amino acid residues and their positions. Besides, keratinase possesses a high-affinity calcium-binding site (Asp, Leu, Asn, Ile, and Val) and a catalytic triad of Asp, His, and Ser, known attributes of serine protease (subtilisin family). Furthermore, a scale-up to 5 L fermenter revealed complete feather hydrolysis (94.5%) within 24 h with high activity (789 U/ml) and total amino acid of 153.97 μmol/ml. Finally, cytotoxicity evaluation of protein hydrolysate resulted in negligible cytotoxic effects (1.02%) on the mammalian hepatoblastoma cell line, signifying its potential biotechnological applications.
微生物角蛋白酶在将角蛋白生物废料转化为极具价值的蛋白质补充剂方面发挥着重要作用。本研究报道了一种来自RSA27的新型、高度稳定的角蛋白酶，用于从鸡毛中生产富含必需氨基酸的纯肽。纯化后的角蛋白酶比活性为38.73 U/mg，纯化倍数为2.58倍，分子量为36 kDa。以鸡毛为底物的动力学研究报告显示，Km和Vmax值分别为5.69 mg/ml和142.40 μg/ml/min，表明酶与底物具有显著的亲和力/生物催化作用。对角蛋白酶的鉴定和结构功能分析发现了不同氨基酸残基的存在及其位置。此外，角蛋白酶具有一个高亲和力的钙结合位点（天冬氨酸、亮氨酸、天冬酰胺、异亮氨酸和缬氨酸）以及由天冬氨酸、组氨酸和丝氨酸组成的催化三联体，这是丝氨酸蛋白酶（枯草杆菌蛋白酶家族）的已知特征。此外，扩大到5 L发酵罐后，在24小时内实现了鸡毛的完全水解（94.5%），酶活性高（789 U/ml），总氨基酸含量为153.97 μmol/ml。最后，对蛋白水解产物的细胞毒性评估显示，对哺乳动物肝癌细胞系的细胞毒性作用可忽略不计（1.02%），这表明其具有潜在的生物技术应用价值。
