alpha-Galactosidases of Penicillium simplicissimum: production, purification and characterization of the gene encoding AGLI.
Production of extracellular a-galactosidases by the filamentous fungus Penicillium simplicissimum (previously P. janthinellum) VTT-D-78090 was studied on different carbon sources. Steam-exploded oat husks were chosen as the best carbon source for enzyme production. Three a-galactosidases (AGL) were purified from the culture filtrate using ion-exchange chromatography, hydrophobic interaction chromatography and gel filtration. The isoelectric points of AGLI, AGLII and AGLIII were 5.2, 4.4 and 7.0, and the molecular masses as determined by SDS/PAGE were 61, 84 and 61 kDa, respectively. All enzymes were glycosylated. The optimum pH for the activity of AGLI and AGLIII was between 3.0 and 4.5 and that of AGLII was between 4.0 and 5.0. AGLII was more stable and more resistant to product inhibition by galactose than the other two enzymes. AGLI and AGLIII were also inhibited by p-nitrophenol-a-D-galactopyranoside, the substrate used for enzyme activity assay. The gene encoding AGLI was cloned and sequenced. The gene, agl1, encodes 435 amino acids including the signal sequence. It showed similarity with the other a-galactosidases belonging to the glycosyl hydrolase family 27. The N-terminal amino acid sequence of AGLIII was also similar to the sequences of other members of family 27, whereas the N-terminus of AGLII was completely different from the sequences of other reported hydrolases.
Luonteri
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VTT Biotechnology and Food Research, P.O. Box 1501, FIN-02044 VTT, Espoo, Finland.
Alatalo
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Siika-Aho
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Penttilä
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Tenkanen
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D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D003001:Cloning, Molecular; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry; D005690:Galactose / Q000494:pharmacology; D006026:Glycoside Hydrolases / Q000737:chemistry; D007527:Isoenzymes / Q000737:chemistry; D007700:Kinetics; D008361:Mannosidases / Q000737:chemistry; D008969:Molecular Sequence Data; D010407:Penicillium / Q000201:enzymology*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D000519:alpha-Galactosidase / Q000737:chemistry*; D044902:beta-Mannosidase
D001426:Bacterial Proteins; D005656:Fungal Proteins; D007527:Isoenzymes; D006026:Glycoside Hydrolases; D008361:Mannosidases; C115462:alpha-galactosidase I; D000519:alpha-Galactosidase; D044902:beta-Mannosidase; D005690:Galactose