Heterologous expression of Melanocarpus albomyces cellobiohydrolase Cel7B, and random mutagenesis to improve its thermostability

Sanni Voutilainen, Harry Boer, Markus Linder, Terhi Puranen, Juha Rouvinen, Jari Vehmaanperä, Anu Koivula

Research output: Contribution to journalArticleScientificpeer-review

35 Citations (Scopus)

Abstract

Fungal cellobiohydrolases from the glycosyl hydrolase family 7 are key enzymes in crystalline cellulose hydrolysis. Difficulties in heterologous expression in a bacterial or yeast host have hampered engineering of these cellulases for industrial application. We report here a successful expression of the single-module cellobiohydrolase Cel7B from a thermophilic fungus Melanocarpus albomyces in Saccharomyces cerevisiae (Sc Cel7B).
An automated, robotic thermostability screening method, based on residual activity measurements on a small soluble substrate methylumbelliferyl-lactoside (MULac), was then set-up to screen the first generation random mutant libraries. Out of the nine positive thermostable mutants, we picked three based on structural considerations, each containing a single amino acid change (A30T, G184D or S290T). Cel7B A30T and S290T mutants showed improved unfolding temperature (Tm) by 1.5 and 3.5 °C, respectively.
In addition, the temperature optimum (Topt) on a soluble substrate had improved by 5 °C for the A30T mutant. Interestingly, the best enzyme variant on microcrystalline cellulose (Avicel) hydrolysis was the Cel7B S290T, which could hydrolyse Avicel at 70 °C two times more effectively than the Sc Cel7B.
Overall the consensus mutation S290T, located in the hydrophobic core of Cel7B, led to a cellobiohydrolase variant having also application potential in hydrolysis of polymeric substrates at elevated temperatures.
Original languageEnglish
Pages (from-to)234-243
JournalEnzyme and Microbial Technology
Volume41
Issue number3
DOIs
Publication statusPublished - 2007
MoE publication typeA1 Journal article-refereed

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Cellulose 1,4-beta-Cellobiosidase
Mutagenesis
Cellulose
Hydrolysis
Yeast
Temperature
Substrates
Enzymes
Hydrolases
Cellulases
Robotics
Fungi
Industrial applications
Saccharomyces cerevisiae
Amino acids
Screening
Yeasts
Crystalline materials
Amino Acids
Mutation

Keywords

  • Cellulase
  • Heterologous expression
  • High-throughput screening
  • Random mutagenesis
  • Thermostability
  • Saccharomyces cerevisiae
  • Melanocarpus albomyces

Cite this

@article{56a9b1ed04814e76aa965e7e43e0d0ad,
title = "Heterologous expression of Melanocarpus albomyces cellobiohydrolase Cel7B, and random mutagenesis to improve its thermostability",
abstract = "Fungal cellobiohydrolases from the glycosyl hydrolase family 7 are key enzymes in crystalline cellulose hydrolysis. Difficulties in heterologous expression in a bacterial or yeast host have hampered engineering of these cellulases for industrial application. We report here a successful expression of the single-module cellobiohydrolase Cel7B from a thermophilic fungus Melanocarpus albomyces in Saccharomyces cerevisiae (Sc Cel7B). An automated, robotic thermostability screening method, based on residual activity measurements on a small soluble substrate methylumbelliferyl-lactoside (MULac), was then set-up to screen the first generation random mutant libraries. Out of the nine positive thermostable mutants, we picked three based on structural considerations, each containing a single amino acid change (A30T, G184D or S290T). Cel7B A30T and S290T mutants showed improved unfolding temperature (Tm) by 1.5 and 3.5 °C, respectively.In addition, the temperature optimum (Topt) on a soluble substrate had improved by 5 °C for the A30T mutant. Interestingly, the best enzyme variant on microcrystalline cellulose (Avicel) hydrolysis was the Cel7B S290T, which could hydrolyse Avicel at 70 °C two times more effectively than the Sc Cel7B. Overall the consensus mutation S290T, located in the hydrophobic core of Cel7B, led to a cellobiohydrolase variant having also application potential in hydrolysis of polymeric substrates at elevated temperatures.",
keywords = "Cellulase, Heterologous expression, High-throughput screening, Random mutagenesis, Thermostability, Saccharomyces cerevisiae, Melanocarpus albomyces",
author = "Sanni Voutilainen and Harry Boer and Markus Linder and Terhi Puranen and Juha Rouvinen and Jari Vehmaanper{\"a} and Anu Koivula",
year = "2007",
doi = "10.1016/j.enzmictec.2007.01.015",
language = "English",
volume = "41",
pages = "234--243",
journal = "Enzyme and Microbial Technology",
issn = "0141-0229",
publisher = "Elsevier",
number = "3",

}

Heterologous expression of Melanocarpus albomyces cellobiohydrolase Cel7B, and random mutagenesis to improve its thermostability. / Voutilainen, Sanni; Boer, Harry; Linder, Markus; Puranen, Terhi; Rouvinen, Juha; Vehmaanperä, Jari; Koivula, Anu.

In: Enzyme and Microbial Technology, Vol. 41, No. 3, 2007, p. 234-243.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Heterologous expression of Melanocarpus albomyces cellobiohydrolase Cel7B, and random mutagenesis to improve its thermostability

AU - Voutilainen, Sanni

AU - Boer, Harry

AU - Linder, Markus

AU - Puranen, Terhi

AU - Rouvinen, Juha

AU - Vehmaanperä, Jari

AU - Koivula, Anu

PY - 2007

Y1 - 2007

N2 - Fungal cellobiohydrolases from the glycosyl hydrolase family 7 are key enzymes in crystalline cellulose hydrolysis. Difficulties in heterologous expression in a bacterial or yeast host have hampered engineering of these cellulases for industrial application. We report here a successful expression of the single-module cellobiohydrolase Cel7B from a thermophilic fungus Melanocarpus albomyces in Saccharomyces cerevisiae (Sc Cel7B). An automated, robotic thermostability screening method, based on residual activity measurements on a small soluble substrate methylumbelliferyl-lactoside (MULac), was then set-up to screen the first generation random mutant libraries. Out of the nine positive thermostable mutants, we picked three based on structural considerations, each containing a single amino acid change (A30T, G184D or S290T). Cel7B A30T and S290T mutants showed improved unfolding temperature (Tm) by 1.5 and 3.5 °C, respectively.In addition, the temperature optimum (Topt) on a soluble substrate had improved by 5 °C for the A30T mutant. Interestingly, the best enzyme variant on microcrystalline cellulose (Avicel) hydrolysis was the Cel7B S290T, which could hydrolyse Avicel at 70 °C two times more effectively than the Sc Cel7B. Overall the consensus mutation S290T, located in the hydrophobic core of Cel7B, led to a cellobiohydrolase variant having also application potential in hydrolysis of polymeric substrates at elevated temperatures.

AB - Fungal cellobiohydrolases from the glycosyl hydrolase family 7 are key enzymes in crystalline cellulose hydrolysis. Difficulties in heterologous expression in a bacterial or yeast host have hampered engineering of these cellulases for industrial application. We report here a successful expression of the single-module cellobiohydrolase Cel7B from a thermophilic fungus Melanocarpus albomyces in Saccharomyces cerevisiae (Sc Cel7B). An automated, robotic thermostability screening method, based on residual activity measurements on a small soluble substrate methylumbelliferyl-lactoside (MULac), was then set-up to screen the first generation random mutant libraries. Out of the nine positive thermostable mutants, we picked three based on structural considerations, each containing a single amino acid change (A30T, G184D or S290T). Cel7B A30T and S290T mutants showed improved unfolding temperature (Tm) by 1.5 and 3.5 °C, respectively.In addition, the temperature optimum (Topt) on a soluble substrate had improved by 5 °C for the A30T mutant. Interestingly, the best enzyme variant on microcrystalline cellulose (Avicel) hydrolysis was the Cel7B S290T, which could hydrolyse Avicel at 70 °C two times more effectively than the Sc Cel7B. Overall the consensus mutation S290T, located in the hydrophobic core of Cel7B, led to a cellobiohydrolase variant having also application potential in hydrolysis of polymeric substrates at elevated temperatures.

KW - Cellulase

KW - Heterologous expression

KW - High-throughput screening

KW - Random mutagenesis

KW - Thermostability

KW - Saccharomyces cerevisiae

KW - Melanocarpus albomyces

U2 - 10.1016/j.enzmictec.2007.01.015

DO - 10.1016/j.enzmictec.2007.01.015

M3 - Article

VL - 41

SP - 234

EP - 243

JO - Enzyme and Microbial Technology

JF - Enzyme and Microbial Technology

SN - 0141-0229

IS - 3

ER -