N端二硫键对11家族木聚糖酶热稳定性的影响

Effect of N-terminal disulfide bridge on thermostability of family 11 xylanases

以来源于米曲霉Aspergillus oryzae的11家族常温木聚糖酶AoXyn11A为母本,将其N端替换成同一家族耐热木聚糖酶EvXyn11TS的对应片段,构建出耐热杂合木聚糖酶AEx11A。将AoXyn11A和AEx11A基因分别在毕赤酵母GS115中进行表达并分析比较温度对表达产物酶活性的影响。结果表明,AEx11A的最适温度Topt为75℃,在70℃的半衰期t1/270为197 min,较AoXyn11A(Topt=50℃,t1/270=1.0 min)显著提高。通过对AEx11A结构的同源建模及其与AoXyn11A结构的比对,发现在AEx11A的N端引入了一个二硫键(Cys5–Cys32)。利用定点突变法将其5位的半胱氨酸突变为苏氨酸(C5T),去除该二硫键,以探讨其对AEx11A热稳定性的影响。分析表明,突变酶(AEx11AC5T)的Topt由突变前的75℃降为60℃,其t1/270和t1/280也分别由197 min和25 min缩短为3.0 min和1.0 min。

A mesophilic xylanase from Aspergillus oryzae, abbreviated to AoXynllA, belongs to glycoside hydrolase family 11. Using AoXynl I A as the parent, the thermotolerant hybrid xylanase, we constructed AExl I A by substituting itsN-terminus with the corresponding region of a hyperthermostable family 11 xylanase, EvXynllxs. AoXynllA- and AExl 1A-encoding genes were expressed in Pichia pastoris GSl15 separately, and effects of temperatures on expressed products were determined and compared. The optimum temperature （Topt） of AExl 1A was 75 ℃ and its half-life at 70 ℃ （tl/270） was 197 min, improved as compared with those （Topt = 50 ℃, tl/270 = 1.0 min） of AoXynl 1A. Homology modeling of the AExl 1A＇s structure and comparison between structures of AExl 1A and AoXynl 1A revealed that one disulfide bridge （CysS-Cys32） was introduced into AExl 1A resulted from N-terminus substitution. To explore the effect of the disulfide bridge on the thermostability of AExl 1A, it was removed from AExl 1A by site-directed rnutagenesis （C5T）. Analytical results show that the Topt of the mutant AExl 1A （AExl 1AcST） dropped to 60 ℃ from 75 ℃ of AExl 1A, and its t1/270 and tl/280also decreased to 3.0 and 1.0 min from 197 and 25 min.