| 玉米半胱氨酸蛋白酶的原核表达及酶学性质表征 |
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| 引用本文： | 刘回民,陈方奇,郑明珠,程国栋,詹冬玲,刘景圣.玉米半胱氨酸蛋白酶的原核表达及酶学性质表征[J].吉林大学学报(理学版),2015,53(5):1054-1059. |
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| 作者姓名： | 刘回民 陈方奇 郑明珠 程国栋 詹冬玲 刘景圣 |
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| 作者单位： | 1. 吉林农业大学 生命科学学院, 长春 130118； 2. 吉林农业大学 小麦和玉米深加工国家工程实验室, 长春 130118；3. 吉林农业大学 食品科学与工程学院,
长春 130118 |
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| 摘 要： | 以玉米基因组DNA为模板扩增获得玉米半胱氨酸蛋白酶基因(zmCP1),先将其克隆至pET-28a(+)原核表达载体中,再转化至大肠杆菌BL21(DE3)中.对重组酶进行诱导表达后经聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白免疫印迹(Western blotting)鉴定,经Ni柱纯化后其质量分数大于95%.利用二肽荧光底物(R-AMC)和四肽荧光底物(LAFR-AMC)进行酶反应动力学实验,证明该酶对小底物R-AMC具有更好的亲和力和催化活性.重组酶的酶学性质研究表明,该酶最适温度为55℃,最适pH值为6.0,在90℃下的半衰期为39.82min.
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| 关 键 词： | 玉米 半胱氨酸蛋白酶 原核表达 酶学性质 |
| 收稿时间： | 2014-12-09 |
Prokaryotic Expression and Characterization of Cysteine Protease from Zea mays |
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| LIU Huimin,CHEN Fangqi,ZHENG Mingzhu,CHENG Guodong,ZHAN Dongling,LIU Jingsheng.Prokaryotic Expression and Characterization of Cysteine Protease from Zea mays[J].Journal of Jilin University: Sci Ed,2015,53(5):1054-1059. |
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| Authors: | LIU Huimin CHEN Fangqi ZHENG Mingzhu CHENG Guodong ZHAN Dongling LIU Jingsheng |
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| Institution: | 1. College of Life Science, Jilin Agricultural University, Changchun 130118, China; 2. National Engineering Laboratory for Wheat and Corn Deep Processing, Jilin Agricultural University, Changchun 130118, China;3. College of Food Science and Engineering, Jilin Agricultural University, Changchun 130118, China |
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| Abstract: | Cysteine protease from Zea mays (zmCP1) was cloned by PCR with corn genome DNA as template, and ligated to pET28a(+), expressed in E.coli BL21(DE3). The recombinant enzyme was identified by SDS PAGE and Western blotting, finally purified through Ni chelating affinity chromatography. The purity can reach 95%. The properties characterization of zmCP1 showed that the optimal temperature was 55 ℃, and the optimal pH was 6.0, the half time was 39.82 min at 90 ℃. The enzyme kinetics researches with R AMC and LAFR-AMC as substrate show that zmCP1 has better affinity and catalytic activity to small substrate. |
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| Keywords: | Zea mays cysteine protease prokaryotic expression enzyme properties |
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