摘要
糖苷水解酶第七家族(GH7)的外切葡聚糖苷酶(exo-1,4-β-D-glucanase),又称为纤维素生物降解酶(CBHs),在其催化中心活性通道的顶端有1个环状结构,嗜热毁丝菌(Myceliophthora thermophila)第七家族外切葡聚糖苷酶CBH1的环状结构(LS,Loop Structure)由9个氨基酸组成,即G245-Y253,但其对酶特性影响机制尚不清楚。本文构建环状结构缺失突变体CBH1-DM﹝Δ(G245-Y253)﹞以研究该结构对酶特性的影响。野生酶WT与突变酶CBH1-DM通过毕赤酵母表达并纯化,将纯化产物进行酶学性质的测定及分析。研究结果显示野生酶和突变酶的最适反应温度和最适p H值均相同,分别为50℃和5.0。CBH1-DM的比活力为2.42 U/mg,是WT(1.17 U/mg)的2.1倍。野生酶与突变酶在70℃处理1 h后,野生酶仅剩余35%的酶活力,而突变酶剩余70%的酶活力,具有较好的热稳定性。研究表明环状结构对酶的活性及热稳定性具有显著影响。
The exo-1,4-β-D-glucanases,also called cellobiohydrolases（ CBHs）,in glycoside hydrolase family 7（ GH7） have a loop structure,which is located on the top of the active site tunnel at the catalytic centre. The loop structure of the GH7 CBHs from the Myceliophthora thermophila consists of 9 amino acids,G245 to Y253. In this paper,we employed the deletion mutant CBH1-DM（ Δ（ G245-Y253） to identify the function of the loop structure acting on cellulose hydrolysis. Wild type and mutant enzymes were expressed in Pichia pastoris. The enzymatic properties were determined. The optimal reaction temperature and p H values of wild type and mutant enzymes were 50℃ and 5,respectively. The specific activity of the mutant enzyme was 2.42 U/mg,which was 2.1-fold higher than that of the wild type（1.17 U/mg）.After the wild type and mutant enzymes were treated at 70℃for 1 hour,the remained activity of wild type enzyme was 35%,the remained activity of CBH1-DM was 70%,the mutant enzyme has better thermostability.This result revealed that the loop structure negatively affects the enzyme activity and thermostability.
作者
杨志芹
高金鹏
郭秀娜
李多川
YANG Zhi-qin;GAO Jin-peng;GUO Xiu-na;LI Duo-chuan(Department of Mycology,College of Plant Protection,Shandong Agricultural University,Tai＇ an 271018,China)
出处
《菌物研究》
CAS
2018年第2期95-101,共7页
Journal of Fungal Research
基金
国家科技支撑计划课题(2015BAD15B05)
国家863计划课题(2012AA10180402)
国家自然科学基金项目(31571949)
关键词
外切葡聚糖苷酶
酶活力
敲除
环状结构
热稳定性
cellobiohydrolase
enzyme activity
deletion
loop structure
thermostability
