突变木聚糖酶基因xynⅡC~*在毕赤酵母中的表达及酶学性质研究

Engineering of Multiple Arginines into the Ser/Thr Surface of Xylanase Xyn Ⅱ Increases the Thermo-tolerance

对来源于宇佐美曲霉的高比活木聚糖酶xyn Ⅱ基因进行热稳定性改造,获得突变木聚糖酶基因xynI-IC-*ST4、xynⅡC-*ST5,将其分别克隆到毕赤酵母表达载体pPIC9K上,得到重组质粒,经SalI线性化后分别转化毕赤酵母GS115,xynⅡC*基因通过同源重组被整合到毕赤酵母染色体上,并处于酵母α因子的下游,经筛选获得阳性重组菌。获得的2个突变酶在热稳定性上比野生型酶都有不同程度的提高。突变酶ST4和突变酶ST5使酶的最适温度分别由原酶的50℃提高为52℃和55℃。55℃保温15min,ST4、ST5的残留酶活性由原酶的20%提高为65%和75%;保温1h,ST4、ST5的残留酶活性由原酶的15%提高为50%和65%。

The endo-1,4-xylanase Xyn Ⅱ gene from Aspergillus usamii E001 was modified to improve its thermostability. The mutant gene xynIIC^＊ was cloned into the Pichia pastoris expression vector, pPIC9K. The recombinant plasmid pPIC9K-xynIIC^＊ was linearized with Sal I and then transformed into Pichia pastoris GS1 15. The xynIIC^＊ gene was in frame integrated into the Pichia genome through homologous recombination. That led to an increase in optimal temperature of the enzymes by 2℃ and 5℃ for the ST4 and ST5, respectively. The modified enzymes ST4 and ST5 showed 65 % and 75 % of maximal activities after incubated for 15 min at 55 ℃ compared to only 20 % activity for wild-type enzyme. After incubated for 1 h at 55 ℃ , ST4 and ST5 showed 50 % and 65 % of maximal activity compared to only 15 % activity for wild-type enzyme. Having the good properties of Xyn 11 , the mutants with higher thermostability are potentially useful in industrial applications.