地衣芽胞杆菌来源角蛋白酶N端对活力及其热稳定性的影响

Effects of the N-terminus of keratinase from Bacillus licheniformis on activity and thermostability

摘要:

【目的】通过对一株地衣芽孢杆菌来源的角蛋白酶N端进行分子改造，研究其对角蛋白酶活力和热稳定性的影响，进而提高角蛋白酶的热稳定性。【方法】将角蛋白酶N端前5个氨基酸进行分段缺失，并通过序列比对将N端的前5个氨基酸替换为来源于Thermoactinomyces vulgaris的嗜热蛋白酶的N端，将野生型和突变体角蛋白酶基因在枯草芽孢杆菌WB600中进行表达，并对重组酶进行纯化与酶学性质研究。【结果】角蛋白酶N端不同长度的缺失大幅度地降低了角蛋白酶的活力，其中缺失前5个氨基酸完全丧失了酶活力。将角蛋白酶N端前5个氨基酸替换为嗜热蛋白酶N端前12个氨基酸，虽然降低了近70%的活力，但是却增加了角蛋白酶的热稳定性，60 °C条件下的半衰期t1/2由原来的9 min提高到20 min。【结论】角蛋白酶的N端对其酶活力具有较大的影响，与嗜热蛋白酶来源的N端进行替换可以有效提高角蛋白酶的热稳定性。

Abstract:

[Objective] In order to functional analysis the N-terminus of keratinase from Bacillus licheniformis and its effect on the activity and thermostability of keratinase, the N-terminus of keratinase was reconstructed by molecular modification. [Methods] The N-terminus residues of keratinase were deleted individually and replaced by an N-terminus of thermitase from Thermoactinomyces vulgaris through sequence alignment. The recombinant keratinases of wild-type and mutants were production in Bacillus subtilis WB600 and then purified for characterization. [Results] Deletion of N-terminus of keratinase resulted in enzyme activity decreased prominently, when deficiency of five residues the activity of enzyme was completely abolished. Although replacement of N-terminus from thermitase decreased the activity of keratinase, the thermostability of mutant was enhanced compared with wild-type. The half-live of thermal inactivation (t1/2) was enhanced from 9 to 20 min at 60 °C. [Conclusion] The N-terminus of keratinase was important for enzyme activity, replacement of N-terminus between keratinase and thermitase could efficient enhanced thermostability of keratinase.