脱毛碱性蛋白酶(DHAP)的饱和突变
Saturation mutagenesis of the dehairing alkaline protease
作者：黄 蓉(四川大学生命科学学院, 四川省分子生物学与生物技术重点实验室)；冯 红(四川大学生命科学学院, 四川省分子生物学与生物技术重点实验室)
Author：HUANG Rong(Key Laboratory of Molecular Biology and Biotechnology of Sichuan Province, College of Life Sciences, Sichuan University)；FENG Hong(Key Laboratory of Molecular Biology and Biotechnology of Sichuan Province, College of Life Sciences, Sichuan University)
收稿日期：2014-05-07 年卷（期）页码：2015,52(5):1177-1181
期刊名称：四川大学学报: 自然科学版
Journal Name：Journal of Sichuan University (Natural Science Edition)
关键字：脱毛碱性蛋白酶; 短小芽胞杆菌; 饱和突变; 冷适应; 热稳定性; 酶活性
Key words：Alkaline protease; Bacillus pumilus; Site directed mutagenesis; Cold adaptation; Thermostability; Protease activity
基金项目：国家863专题项目（2006AA02Z221）
中文摘要
本实验在同源建模和氨基酸序列比对的基础上, 选取脱毛碱性蛋白酶（DHAP）8个氨基酸位点进行饱和突变. 通过牛奶平板和96孔板发酵筛选， 获得了4个催化特性改良的突变体, 测序鉴定为D248F, D248S, M233L和W214K. 利用酪蛋白和合成肽(AAPL pN)作为底物, 对这些突变体进行了评价, 结果表明D248F、D248S、W214K在常温(28℃)下的酪蛋白水解活性提高; M233L在常温、高温(50℃)的酶活以及热稳定性都有增加. 同时, M233L和W214K在25℃和50℃水解AAPL pN的酶活也分别得到了不同程度的提高.
英文摘要
From Bacillus pumilus BA06 an dehairing akaline protease（DHAP） has a good dehairing function and can be applied to relevant industries. On the basis of the homolog modeling and the multiple amino acids alignments, 8 amino acid residues of DHAP were selected for saturation mutagenesis. After screening on the skim milk plates and fermentation in the 96 well plates, 4 mutants were obtained and identified as D248F, D248S, M233L and W214K by DNA sequencing. The catalytic activities of D248F, D248S and W214K towards casein were improved at 28℃. And the caseinolytic activities of M233L were also improved at 50℃, 28℃, and after treatment at 65℃ for 10 minutes. The hydrolytic activities of M233L and W214K towards AAPL pN were enhanced at 50℃ and 25℃.
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