Homologous expression and characterization of Cel61A (EG IV) of Trichoderma reesei

Johan Karlsson, Markku Saloheimo, Matti Siika-Aho, Maija Tenkanen, Merja Penttilä, Folke Tjerneld

Research output: Contribution to journalArticleScientificpeer-review

100 Citations (Scopus)

Abstract

There are currently four proteins in family 61 of the glycoside hydrolases, from Trichoderma reesei, Agaricus bisporus, Cryptococcus neoformans and Neurospora crassa. The enzymatic activity of these proteins has not been studied thoroughly. We report here the homologous expression and purification of T. reesei Cel61A [previously named endoglucanase (EG) IV]. The enzyme was expressed in high amounts with a histidine tag on the C-terminus and purified by metal affinity chromatography. This is the first time that a histidine tag has been used as a purification aid in the T. reesei expression system. The enzyme activity was studied on a series of carbohydrate polymers. The only activity exhibited by Cel61A was an endoglucanase activity observed on substrates containing β-1,4 glycosidic bonds, e.g. carboxymethylcellulose (CMC), hydroxyethylcellulose (HEC) and β-glucan. The endoglucanase activity on CMC and β-glucan was determined by viscosity analysis, by measuring the production of reducing ends and by following the degradation of the polymer on a size exclusion chromatography system. The formation of soluble sugars by Cel61A from microcrystalline cellulose (Avicel; Merck), phosphoric acid swollen cellulose (PASC), and CMC were analysed on a HPLC system. Cel61A produced small amounts of oligosaccharides from these substrates. Furthermore, Cel61A showed activity against cellotetraose and cellopentaose. The activity of Cel61A was several orders of magnitude lower compared to Cel7B (previously EG I) of T. reesei on all substrates. One significant difference between Cel61A and Cel7B was that cellotriose was a poor substrate for Cel61A but was readily hydrolysed by Cel7B. The enzyme activity for Cel61A was further studied on a large number of carbohydrate substrates but the enzyme showed no activity towards any of these substrates.

Original languageEnglish
Pages (from-to)6498-6507
Number of pages10
JournalEuropean Journal of Biochemistry
Volume268
Issue number24
DOIs
Publication statusPublished - 1 Dec 2001
MoE publication typeA1 Journal article-refereed

Fingerprint

Trichoderma
Cellulase
Carboxymethylcellulose Sodium
Glucans
Substrates
Enzymes
Histidine
Cellulose
Polymers
Carbohydrates
Enzyme activity
Agaricus
Cellulases
Neurospora crassa
Cryptococcus neoformans
Purification
Glycoside Hydrolases
Oligosaccharides
Affinity Chromatography
Viscosity

Keywords

  • Cellulase
  • Endoglucanase
  • Histidine tag
  • Trichoderma reesei

Cite this

@article{dddf4e6d2ef34c328a9f0dd59ee2d574,
title = "Homologous expression and characterization of Cel61A (EG IV) of Trichoderma reesei",
abstract = "There are currently four proteins in family 61 of the glycoside hydrolases, from Trichoderma reesei, Agaricus bisporus, Cryptococcus neoformans and Neurospora crassa. The enzymatic activity of these proteins has not been studied thoroughly. We report here the homologous expression and purification of T. reesei Cel61A [previously named endoglucanase (EG) IV]. The enzyme was expressed in high amounts with a histidine tag on the C-terminus and purified by metal affinity chromatography. This is the first time that a histidine tag has been used as a purification aid in the T. reesei expression system. The enzyme activity was studied on a series of carbohydrate polymers. The only activity exhibited by Cel61A was an endoglucanase activity observed on substrates containing β-1,4 glycosidic bonds, e.g. carboxymethylcellulose (CMC), hydroxyethylcellulose (HEC) and β-glucan. The endoglucanase activity on CMC and β-glucan was determined by viscosity analysis, by measuring the production of reducing ends and by following the degradation of the polymer on a size exclusion chromatography system. The formation of soluble sugars by Cel61A from microcrystalline cellulose (Avicel; Merck), phosphoric acid swollen cellulose (PASC), and CMC were analysed on a HPLC system. Cel61A produced small amounts of oligosaccharides from these substrates. Furthermore, Cel61A showed activity against cellotetraose and cellopentaose. The activity of Cel61A was several orders of magnitude lower compared to Cel7B (previously EG I) of T. reesei on all substrates. One significant difference between Cel61A and Cel7B was that cellotriose was a poor substrate for Cel61A but was readily hydrolysed by Cel7B. The enzyme activity for Cel61A was further studied on a large number of carbohydrate substrates but the enzyme showed no activity towards any of these substrates.",
keywords = "Cellulase, Endoglucanase, Histidine tag, Trichoderma reesei",
author = "Johan Karlsson and Markku Saloheimo and Matti Siika-Aho and Maija Tenkanen and Merja Penttil{\"a} and Folke Tjerneld",
year = "2001",
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language = "English",
volume = "268",
pages = "6498--6507",
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Homologous expression and characterization of Cel61A (EG IV) of Trichoderma reesei. / Karlsson, Johan; Saloheimo, Markku; Siika-Aho, Matti; Tenkanen, Maija; Penttilä, Merja; Tjerneld, Folke.

In: European Journal of Biochemistry, Vol. 268, No. 24, 01.12.2001, p. 6498-6507.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Homologous expression and characterization of Cel61A (EG IV) of Trichoderma reesei

AU - Karlsson, Johan

AU - Saloheimo, Markku

AU - Siika-Aho, Matti

AU - Tenkanen, Maija

AU - Penttilä, Merja

AU - Tjerneld, Folke

PY - 2001/12/1

Y1 - 2001/12/1

N2 - There are currently four proteins in family 61 of the glycoside hydrolases, from Trichoderma reesei, Agaricus bisporus, Cryptococcus neoformans and Neurospora crassa. The enzymatic activity of these proteins has not been studied thoroughly. We report here the homologous expression and purification of T. reesei Cel61A [previously named endoglucanase (EG) IV]. The enzyme was expressed in high amounts with a histidine tag on the C-terminus and purified by metal affinity chromatography. This is the first time that a histidine tag has been used as a purification aid in the T. reesei expression system. The enzyme activity was studied on a series of carbohydrate polymers. The only activity exhibited by Cel61A was an endoglucanase activity observed on substrates containing β-1,4 glycosidic bonds, e.g. carboxymethylcellulose (CMC), hydroxyethylcellulose (HEC) and β-glucan. The endoglucanase activity on CMC and β-glucan was determined by viscosity analysis, by measuring the production of reducing ends and by following the degradation of the polymer on a size exclusion chromatography system. The formation of soluble sugars by Cel61A from microcrystalline cellulose (Avicel; Merck), phosphoric acid swollen cellulose (PASC), and CMC were analysed on a HPLC system. Cel61A produced small amounts of oligosaccharides from these substrates. Furthermore, Cel61A showed activity against cellotetraose and cellopentaose. The activity of Cel61A was several orders of magnitude lower compared to Cel7B (previously EG I) of T. reesei on all substrates. One significant difference between Cel61A and Cel7B was that cellotriose was a poor substrate for Cel61A but was readily hydrolysed by Cel7B. The enzyme activity for Cel61A was further studied on a large number of carbohydrate substrates but the enzyme showed no activity towards any of these substrates.

AB - There are currently four proteins in family 61 of the glycoside hydrolases, from Trichoderma reesei, Agaricus bisporus, Cryptococcus neoformans and Neurospora crassa. The enzymatic activity of these proteins has not been studied thoroughly. We report here the homologous expression and purification of T. reesei Cel61A [previously named endoglucanase (EG) IV]. The enzyme was expressed in high amounts with a histidine tag on the C-terminus and purified by metal affinity chromatography. This is the first time that a histidine tag has been used as a purification aid in the T. reesei expression system. The enzyme activity was studied on a series of carbohydrate polymers. The only activity exhibited by Cel61A was an endoglucanase activity observed on substrates containing β-1,4 glycosidic bonds, e.g. carboxymethylcellulose (CMC), hydroxyethylcellulose (HEC) and β-glucan. The endoglucanase activity on CMC and β-glucan was determined by viscosity analysis, by measuring the production of reducing ends and by following the degradation of the polymer on a size exclusion chromatography system. The formation of soluble sugars by Cel61A from microcrystalline cellulose (Avicel; Merck), phosphoric acid swollen cellulose (PASC), and CMC were analysed on a HPLC system. Cel61A produced small amounts of oligosaccharides from these substrates. Furthermore, Cel61A showed activity against cellotetraose and cellopentaose. The activity of Cel61A was several orders of magnitude lower compared to Cel7B (previously EG I) of T. reesei on all substrates. One significant difference between Cel61A and Cel7B was that cellotriose was a poor substrate for Cel61A but was readily hydrolysed by Cel7B. The enzyme activity for Cel61A was further studied on a large number of carbohydrate substrates but the enzyme showed no activity towards any of these substrates.

KW - Cellulase

KW - Endoglucanase

KW - Histidine tag

KW - Trichoderma reesei

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U2 - 10.1046/j.0014-2956.2001.02605.x

DO - 10.1046/j.0014-2956.2001.02605.x

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SN - 1742-464X

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