Použití α-N-Acetylgalactosaminidasy jako nástroje vhodného pro syntézu komplexních oligosacharidových stimulátorů imunitního systému

Mrázek, Hynek

α-N-Acetylgalactosaminidase as a tools in the synthesis of complex oligosaccharide immune stimulators Introduction: Alpha-N-acetylgalactosaminidases are exoglycosidases specific for the hydrolysis of terminal α-linked N-acetylgalactosamine in various sugar chain. They occur widely in microorganisms, plants and animals, and have considerable potential in various industrial application. Stability and activity at high temperatures are important properties of α- N-acetylgalactosaminidases. A large screening study for extracellular α-N- acetylgalactosaminidase activity in a library of filamentous fungi (42 strains), led to the identification of the best constitutive producer, Aspergillus niger CCIM K2. The enzyme from Aspergillus niger has certain unique properties, and it was chosen for further investigations. Methods: Alpha-N-acetylgalactosaminidase from Aspergillus niger CCIM K2 was partially sequenced by Edman degradation and MALDI MS. Degenerated PCR primers were designed based on the obtained N-terminal and internal sequences. A gene fragment encoding a putative part of the α-N-acetylgalactosaminidase was amplified using cDNA prepared from Aspergillus niger CCIM K2. The full-length coding sequence of α-N- acetylgalactosaminidase was cloned into pYES-2CT vector and the recombinant protein was expressed in...

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