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Heterologous expression and characterization of processing α-glucosidase I from Aspergillus brasiliensis ATCC 9642

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Abstract

A gene for processing α-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 Glc3Man9GlcNAc2-PA and Glc3Man4-PA and the products were identified as Glc2Man9GlcNAc2-PA and Glc2Man4-PA, respectively. Saturation curves were obtained for both Glc3Man9GlcNAc2-PA and Glc3Man4-PA, and the K m values for both substrates were estimated in the micromolar range. When 1 μM Glc3Man4-PA was used as a substrate, the inhibitors kojibiose and 1-deoxynojirimycin had similar effects on the enzyme; at 20 μM concentration, both inhibitors reduced activity by 50%.

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Abbreviations

GST:

glutathione S-transferase

PA:

pyridylaminated

AbPGI:

Aspergillus brasiliensis processing α-glucosidase I

IC50 :

concentration for 50% inhibition

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Acknowledgements

We would like to thank Tomoko Fujii for her technical assistance. This study was supported by a grant-in-aid for Scientific Research (20570103 and 23570132) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. We thank Hayashibara Biochemical Laboratories Inc. for providing kojibiose and nigerose.

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Authors and Affiliations

  1. Department of Applied Biological Science, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo, 183-8509, Japan

    Takatsugu Miyazaki, Yuji Matsumoto, Kana Matsuda, Yuma Kurakata, Atsushi Nishikawa & Takashi Tonozuka

  2. Department of Chemistry and Chemical Biology, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma, 376-8515, Japan

    Ichiro Matsuo

  3. RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan

    Yukishige Ito

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  1. Takatsugu Miyazaki
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  2. Yuji Matsumoto
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  3. Kana Matsuda
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  4. Yuma Kurakata
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  5. Ichiro Matsuo
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  8. Takashi Tonozuka
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Correspondence to Takashi Tonozuka.

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Miyazaki, T., Matsumoto, Y., Matsuda, K. et al. Heterologous expression and characterization of processing α-glucosidase I from Aspergillus brasiliensis ATCC 9642. Glycoconj J 28, 563–571 (2011). https://doi.org/10.1007/s10719-011-9356-z

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  • DOI: https://doi.org/10.1007/s10719-011-9356-z

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