重组木聚糖酶的安全高效表达与应用研究

Study on high efficient and safe expression of recombinant xylanse and its application

以pPIC9为载体能够实现里氏木霉木聚糖酶基因(xyn2)在巴斯德毕赤酵母(Pichia pastoris)中的高效表达,但是却需要甲醇诱导,从而限制其在食品加工等领域的应用。本研究以毕赤酵母组成型启动子——GAP启动子替换pPIC9上的甲醇诱导型启动子AOX,成功地表达了木聚糖酶。在此基础上以经密码子优化后的INU信号肽替换载体上原有的α-Factor信号肽,同时根据已发表的基因序列和AOX1基因的氨基酸序列,按照毕赤酵母的酵母偏爱密码子,对毕赤酵母表达载体pPIC9上的α信号肽序列进行改造,结果表明,两种信号肽均能够引导木聚糖酶的分泌。通过添加0.01%的Triton,可以有效增强细胞膜的通透性,提高外源蛋白的分泌能力。薄层层析结果表明,木聚糖酶的酶解产物为木二糖和木三糖为主的低聚木糖及少量的木糖。

Xylanase gene xyn2 from Trichoderma reesei can be expressed efficiently in Pichia pastoris by pPIC9 vector,but it is limited to applicate in food processing for inducing expressing by methanol. Xylanase was expressesed under the controled of GAP promoter instead of AOX promoter of pPIC9 vector.The optimized INU signal peptide and modified co-Factor signal peptide according to the optimal condon of Pichia pastoris were used to replace the α-Factor signal peptide of pPIC9 vector.The result indicated that the two signal peptides can lead the secrete of xylanase. The secrete ability of foreign protein raised by the addition of 0.01% Triton which could strengthen the permeability of cellular membrance.Result of TLC showed that the enzymolysis product of xylan by the recombinant xylanase were xylobiose,xylotriose and little xylose.