Matkawala Fatema, Nighojkar Sadhana, Kumar Anil, Nighojkar Anand
School of Biotechnology, Devi Ahilya University, Khandwa Road, Indore, 452001, India.
Mata Gujri College of Professional Studies, A.B. Road, Indore, 452001, India.
Heliyon. 2019 Aug 9;5(8):e02246. doi: 10.1016/j.heliyon.2019.e02246. eCollection 2019 Aug.
Alkaline proteases have several industrial applications. In the present study, newly isolated sp. N1 was screened as hyper producer of serine protease A multimeric protease of the fungus was purified to homogeneity till 96.78 fold purification with 22.51% recovery. The homogeneity of purified enzyme was checked by native PAGE and its molecular weight was found to be 198.03 kDa by MALDI-TOF. On SDS-PAGE analysis, enzyme was found to be a hetero oligomer of 17.66 kDa and 20.89 kDa subunits. The purified enzyme showed maximum activity with casein as substrate at 60 °C and pH 8.5. The K and V values were found to be 0.015 mg/ml and 454.45 U/ml, respectively. The enzyme was completely inhibited by PMSF, while the activity was 40% enhanced using β-mercaptoethanol, suggesting that it is a thiol-dependent serine protease. The purified protease was active over an alkaline pH range from 7 to 12 and temperatures from 20 °C to 60 °C. The enzyme exhibited excellent stability, almost 100% towards organic solvents such as toluene, benzene and hexane, surfactants such as Triton X-100, Tween-20, Tween-80 and SDS, as well as commercial detergents. The significant properties of purified enzyme assure that it could be a potential candidate for commercial purposes.
碱性蛋白酶有多种工业应用。在本研究中，新分离出的菌株N1被筛选为丝氨酸蛋白酶的高产菌株。该真菌的一种多聚体蛋白酶被纯化至同质，纯化倍数达96.78倍，回收率为22.51%。通过非变性聚丙烯酰胺凝胶电泳（native PAGE）检测纯化酶的同质性，通过基质辅助激光解吸电离飞行时间质谱（MALDI-TOF）测定其分子量为198.03 kDa。在十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）分析中，该酶被发现是由17.66 kDa和20.89 kDa亚基组成的杂合寡聚体。纯化后的酶以酪蛋白为底物时，在60℃和pH 8.5条件下表现出最大活性。其米氏常数（K）和最大反应速度（V）分别为0.015 mg/ml和454.45 U/ml。该酶被苯甲基磺酰氟（PMSF）完全抑制，而使用β-巯基乙醇时活性增强40%，这表明它是一种硫醇依赖性丝氨酸蛋白酶。纯化后的蛋白酶在pH值7至12的碱性范围内以及20℃至60℃的温度范围内均有活性。该酶对甲苯、苯和己烷等有机溶剂、吐温X-100、吐温-20、吐温-80和十二烷基硫酸钠（SDS）等表面活性剂以及商用洗涤剂表现出优异的稳定性，几乎达到100%。纯化酶的这些显著特性确保它可能成为具有商业用途的潜在候选物。
