Molecular cloning of Mucor hiemalis endo-beta-N-acetylglucosaminidase and some properties of the recombinant enzyme |
| |
Authors: | Fujita Kiyotaka Kobayashi Kazuo Iwamatsu Akihiro Takeuchi Makoto Kumagai Hidehiko Yamamoto Kenji |
| |
Affiliation: | Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan. kfujita@lif.kyoto-u.ac.jp |
| |
Abstract: | Endo-M, endo-beta-N-acetylglucosaminidase from Mucor hiemalis, is known as a useful enzyme for the synthesis of neoglycopeptides due to its transglycosylation activity. We cloned the Endo-M gene encoding a putative 744 amino acids, which shows high identity to glycoside hydrolase family 85 endo-beta-N-acetylglucosaminidases. The gene encoding Endo-M was expressed in protease-deficient Candida boidinii with a molecular mass of 85 kDa as a monomeric form. Recombinant Endo-M could liberate both high-mannose type and biantennary complex type oligosaccharides from glycopeptides, which was same as the native enzyme. The Km and Kcat values for DNS-Man6GlcNAc2Asn were 0.51 mM and 8.25 s(-1), respectively. Recombinant Endo-M also exhibited transglycosylation activity toward high-mannose type and biantennary complex type oligosaccharides, which were transferred to alcohols, monosaccharides, oligosaccharides, and glycosides. To investigate about the catalytically essential amino acids of Endo-M, site-directed mutagenesis was performed, and it was found that mutants E177G and E177Q completely abolished the hydrolytic activity and W228R partially abolished the transglycosylation activity. |
| |
Keywords: | Endo-β-N-acetylglucosaminidase Transglycosylation Cloning N-linked oligosaccaharides Mucor hiemalis |
本文献已被 ScienceDirect PubMed 等数据库收录! |
|