Three alfa-galactosidase genes of Trichoderma reesei cloned by expression in yeast

Emilio Margolles-Clark, Maija Tenkanen, Elina Luonteri, Merja Penttilä

Research output: Contribution to journalArticleScientificpeer-review

72 Citations (Scopus)

Abstract

Three alpha-galactosidase genes, agl1, agl2 and agl3, were isolated from a cDNA expression library of Trichoderma reesei RutC-30 constructed in the yeast Saccharomyces cerevisiae by screening the library on plates containing the substrate 5-bromo-4-chloro-3-indolyl-alpha-D-galactopyranoside. The genes agl1, agl2 and agl3 encode 444, 746 and 624 amino acids, respectively, including the signal sequences. The deduced amino acid sequences of AGLI and AGLIII showed similarity with the alpha-galactosidases of plant, animal, yeast and filamentous fungal origin classified into family 27 of glycosyl hydrolases whereas the deduced amino acid sequence of AGLII showed similarity with the bacterial alpha-galactosidases of family 36. The enzymes produced by yeast were analysed for enzymatic activity against different substrates. AGLI, AGLII and AGLIII were able to hydrolyse the synthetic substrate p-nitrophenyl-alpha-D-galactopyranoside and the small galactose-containing oligosaccharides, melibiose and raffinose. They liberated galactose from polymeric galacto(gluco)mannan with different efficiencies. The action of AGLI towards polymeric substrates was enhanced by the presence of the endo-1,4-beta-mannanase of T. reesei. AGLII and AGLIII showed synergy in galacto(gluco)mannan hydrolysis with the endo-1,4-beta-mannanase of T. reesei and a beta-mannosidase of Aspergillus niger. The calculated molecular mass and the hydrolytic properties of AGLI indicate that it corresponds to the alpha-galactosidase previously purified from T. reesei.
Original languageEnglish
Pages (from-to)104-111
JournalEuropean Journal of Biochemistry
Volume240
Issue number1
DOIs
Publication statusPublished - 1996
MoE publication typeA1 Journal article-refereed

Fingerprint

alpha-Galactosidase
Trichoderma
Yeast
Genes
Yeasts
Galactose
Mannans
Substrates
Amino Acids
Amino Acid Sequence
beta-Mannosidase
Melibiose
Raffinose
Aspergillus niger
Aspergillus
Molecular mass
Hydrolases
Protein Sorting Signals
Oligosaccharides
Gene Library

Cite this

Margolles-Clark, Emilio ; Tenkanen, Maija ; Luonteri, Elina ; Penttilä, Merja. / Three alfa-galactosidase genes of Trichoderma reesei cloned by expression in yeast. In: European Journal of Biochemistry. 1996 ; Vol. 240, No. 1. pp. 104-111.
@article{c24622f8c5fb49889782ee4200ca6d1c,
title = "Three alfa-galactosidase genes of Trichoderma reesei cloned by expression in yeast",
abstract = "Three alpha-galactosidase genes, agl1, agl2 and agl3, were isolated from a cDNA expression library of Trichoderma reesei RutC-30 constructed in the yeast Saccharomyces cerevisiae by screening the library on plates containing the substrate 5-bromo-4-chloro-3-indolyl-alpha-D-galactopyranoside. The genes agl1, agl2 and agl3 encode 444, 746 and 624 amino acids, respectively, including the signal sequences. The deduced amino acid sequences of AGLI and AGLIII showed similarity with the alpha-galactosidases of plant, animal, yeast and filamentous fungal origin classified into family 27 of glycosyl hydrolases whereas the deduced amino acid sequence of AGLII showed similarity with the bacterial alpha-galactosidases of family 36. The enzymes produced by yeast were analysed for enzymatic activity against different substrates. AGLI, AGLII and AGLIII were able to hydrolyse the synthetic substrate p-nitrophenyl-alpha-D-galactopyranoside and the small galactose-containing oligosaccharides, melibiose and raffinose. They liberated galactose from polymeric galacto(gluco)mannan with different efficiencies. The action of AGLI towards polymeric substrates was enhanced by the presence of the endo-1,4-beta-mannanase of T. reesei. AGLII and AGLIII showed synergy in galacto(gluco)mannan hydrolysis with the endo-1,4-beta-mannanase of T. reesei and a beta-mannosidase of Aspergillus niger. The calculated molecular mass and the hydrolytic properties of AGLI indicate that it corresponds to the alpha-galactosidase previously purified from T. reesei.",
author = "Emilio Margolles-Clark and Maija Tenkanen and Elina Luonteri and Merja Penttil{\"a}",
note = "LIS: A1 CA2: 1503 CA2: 1501 CA: BEL Project code: BEL4103",
year = "1996",
doi = "10.1111/j.1432-1033.1996.0104h.x",
language = "English",
volume = "240",
pages = "104--111",
journal = "FEBS Journal",
issn = "1742-464X",
publisher = "Wiley",
number = "1",

}

Three alfa-galactosidase genes of Trichoderma reesei cloned by expression in yeast. / Margolles-Clark, Emilio; Tenkanen, Maija; Luonteri, Elina; Penttilä, Merja.

In: European Journal of Biochemistry, Vol. 240, No. 1, 1996, p. 104-111.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Three alfa-galactosidase genes of Trichoderma reesei cloned by expression in yeast

AU - Margolles-Clark, Emilio

AU - Tenkanen, Maija

AU - Luonteri, Elina

AU - Penttilä, Merja

N1 - LIS: A1 CA2: 1503 CA2: 1501 CA: BEL Project code: BEL4103

PY - 1996

Y1 - 1996

N2 - Three alpha-galactosidase genes, agl1, agl2 and agl3, were isolated from a cDNA expression library of Trichoderma reesei RutC-30 constructed in the yeast Saccharomyces cerevisiae by screening the library on plates containing the substrate 5-bromo-4-chloro-3-indolyl-alpha-D-galactopyranoside. The genes agl1, agl2 and agl3 encode 444, 746 and 624 amino acids, respectively, including the signal sequences. The deduced amino acid sequences of AGLI and AGLIII showed similarity with the alpha-galactosidases of plant, animal, yeast and filamentous fungal origin classified into family 27 of glycosyl hydrolases whereas the deduced amino acid sequence of AGLII showed similarity with the bacterial alpha-galactosidases of family 36. The enzymes produced by yeast were analysed for enzymatic activity against different substrates. AGLI, AGLII and AGLIII were able to hydrolyse the synthetic substrate p-nitrophenyl-alpha-D-galactopyranoside and the small galactose-containing oligosaccharides, melibiose and raffinose. They liberated galactose from polymeric galacto(gluco)mannan with different efficiencies. The action of AGLI towards polymeric substrates was enhanced by the presence of the endo-1,4-beta-mannanase of T. reesei. AGLII and AGLIII showed synergy in galacto(gluco)mannan hydrolysis with the endo-1,4-beta-mannanase of T. reesei and a beta-mannosidase of Aspergillus niger. The calculated molecular mass and the hydrolytic properties of AGLI indicate that it corresponds to the alpha-galactosidase previously purified from T. reesei.

AB - Three alpha-galactosidase genes, agl1, agl2 and agl3, were isolated from a cDNA expression library of Trichoderma reesei RutC-30 constructed in the yeast Saccharomyces cerevisiae by screening the library on plates containing the substrate 5-bromo-4-chloro-3-indolyl-alpha-D-galactopyranoside. The genes agl1, agl2 and agl3 encode 444, 746 and 624 amino acids, respectively, including the signal sequences. The deduced amino acid sequences of AGLI and AGLIII showed similarity with the alpha-galactosidases of plant, animal, yeast and filamentous fungal origin classified into family 27 of glycosyl hydrolases whereas the deduced amino acid sequence of AGLII showed similarity with the bacterial alpha-galactosidases of family 36. The enzymes produced by yeast were analysed for enzymatic activity against different substrates. AGLI, AGLII and AGLIII were able to hydrolyse the synthetic substrate p-nitrophenyl-alpha-D-galactopyranoside and the small galactose-containing oligosaccharides, melibiose and raffinose. They liberated galactose from polymeric galacto(gluco)mannan with different efficiencies. The action of AGLI towards polymeric substrates was enhanced by the presence of the endo-1,4-beta-mannanase of T. reesei. AGLII and AGLIII showed synergy in galacto(gluco)mannan hydrolysis with the endo-1,4-beta-mannanase of T. reesei and a beta-mannosidase of Aspergillus niger. The calculated molecular mass and the hydrolytic properties of AGLI indicate that it corresponds to the alpha-galactosidase previously purified from T. reesei.

U2 - 10.1111/j.1432-1033.1996.0104h.x

DO - 10.1111/j.1432-1033.1996.0104h.x

M3 - Article

VL - 240

SP - 104

EP - 111

JO - FEBS Journal

JF - FEBS Journal

SN - 1742-464X

IS - 1

ER -