Expression, purification, and characterization of human intestinal maltase secreted from Pichia pastoris
| Autor: | Hee-Kyoung Kang, Young-Min Kim, Doman Kim, Eun-Seong Seo, Hwa-Ja Ryu |
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| Rok vydání: | 2011 |
| Předmět: |
Maltose
Biology biology.organism_classification Applied Microbiology and Biotechnology Molecular biology Pichia pastoris law.invention Alcohol oxidase chemistry.chemical_compound Diafiltration Endoglycosidase H chemistry Affinity chromatography Biochemistry law biology.protein Recombinant DNA Maltase Food Science Biotechnology |
| Zdroj: | Food Science and Biotechnology. 20:561-565 |
| ISSN: | 2092-6456 1226-7708 |
| DOI: | 10.1007/s10068-011-0079-5 |
| Popis: | A gene encoding human intestinal maltase (HMA) was successfully expressed in Pichia pastoris under the control of the methanol-induced alcohol oxidase (AOX1) promoter. The secreted recombinant HMA fused with a His6-tag was produced (150 U/L) and was easily purified from culture supernatants in a 3-step diafiltration, ultrafiltration, and affinity column chromatography protocol. The specific activity of the purified HMA was 16.8 U/mg. Endoglycosidase H digestion of the protein showed that the recombinant HMA was N-glycosylated. The purified HMA was maximally active at pH 6.5 and stable (≥90%) up to 65°C. The kinetic parameters Km and Vmax were 3.3±0.25 mM maltose and 61.9±2 U/mg, respectively. |
| Databáze: | OpenAIRE |
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