在大肠杆菌中表达地衣芽孢杆菌 ER-15 的角蛋白酶。
Extracellular expression of keratinase from Bacillus licheniformis ER-15 in Escherichia coli.
机构信息
Department of Microbiology, University of Delhi, New Delhi, India.
出版信息
J Agric Food Chem. 2010 Jul 28;58(14):8380-5. doi: 10.1021/jf100803g.
Keratinase (ker BL) from Bacillus licheniformis ER-15 was cloned into vector pEZZ18 for extracellular expression in Escherichia coli HB101. Recombinant keratinase was secreted with high specific activity (75 units/mg) under non-inducible conditions after 36 h at 37 degrees C and 300 rpm in a shake flask. Protein was concentrated and, subsequently, purified by ion-exchange chromatography using Q-sepharose with 95.8% yield. The recombinant keratinase was a serine protease and most active in the pH range of 8-12 and at 60 degrees C. The enzyme was stable over a wide pH range of 4-12 for 3 h. ker BL degraded bovine serum albumin, casein, azocasein, gelatin, and feather. E. coli HB101 harboring pEZZ18 ker BL2 degraded chicken feather completely within 24 h at 37 degrees C.
来自地衣芽孢杆菌 ER-15 的角蛋白酶（ker BL）被克隆到载体 pEZZ18 中，用于在大肠杆菌 HB101 中外源表达。在摇瓶中 37°C 和 300rpm 下培养 36 小时后，在非诱导条件下，重组角蛋白酶以高比活（75 单位/mg）分泌。蛋白质经过浓缩，随后通过 Q-琼脂糖离子交换层析进行纯化，收率为 95.8%。重组角蛋白酶是一种丝氨酸蛋白酶，在 pH 8-12 范围内和 60°C 时最活跃。该酶在 pH4-12 的宽范围内稳定 3 小时。ker BL 可降解牛血清白蛋白、酪蛋白、偶氮酪蛋白、明胶和羽毛。携带 pEZZ18 ker BL2 的大肠杆菌 HB101 在 37°C 下 24 小时内可完全降解鸡毛。
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