Low-level endoglucanase contamination in a Trichoderma reesei cellobiohydrolase II preparation affects its enzymatic activity on beta-glucan

Tapani Reinikainen, Kirsti Henriksson, Matti Siika-aho, Olle Teleman, Kaisa Poutanen

Research output: Contribution to journalArticleScientificpeer-review

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Abstract

A routinely employed and specific affinity chromatography purification method was used to isolate cellobiohydrolase II (CBHII) from a Trichoderma reesei culture supernatant.
Two different identically purified batches of CBHII were found to have distinctly different properties in barley β-glucan hydrolysis. The abilities of the two preparations to produce small oligosaccharides and reduce the viscosity of β-glucan were substantially different, but no contamination or heterogeneity was detected in sodium dodecyl sulfate-electrophoresis used to assess the protein purity. Furthermore, the specific activities of the preparations on cellotetraose substrate were similar.
Careful analysis with specific chromogenic oligosaccharide substrates showed that the considerable variation in β-glucan hydrolysis was caused by a minor endoglucanase contamination consisting of only 0.4% of the total protein.
The contamination is possibly caused by nonspecific interactions between the proteins and chromatographic column material or by unfavorable protein-protein interactions during the chromatographic separation.
The data demonstrate clearly the caution needed in the interpretation of hydrolysis studies on polymeric substrates with cellulolytic enzyme preparations.

Original languageEnglish
Pages (from-to)888-892
JournalEnzyme and Microbial Technology
Volume17
Issue number10
DOIs
Publication statusPublished - 1995
MoE publication typeA1 Journal article-refereed

Fingerprint

Cellulose 1,4-beta-Cellobiosidase
beta-Glucans
Trichoderma
Cellulase
Contamination
Glucans
Proteins
Hydrolysis
Oligosaccharides
Substrates
Chromogenics
Affinity chromatography
Chromogenic Compounds
Sodium dodecyl sulfate
Hordeum
Electrophoresis
Affinity Chromatography
Viscosity
Sodium Dodecyl Sulfate
Purification

Cite this

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title = "Low-level endoglucanase contamination in a Trichoderma reesei cellobiohydrolase II preparation affects its enzymatic activity on beta-glucan",
abstract = "A routinely employed and specific affinity chromatography purification method was used to isolate cellobiohydrolase II (CBHII) from a Trichoderma reesei culture supernatant. Two different identically purified batches of CBHII were found to have distinctly different properties in barley β-glucan hydrolysis. The abilities of the two preparations to produce small oligosaccharides and reduce the viscosity of β-glucan were substantially different, but no contamination or heterogeneity was detected in sodium dodecyl sulfate-electrophoresis used to assess the protein purity. Furthermore, the specific activities of the preparations on cellotetraose substrate were similar. Careful analysis with specific chromogenic oligosaccharide substrates showed that the considerable variation in β-glucan hydrolysis was caused by a minor endoglucanase contamination consisting of only 0.4{\%} of the total protein. The contamination is possibly caused by nonspecific interactions between the proteins and chromatographic column material or by unfavorable protein-protein interactions during the chromatographic separation. The data demonstrate clearly the caution needed in the interpretation of hydrolysis studies on polymeric substrates with cellulolytic enzyme preparations.",
author = "Tapani Reinikainen and Kirsti Henriksson and Matti Siika-aho and Olle Teleman and Kaisa Poutanen",
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Low-level endoglucanase contamination in a Trichoderma reesei cellobiohydrolase II preparation affects its enzymatic activity on beta-glucan. / Reinikainen, Tapani; Henriksson, Kirsti; Siika-aho, Matti; Teleman, Olle; Poutanen, Kaisa.

In: Enzyme and Microbial Technology, Vol. 17, No. 10, 1995, p. 888-892.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Low-level endoglucanase contamination in a Trichoderma reesei cellobiohydrolase II preparation affects its enzymatic activity on beta-glucan

AU - Reinikainen, Tapani

AU - Henriksson, Kirsti

AU - Siika-aho, Matti

AU - Teleman, Olle

AU - Poutanen, Kaisa

N1 - Project code: BIO1004

PY - 1995

Y1 - 1995

N2 - A routinely employed and specific affinity chromatography purification method was used to isolate cellobiohydrolase II (CBHII) from a Trichoderma reesei culture supernatant. Two different identically purified batches of CBHII were found to have distinctly different properties in barley β-glucan hydrolysis. The abilities of the two preparations to produce small oligosaccharides and reduce the viscosity of β-glucan were substantially different, but no contamination or heterogeneity was detected in sodium dodecyl sulfate-electrophoresis used to assess the protein purity. Furthermore, the specific activities of the preparations on cellotetraose substrate were similar. Careful analysis with specific chromogenic oligosaccharide substrates showed that the considerable variation in β-glucan hydrolysis was caused by a minor endoglucanase contamination consisting of only 0.4% of the total protein. The contamination is possibly caused by nonspecific interactions between the proteins and chromatographic column material or by unfavorable protein-protein interactions during the chromatographic separation. The data demonstrate clearly the caution needed in the interpretation of hydrolysis studies on polymeric substrates with cellulolytic enzyme preparations.

AB - A routinely employed and specific affinity chromatography purification method was used to isolate cellobiohydrolase II (CBHII) from a Trichoderma reesei culture supernatant. Two different identically purified batches of CBHII were found to have distinctly different properties in barley β-glucan hydrolysis. The abilities of the two preparations to produce small oligosaccharides and reduce the viscosity of β-glucan were substantially different, but no contamination or heterogeneity was detected in sodium dodecyl sulfate-electrophoresis used to assess the protein purity. Furthermore, the specific activities of the preparations on cellotetraose substrate were similar. Careful analysis with specific chromogenic oligosaccharide substrates showed that the considerable variation in β-glucan hydrolysis was caused by a minor endoglucanase contamination consisting of only 0.4% of the total protein. The contamination is possibly caused by nonspecific interactions between the proteins and chromatographic column material or by unfavorable protein-protein interactions during the chromatographic separation. The data demonstrate clearly the caution needed in the interpretation of hydrolysis studies on polymeric substrates with cellulolytic enzyme preparations.

U2 - 10.1016/0141-0229(95)00008-S

DO - 10.1016/0141-0229(95)00008-S

M3 - Article

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JO - Enzyme and Microbial Technology

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SN - 0141-0229

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