Expression in Trichoderma reesei and characterisation of a thermostable family 3 β-glucosidase from the moderately thermophilic fungus Talaromyces emersonii

Patrick Murray, Nina Aro, Catherine Collins, Alice Grassick, Merja Penttilä, Markku Saloheimo, Maria Tuohy

Research output: Contribution to journalArticleScientificpeer-review

102 Citations (Scopus)

Abstract

The gene encoding a thermostable β-glucosidase (cel3a) was isolated from the thermophilic fungus Talalaromyces emersonii by degenerate PCR and expressed in the filamentous fungus Trichoderma reesei. The cel3a gene encodes an 857 amino acid long protein with a calculated molecular weight of 90.59 kDa. Tal. emersonii β-glucosidase falls into glycosyl hydrolase family 3, showing approximately 56 and 67% identity with Cel3b (GenBank AAP57755) from T. reesei, and a β-glucosidase from Aspergillus Niger (GenBank CAB75696), respectively. The heterologously expressed enzyme, Cel3a, was a dimer equal to 130 kDa subunits with 17 potential N-glycosylation sites and a previously unreported β-glucosidase activity produced extracellularly by Tal. emersonii. Cel3a was thermostable with an optimum temperature of 71.5°C and half life of 62 min at 65°C and was a specific β-glucosidase with no β-galactosidase side activity. Cel3a had a high specific activity against p-nitrophenyl-β-d-glucopyranoside (V max, 512 IU/mg) and was competitively inhibited by glucose (k i, 0.254 mM). Cel3a was also active against natural cellooligosacharides with glucose being the product of hydrolysis. It displayed transferase activity producing mainly cellobiose from glucose and cellotetrose from cellobiose.

Original languageEnglish
Pages (from-to)248-257
Number of pages10
JournalProtein Expression and Purification
Volume38
Issue number2
DOIs
Publication statusPublished - 1 Dec 2004
MoE publication typeA1 Journal article-refereed

Fingerprint

Talaromyces
Glucosidases
Trichoderma
Fungi
Cellobiose
Nucleic Acid Databases
Glucose
Galactosidases
Glycosylation
Gene encoding
Aspergillus niger
Aspergillus
Hydrolases
Transferases
Dimers
Genes
Half-Life
Hydrolysis
Molecular Weight
Molecular weight

Keywords

  • β-glucosidase
  • Fungus
  • Gene cloning
  • Glycosyl hydrolase
  • Heterologous expression
  • Secondary structure prediction
  • Thermostable

Cite this

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title = "Expression in Trichoderma reesei and characterisation of a thermostable family 3 β-glucosidase from the moderately thermophilic fungus Talaromyces emersonii",
abstract = "The gene encoding a thermostable β-glucosidase (cel3a) was isolated from the thermophilic fungus Talalaromyces emersonii by degenerate PCR and expressed in the filamentous fungus Trichoderma reesei. The cel3a gene encodes an 857 amino acid long protein with a calculated molecular weight of 90.59 kDa. Tal. emersonii β-glucosidase falls into glycosyl hydrolase family 3, showing approximately 56 and 67{\%} identity with Cel3b (GenBank AAP57755) from T. reesei, and a β-glucosidase from Aspergillus Niger (GenBank CAB75696), respectively. The heterologously expressed enzyme, Cel3a, was a dimer equal to 130 kDa subunits with 17 potential N-glycosylation sites and a previously unreported β-glucosidase activity produced extracellularly by Tal. emersonii. Cel3a was thermostable with an optimum temperature of 71.5°C and half life of 62 min at 65°C and was a specific β-glucosidase with no β-galactosidase side activity. Cel3a had a high specific activity against p-nitrophenyl-β-d-glucopyranoside (V max, 512 IU/mg) and was competitively inhibited by glucose (k i, 0.254 mM). Cel3a was also active against natural cellooligosacharides with glucose being the product of hydrolysis. It displayed transferase activity producing mainly cellobiose from glucose and cellotetrose from cellobiose.",
keywords = "β-glucosidase, Fungus, Gene cloning, Glycosyl hydrolase, Heterologous expression, Secondary structure prediction, Thermostable",
author = "Patrick Murray and Nina Aro and Catherine Collins and Alice Grassick and Merja Penttil{\"a} and Markku Saloheimo and Maria Tuohy",
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language = "English",
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Expression in Trichoderma reesei and characterisation of a thermostable family 3 β-glucosidase from the moderately thermophilic fungus Talaromyces emersonii. / Murray, Patrick; Aro, Nina; Collins, Catherine; Grassick, Alice; Penttilä, Merja; Saloheimo, Markku; Tuohy, Maria.

In: Protein Expression and Purification, Vol. 38, No. 2, 01.12.2004, p. 248-257.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Expression in Trichoderma reesei and characterisation of a thermostable family 3 β-glucosidase from the moderately thermophilic fungus Talaromyces emersonii

AU - Murray, Patrick

AU - Aro, Nina

AU - Collins, Catherine

AU - Grassick, Alice

AU - Penttilä, Merja

AU - Saloheimo, Markku

AU - Tuohy, Maria

PY - 2004/12/1

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N2 - The gene encoding a thermostable β-glucosidase (cel3a) was isolated from the thermophilic fungus Talalaromyces emersonii by degenerate PCR and expressed in the filamentous fungus Trichoderma reesei. The cel3a gene encodes an 857 amino acid long protein with a calculated molecular weight of 90.59 kDa. Tal. emersonii β-glucosidase falls into glycosyl hydrolase family 3, showing approximately 56 and 67% identity with Cel3b (GenBank AAP57755) from T. reesei, and a β-glucosidase from Aspergillus Niger (GenBank CAB75696), respectively. The heterologously expressed enzyme, Cel3a, was a dimer equal to 130 kDa subunits with 17 potential N-glycosylation sites and a previously unreported β-glucosidase activity produced extracellularly by Tal. emersonii. Cel3a was thermostable with an optimum temperature of 71.5°C and half life of 62 min at 65°C and was a specific β-glucosidase with no β-galactosidase side activity. Cel3a had a high specific activity against p-nitrophenyl-β-d-glucopyranoside (V max, 512 IU/mg) and was competitively inhibited by glucose (k i, 0.254 mM). Cel3a was also active against natural cellooligosacharides with glucose being the product of hydrolysis. It displayed transferase activity producing mainly cellobiose from glucose and cellotetrose from cellobiose.

AB - The gene encoding a thermostable β-glucosidase (cel3a) was isolated from the thermophilic fungus Talalaromyces emersonii by degenerate PCR and expressed in the filamentous fungus Trichoderma reesei. The cel3a gene encodes an 857 amino acid long protein with a calculated molecular weight of 90.59 kDa. Tal. emersonii β-glucosidase falls into glycosyl hydrolase family 3, showing approximately 56 and 67% identity with Cel3b (GenBank AAP57755) from T. reesei, and a β-glucosidase from Aspergillus Niger (GenBank CAB75696), respectively. The heterologously expressed enzyme, Cel3a, was a dimer equal to 130 kDa subunits with 17 potential N-glycosylation sites and a previously unreported β-glucosidase activity produced extracellularly by Tal. emersonii. Cel3a was thermostable with an optimum temperature of 71.5°C and half life of 62 min at 65°C and was a specific β-glucosidase with no β-galactosidase side activity. Cel3a had a high specific activity against p-nitrophenyl-β-d-glucopyranoside (V max, 512 IU/mg) and was competitively inhibited by glucose (k i, 0.254 mM). Cel3a was also active against natural cellooligosacharides with glucose being the product of hydrolysis. It displayed transferase activity producing mainly cellobiose from glucose and cellotetrose from cellobiose.

KW - β-glucosidase

KW - Fungus

KW - Gene cloning

KW - Glycosyl hydrolase

KW - Heterologous expression

KW - Secondary structure prediction

KW - Thermostable

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U2 - 10.1016/j.pep.2004.08.006

DO - 10.1016/j.pep.2004.08.006

M3 - Article

VL - 38

SP - 248

EP - 257

JO - Protein Expression and Purification

JF - Protein Expression and Purification

SN - 1046-5928

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ER -