XXII. BIOCHEMICKÝ ZJAZD - Jesseniova lekárska fakulta

Lectures
Fungal ɑ-N-aCETYLGalaCTOSaMINIDaSE frOM aSPergILLus niger:
CLONING aND EXPrESSION IN YEaST
H.Mrázek 1,2 , L. Weignerová 2 , D. Manglová 1,2 , D. Kavan 1,2 , V. Křen 2 and K. Bezouška 2
1
Department of Biochemistry, Faculty of Science, Charles University in Prague, Prague,
Czech Republic, 2 Institute of Microbiology v.v.i., Academy of Sciences
of Czech Republic, Prague, Czech Republic
Alpha-N-acetylgalactosaminidase is an exoglycosidase specific for the hydrolysis of terminal
ɑ-linked N-acetylgalactosamine in various sugar chain. A large screening study of extracellular
ɑ-N-acetylgalactosaminidase activity of a library of filamentous fungi (42 strains), led
to the identification of the best constitutive producer Aspergillus niger CCIM K2.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 ɑ-N-acetylgalactosaminidases. This enzyme from Aspergillus niger has certain unique
properties, and it was thus interesting to clone it for further structural investigations.
Alpha-N-acetylgalactosaminidase from Aspergillus niger CCIM K2 was partially sequenced
by Edman degradation and MALDI MS. A gene fragment encoding a putative part of the
ɑ-N-acetylgalactosaminidase was amplified using cDNA prepared from Aspergillus niger
CCIM K2. Degenerated PCR primers were designed according to amino acids found in ɑ-Nacetylgalactosaminidase
from Aspergillus niger CCIM K2. The full-length coding sequence
of ɑ-N-acetylgalactosaminidase was cloned into pPICZɑ and the recombinant protein was
expressed in yeast. The cloned DNA consists of 1450 base pair, and the deduced amino acid
sequence (480 amino acid residues with molecular mass 54,814kDa) is almost identical to that
of purified enzymes (as determined by SDS-PAGE). Comparison of this amino acid sequence
with the GenBank database revealed significant homology (96% identity) with sequence of
ɑ-galactosidase (EC3.2.1.22) from Aspergillus niger. Analysis of the identified sequences
shows that ɑ-N-acetylgalactosaminidase is composed of three domains.
The ɑ-N-acetylgalactosaminidase from Aspergillus niger CCIM K2 was expressed in yeast. In
further experiments we would like to express the individual domains of this complex enzyme,
and to evaluate, if there is an active carbohydrate-binding (lectin) domain within this enzyme.
Acknowledgements: This work was supported, by Grant agency of Charles University in
Prague (19309) and the Institutional Research Concept for the Institute of Microbiology
(AVOZ5020051), and grants from the Czech Science Foundations
(203/05/0172, 204/06/0771, I)
XXII. Biochemistry Congress, Martin
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