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Biotechnology and Applied Biochemistry (1998) 28, (179–188) (Printed in Great Britain)

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a-Galactosidases of Penicillium simplicissimum: production, purification and characterization of the gene encoding AGLI

Elina Luonteri¹, Edward Alatalo, Matti Siika-aho, Merja Penttilä and Maija Tenkanen

VTT Biotechnology and Food Research, P.O. Box 1501, FIN-02044 VTT, Espoo, Finland

¹ To whom correspondence should be addressed.

The nucleotide sequence data reported for the agl1 gene will appear in EMBL Nucleotide Sequence Database under the accession number AJ009956.

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.

Received 5 January 1998/24 June 1998; accepted 25 June 1998

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