Expression of Bacillus licheniformis α-amylase in Pichia pastoris without antibiotics-resistant gene and effects of glycosylation on the enzymatic thermostability
Expression of Bacillus licheniformis α-amylase in Pichia pastoris without antibiotics-resistant gene and effects of glycosylation on the enzymatic thermostability
复制标题
地衣芽孢杆菌α-淀粉酶在无耐药基因的毕赤酵母中的表达及糖基化对酶热稳定性的影响
DOI：
10.1007/s13205-019-1943-x
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发表时间：
2019
期刊：
影响因子：
--
通讯作者：
Guimin Zhang
中科院分区：
文献类型：
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作者：
Xinlin Hu;Xin Yuan;Nisha He;Tony Z. Zhuanng;Pan Wu;Guimin Zhang
耐热α-淀粉酶在工业上有着广泛的应用。地衣芽孢杆菌（Bacillus licheniformis，BLA）α-淀粉酶具有6个潜在的糖基化位点，具有良好的热稳定性和pH稳定性，酶活力高。在此，它在毕赤酵母中表达。对不含抗真菌基因的Pic-BLA酵母进行摇瓶培养，发酵上清液中淀粉酶活力达到900 U/mL。重组α-淀粉酶Pic-BLA在P. pastoris被深度糖基化，分子量（MM）增加了30%。通过内切糖苷酶H（Endo H）的去糖基化处理减少了Pic-BLA的MM。热稳定性分析表明，Pic-BLA和去糖基化Pic-BLA的耐热性相似。为了消除Endo H的额外影响，还在大肠杆菌中表达了BLA，得到非糖基化的Eco-BLA。非糖基化Eco-BLA和糖基化Pic-BLA的热稳定性比较研究显示无明显差异。推测糖基化对α-淀粉酶BLA的热稳定性影响不大。
Thermostable α-amylases are widely used in industry. The α-amylase fromBacillus licheniformis(BLA) with six potential glycosylation sites possessed excellent thermal and pH stability and high activity. Here, it was expressed inPichia pastoris. The Pic-BLA-producing yeast without any antibiotics-resistant gene was cultivated in flasks and the amylase activity in fermentation supernatant reached 900 U/mL. The recombinant α-amylase Pic-BLA produced inP. pastoriswas deeply glycosylated with 30% increase in molecular mass (MM). The deglycosylation treatment by Endoglycosidase H (Endo H) reduced the MM of Pic-BLA. Thermostability analysis showed that Pic-BLA and deglycosylated Pic-BLA were similar in heat tolerance. In order to eliminate the extra impact of Endo H, the BLA was also expressed inEscherichia colito get non-glycosylated Eco-BLA. A comparative study between non-glycosylated Eco-BLA and glycosylated Pic-BLA showed no obvious difference in thermostability. It is speculated that the glycosylation has little effect on the thermostability of α-amylase BLA.