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

作　　者：刘柏宏[1,2] 张娟[3,2] 堵国成[4,2] 陈坚[5,2] 廖祥儒[3,2] 

机构地区：[1]食品安全与营养协同创新中心,江苏无锡214122 [2]江南大学生物工程学院,江苏无锡214122 [3]江南大学工业生物技术教育部重点实验室,江苏无锡214122 [4]江南大学糖化学与生物技术教育部重点实验室,江苏无锡214122 [5]江南大学粮食发酵工艺与技术国家工程实验室,江苏无锡214122

出　　处：《微生物学通报》2014年第8期1491-1497,共7页Microbiology China

基　　金：国家863计划项目(No.2011AA100905);国家十二五关键技术研究发展计划项目(No.2011BAK10B03);教育部长江学者和创新团队发展计划项目(No.IRT1135);江苏省自然科学基金项目(No.BK2012553)

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。[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 ℃. ]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.

关 键 词：角蛋白酶 N端 热稳定性 

分 类 号：Q936[生物学—微生物学]