Heterologous expression and characterization of processing α-glucosidase I from Aspergillus brasiliensis ATCC 9642
Heterologous expression and characterization of processing α-glucosidase I from Aspergillus brasiliensis ATCC 9642
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DOI：
10.1007/s10719-011-9356-z
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发表时间：
2011-12-01
影响因子：
3
通讯作者：
Tonozuka, Takashi
中科院分区：
文献类型：
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作者：
Miyazaki, Takatsugu;Matsumoto, Yuji;Tonozuka, Takashi
从丝状真菌巴西曲霉（Aspergillus brasiliensis，以前称为黑曲霉）ATCC 9642中克隆了一个加工α -葡萄糖苷酶I的基因，并将其融合到谷胱甘肽s -转移酶标签上。产量最高的活性构建体是一个截断突变体，它删除了疏水n端结构域的前16个残基。该融合酶可水解pyridylaminated （PA-）寡糖Glc(3)Man(9)GlcNAc(2)-PA和Glc(3)Man(4)-PA，产物分别鉴定为Glc(2)Man(9)GlcNAc(2)-PA和Glc(2)Man(4)-PA。得到了Glc(3)Man(9)GlcNAc(2)-PA和Glc(3)Man(4)-PA的饱和曲线，并在微摩尔范围内估计了两种底物的K (m)值。以1 μ M Glc(3)Man(4)-PA为底物时，抑制剂曲二糖和1-脱氧诺吉霉素对酶的影响相似；在20 μ M浓度下，两种抑制剂的活性均降低50%。
A gene for processing alpha-glucosidase I from a filamentous fungus, Aspergillus brasiliensis (formerly called Aspergillus niger) ATCC 9642 was cloned and fused to a glutathione S-transferase tag. The active construct with the highest production level was a truncation mutant deleting the first 16 residues of the hydrophobic N-terminal domain. This fusion enzyme hydrolyzed pyridylaminated (PA-) oligosaccharides Glc(3)Man(9)GlcNAc(2)-PA and Glc(3)Man(4)-PA and the products were identified as Glc(2)Man(9)GlcNAc(2)-PA and Glc(2)Man(4)-PA, respectively. Saturation curves were obtained for both Glc(3)Man(9)GlcNAc(2)-PA and Glc(3)Man(4)-PA, and the K (m) values for both substrates were estimated in the micromolar range. When 1 mu M Glc(3)Man(4)-PA was used as a substrate, the inhibitors kojibiose and 1-deoxynojirimycin had similar effects on the enzyme; at 20 mu M concentration, both inhibitors reduced activity by 50%.