Abstract
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 language | English |
|---|---|
| Pages (from-to) | 234-243 |
| Journal | Enzyme and Microbial Technology |
| Volume | 41 |
| Issue number | 3 |
| DOIs | |
| Publication status | Published - 2007 |
| MoE publication type | A1 Journal article-refereed |
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Keywords
- Cellulase
- Heterologous expression
- High-throughput screening
- Random mutagenesis
- Thermostability
- Saccharomyces cerevisiae
- Melanocarpus albomyces
Cite this
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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 journal › Article › Scientific › peer-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 -