贝莱斯芽孢杆菌角蛋白酶的克隆表达、纯化及酶学性质

Gene Cloning, Expression, Purification, and Enzymatic Properties Study of Keratinase from Bacillus velezensis

摘要:

为了获得更好的羽毛粉降解酶，作者从贝莱斯芽孢杆菌基因组中筛选到一个角蛋白酶基因（baker1），对其克隆并在大肠杆菌BL21（DE3）中异源表达，最后对重组角蛋白酶（BaKer1）进行了分离纯化、表征及应用研究。结果表明，BaKer1的最适反应pH为9.5，最适反应温度为55 ℃，在中温和碱性条件下具有较好的稳定性。1 mmol/L Ca²⁺对BaKer1有显著促进作用，5 mmol/L Mn²⁺对BaKer1有轻微抑制作用，其他金属离子对BaKer1影响不大。PMSF对BaKer1的活性具有显著抑制作用，表明BaKer1是典型的丝氨酸蛋白酶。分别以羽毛粉、偶氮酪蛋白、酪蛋白为底物测定了BaKer1的动力学参数，K_m值分别为5.06、1.09、1.39 mmol/L，k_cat/K_m值分别为1 058.24、2 536.54、3 733.79 s·mmol/L。BaKer1处理羽毛粉，能达到酸水解效果的 33.51%，说明它在羽毛粉降解过程中具有潜在的应用价值。

Abstract:

In order to obtain a better feather meal degrading enzyme, a keratinase gene (baker1) was screened from the genome of Bacillus velezensis SYBC H47. Subsequently, baker1 was cloned and its heterologous expression was achieved in Escherichia coli BL21 (DE3). Finally, the recombinant keratinase (BaKer1) was isolated and purified, characterized, and was studied for its practical application. The results showed that the optimal pH and temperature for BaKer1 were 9.5 and 55 ℃, respectively, and it exhibited good stability under moderately-temperature and alkaline conditions. BaKer1 was significantly activated by 1 mmol/L Ca²⁺ ion, slightly inhibited by 5 mmol/L Mn²⁺ ion, and insignificantly affected by other metal ions. BaKer1 had significant inhibitory effect on the activity of PMSF, indicating that BaKer1 is a typical serine protease. The kinetic parameters of BaKer1 were determined using feather meal, azocasein and casein as substrates. The K_m values were 5.06, 1.09, 1.39 mmol/L, respectively, and their k_cat/K_m values were 1 058.24, 2 536.54, 3 733.79 s·mmol/L, respectively. An acid hydrolysis efficiency of 33.51% could be achieved during the degradation of feather meal by BaKer1, indicating its potential application value in the degradation of feather meal.