α-Galactosidases of Penicillium simplicissimum: Production, purification and characterization of the gene encoding AGLI

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

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Abstract

Production of extracellular α-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 α-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-α-D-galactopyranoside, the substrate used for enzyme activity assay. The gene encoding AGLI was cloned and sequenced. The gene, agII, encodes 435 amino acids including the signal sequence. It showed similarity with the other α-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.

Original languageEnglish
Pages (from-to)179-188
Number of pages10
JournalBiotechnology and Applied Biochemistry
Volume28
Issue number2
Publication statusPublished - 26 Oct 1998
MoE publication typeA1 Journal article-refereed

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Galactosidases
Gene encoding
Penicillium
Purification
Hydrolases
Enzymes
Galactose
Hydrophobic chromatography
Amino acids
Carbon
Genes
Enzyme inhibition
Amino Acids
Ion Exchange Chromatography
Steam
Isoelectric Point
Enzyme Assays
Enzyme activity
Molecular mass
Protein Sorting Signals

Cite this

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title = "α-Galactosidases of Penicillium simplicissimum: Production, purification and characterization of the gene encoding AGLI",
abstract = "Production of extracellular α-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 α-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-α-D-galactopyranoside, the substrate used for enzyme activity assay. The gene encoding AGLI was cloned and sequenced. The gene, agII, encodes 435 amino acids including the signal sequence. It showed similarity with the other α-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.",
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α-Galactosidases of Penicillium simplicissimum : Production, purification and characterization of the gene encoding AGLI. / Luonteri, Elina; Alatalo, Edward; Siika-aho, Matti; Penttilä, Merja; Tenkanen, Maija.

In: Biotechnology and Applied Biochemistry, Vol. 28, No. 2, 26.10.1998, p. 179-188.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - α-Galactosidases of Penicillium simplicissimum

T2 - Production, purification and characterization of the gene encoding AGLI

AU - Luonteri, Elina

AU - Alatalo, Edward

AU - Siika-aho, Matti

AU - Penttilä, Merja

AU - Tenkanen, Maija

PY - 1998/10/26

Y1 - 1998/10/26

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AB - Production of extracellular α-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 α-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-α-D-galactopyranoside, the substrate used for enzyme activity assay. The gene encoding AGLI was cloned and sequenced. The gene, agII, encodes 435 amino acids including the signal sequence. It showed similarity with the other α-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.

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