热稳定性
化学
二硫键
木聚糖酶
中层
米曲霉
同源建模
生物化学
酶
立体化学
突变体
基因
糖苷水解酶
毕赤酵母
生物
遗传学
重组DNA
细菌
作者
Siyu Gao,Junqing Wang,Minchen Wu,Cun-Duo Tang,Jing Wu
出处
期刊:PubMed
日期:2012-12-01
卷期号:28 (12): 1441-9
被引量:1
摘要
A mesophilic xylanase from Aspergillus oryzae, abbreviated to AoXyn11A, belongs to glycoside hydrolase family 11. Using AoXyn11A as the parent, the thermotolerant hybrid xylanase, we constructed AEx11A by substituting its N-terminus with the corresponding region of a hyperthermostable family 11 xylanase, EvXyn11(TS). AoXyn11A- and AEx11A-encoding genes were expressed in Pichia pastoris GS115 separately, and effects of temperatures on expressed products were determined and compared. The optimum temperature (T(opt)) of AEx11A was 75 degrees C and its half-life at 70 degrees C (t1/2(70)) was 197 min, improved as compared with those (T(opt) = 50 degrees C, t1/2(70) = 1.0 min) of AoXyn11A. Homology modeling of the AEx11A's structure and comparison between structures of AEx11A and AoXyn11A revealed that one disulfide bridge (Cys5-Cys32) was introduced into AEx11A resulted from N-terminus substitution. To explore the effect of the disulfide bridge on the thermostability of AEx11A, it was removed from AEx11A by site-directed mutagenesis (C5T). Analytical results show that the T(opt) of the mutant AEx11A (AEx11A(C5T)) dropped to 60 degrees C from 75 degrees C of AEx11A, and its t1/2(70) and t1/2(80) also decreased to 3.0 and 1.0 min from 197 and 25 min.
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