Loop结构引入半胱氨酸对木聚糖酶XynASP热稳定性的影响  

Effects of introducing cysteine into the Loop structure on the thermal stability of xylanase XynASP

作　　者：陈康太 杨思文 刁梦月 王宵宵 郭双 李恩中 李同彪 CHEN Kangtai;YANG Siwen;DIAO Mengyue;WANG Xiaoxiao;GUO Shuang;LI Enzhong;LI Tongbiao(College of Biological and Food Engineering,Huanghuai University,Zhumadian 463000,China)

机构地区：[1]黄淮学院生物与食品工程学院,河南驻马店463000

出　　处：《中国酿造》2022年第10期106-112,共7页China Brewing

基　　金：河南省省级重大科技专项项目(191110110600);河南省重点研发与推广专项(科技攻关)项目(212102110274;222102110372);河南省高等学校重点科研项目(22A180022);黄淮学院博士科研项目启动基金(12011947);黄淮学院国家级科研项目培育基金(XKPY-2021003)。

摘　　要：为了探究Loop结构引入半胱氨酸对GH11家族木聚糖酶热稳定性的影响,以溶糖曲霉(Aspergillus saccharolyticus)JOP 1030-1木聚糖酶XynASP Loop结构为研究对象,通过定点突变技术,将第95位点天冬酰胺(Asn)突变成半胱氨酸(Cys),获得突变体XynN95C,并在大肠杆菌(Escherichia coli)BL21(DE3)中诱导表达。酶学性质分析结果表明,突变体XynN95C最适温度为50℃,酶活性半衰期t_(1/2)^(40℃)为38 min,相比野生型XynASP分别提高了5℃、18 min,最适pH从6.0降至5.0。另外,XynN95C的金属离子耐受性较强,经Fe^(3+)处理1 h,相对酶活性为93.9%,较野生型(65.9%)明显提高。由此得出,在Loop结构引入半胱氨酸可有效提高木聚糖酶的热稳定性,这为GH11家族木聚糖酶的热稳定性改造又提供一新思路。In order to explore the effects of introducing cysteine into the Loop structure on the thermal stability of GH11 family xylanase,using the Loop structure of xylanase XynASP from Aspergillus saccharolyticus JOP 1030-1 as the research object,the mutant XynN95C was obtained by mutating asparagine(Asn)at site 95 to cysteine(Cys)by site-directed mutagenesis,and induced expression in Escherichia coli BL21(DE3).Enzymatic property analysis results showed that the optimum temperature of mutant XynN95C was 50℃,the half-life of the enzyme activity t_(1/2)^(40℃)was 38 min,which was 5℃and 18 min higher than that of wild-type XynASP,and the optimum pH decreased from 6.0 to 5.0.In addition,the metal ion tolerance of XynN95C was stronger,the relative enzyme activity was 93.9%after treatment with Fe^(3+)for 1 h,which was significantly higher than that of wild type(65.9%).In conclusion,the introduction of cysteine into Loop structure could effectively improve the thermal stability of xylanases,which provided a new idea for the thermal stability modification of GH11 family xylanases.

关 键 词：木聚糖酶 热稳定性 定点突变 半胱氨酸 Loop结构 

分 类 号：Q814[生物学—生物工程]