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

摘要:

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。

Abstract:

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, EvXyn11TS. 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 (Topt) of AEx11A was 75 ℃ and its half-life at 70 ℃ (t1/270) was 197 min, improved as compared with those (Topt = 50 ℃, t1/270 = 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 Topt of the mutant AEx11A (AEx11AC5T) dropped to 60 ℃ from 75 ℃ of AEx11A, and its t1/270 and t1/280 also decreased to 3.0 and 1.0 min from 197 and 25 min.