米曲霉木聚糖酶N端引入二硫键对其热稳定性的影响

Effect of Engineering a Disulfide Bridge into the N-terminal Region of Aspergillus oryzae Xylanase on its Thermostability

为提高源自米曲霉Aspergillus oryzae 11家族木聚糖酶(AoXyn11A)的热稳定性,对其相关的氨基酸残基进行定点突变改造。通过对野生型酶AoXyn11A和一种超耐热酶EvXyn11TS的N端氨基酸残基序列进行同源比较,在野生型酶N端引入一个二硫键,获得突变酶AoXyn11AM。野生型酶和突变酶的热稳定性通过同源建模和分子动力学模拟分析评估后,其基因分别在毕赤酵母GS115中进行表达并分析温度对表达产物酶活性的影响。结果表明:突变酶的最适温度由野生型酶的55℃提高至60℃;在50℃和55℃保温30 min,突变酶保留94%和45%保温处理前的酶活性,分别较野生型酶(62.5%和1.4%)有大幅度的提高。突变酶在保留了野生酶其它优良性质的基础上,提高了热稳定性,具有潜在的工业应用价值。

Site-directed mutagenesises had been done in this work with the consideration of increasing the thermostability of a family 11 xylanase （AoXynl IA） from Aspergillus oryzae. The mutant,AoXyn11A^M,was obtained by introducing a disulfide bridge into the N-terminal region of AoXyn11A after N-terminal amino acid homology alignment of AoXyn11A with a thermostable family 11 xylanase,EuXyn11^TS. The thermostability of the wild-type enzyme and mutant were analyzed by homology modeling,and assessed by molecular dynamics （MD） simulations. The genes, AoXyn11A and AoXyn11A^M,were expressed in Pichia pastoris GSI 15, respectively. And the effects of temperatures on the thermostability of the expressed AoXyn11A and AoXyn11A^M were analyzed. The results showed the optimal temperature of AoXyn11A^M was raised to 60 ℃ from 55 ℃ of AoXyn11A;after incubated at 50 ℃ and 55 ℃ for 30 min,respectively,the mutant retained 94% and 45% of its original activity while the wild-type enzyme retained 62.5% and 1.4% of its original activity. The mutant not only preserved the major excellent properties of the wild-type enzyme,but also enhanced the thermostalfility which is potentially useful in industrial applications.