嗜碱芽孢杆菌耐热耐碱内切木聚糖酶基因的密码子优化及在毕赤酵母中的表达

The Codon Optimization of Thermostable and Alkali-resisting Endo-xylanase from Bacillus halodurans S7 and Expression in Pichia pastoris

关键词:  嗜碱芽孢杆菌内切木聚糖酶  基因优化  表达  毕赤酵母

Key words:  thermostable and alkali-resisting xylanase  codon optimization  expression  Pichia pastoris

摘要:

[目的]研究极端耐热耐碱的木聚糖酶,使其能够满足造纸行业中碱性高温的苛刻环境。[方法]将一种来源于嗜碱芽孢杆菌S7的耐热耐碱内切木聚糖酶基因(xyn10A)密码子进行优化,基因合成后克隆至pHBM905BDM载体上,并转化毕赤酵母GS115菌株。[结果]木聚糖酶基因(xyn10A)优化后与原始序列比对相似性为76.70%。基因合成后克隆到pHBM905BDM载体上,并成功转化毕赤酵母GS115菌株。通过交联木聚糖底物平板水解圈法初筛及摇瓶复筛得到一株产酶量较高的菌株,且其在摇瓶发酵第10天达到最高酶活力,为495 U/mL。另外,糖基化分析表明该酶在毕赤酵母中表达时被糖基化修饰。[结论]密码子优化后的极端耐热耐碱木聚糖酶基因在毕赤酵母中成功表达。

Abstract:

[Objective] Studying the extreme heat and alkali xylanase can satisfy the harsh environment with high temperature and alkaline in papermaking industry.[Methods] A xylanase gene derived from an alkalophilic Bacillus S7 (xyn10A) is codon optimized, then cloned into pHBM905BDM vector, and transformed into Pichia pastoris strain GS115.[Results] The similarity between the optimized xylanase gene (xyn10A) and the original sequence is 76.70%.Then the gene is cloned into pHBM905BDM vector and successfully transformed into P.pastoris GS115. The transformations are screened on the RBB-xylan plate, then in the shake flask fermentation a high expression strain is obtained. The highest activity is 495 U/mL at the 10th day of the shake fermentation. In addition, glycosylation analysis shows that the enzyme is glycosylated in P.pastoris.[Conclusion] The optimized extreme heat and alkali resistance xylanase gene are successfully expressed in P.pastoris.