Improving expression of thermostable trehalase from Myceliophthora sepedonium in Aspergillus niger mediated by the CRISPR/Cas9 tool and its purification, characterization
Improving expression of thermostable trehalase from Myceliophthora sepedonium in Aspergillus niger mediated by the CRISPR/Cas9 tool and its purification, characterization
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CRISPR/Cas9工具介导的提高黑曲霉中毁丝霉耐热海藻糖酶的表达及其纯化、表征
DOI：
10.1016/j.pep.2019.105482
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发表时间：
2020-01-01
影响因子：
1.6
通讯作者：
Pan, Li
中科院分区：
文献类型：
--
作者：
Dong, Liangbo;Yu, Dou;Pan, Li
海藻糖酶催化一个海藻糖分子转化为两个葡萄糖分子。采用传统同源重组法在黑曲霉中表达了嗜热真菌霉霉的海藻糖酶TreM，其海藻糖酶活性为406.44 U/mL。利用CRISPR/Cas9工具介导的多拷贝敲入表达策略，提高了黑曲霉TreM海藻化酶的产量，在低背景分泌蛋白的情况下，TreM海藻化酶的产量高达1943.06 U/mL，是传统方法的4.8倍。当葡萄糖浓度为1.5%时，摇摇发酵上清液重组海藻糖酶活性最高，达到4268.29 U/mL。经凝胶过滤层析纯化后，重组TreM的比活性为679.09 U/mg。重组TreM在pH 5.6和60℃条件下活性最佳，在45 ~ 50℃和pH 4.0 ~ 7.5条件下稳定性较好。重组TreM的活性被Co2+ (1,5 mM)、Cu2+ (1 mM)、Mn2+ (1,5 mM)和ATP （5 mM）显著增强，被Cu2+ (10 mM)、EDTA （10 mM）和SDS （10 mM）显著抑制。
Trehalase catalyzes the conversion of one molecule of trehalose into two glucose molecules. The trehalase TreM from thermophilic fungus Myceliophthora sepedonium was expressed in Aspergillus niger via traditional homologous recombination with trehalase activity of 406.44 U/mL. The multi-copy knock-in expression strategy mediated by the CRISPR/Cas9 tool was used to improve the production of the TreM trehalase in Aspergillus niger, which was up to 1943.06 U/mL with a low-background of secreted proteins, 4.8-fold than the transformant obtained via the traditional method. The highest recombinant trehalase activity of the shake fermentation supernatant achieved 4268.29 U/mL when 1.5% glucose was added. Activity assaying showed that the recombinant TreM possessed a specific activity of 679.09 U/mg after gel filtration chromatography purification. The recombinant TreM displayed optimal activity at pH 5.6 and 60 degrees C and exhibited prominent stability under the conditions of 45-50 degrees C and pH 4.0-7.5. The activity of recombinant TreM was strongly enhanced by Co2+ (1, 5 mM), Cu2+ (1 mM), Mn2+ (1, 5 mM) and ATP (5 mM), and was greatly inhibited by Cu2+ (10 mM), EDTA (10 mM) and SDS (10 mM).