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

Paper ID Volume ID Publish Year Pages File Format Full-Text
18715 42738 2007 10 PDF Available
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
CBM, cellulose-binding module; CD, circular dichroism; GH, glycosyl hydrolase; Glc2, cellobiose; Ma Cel7B, Melanocarpus albomyces cellobiohydrolase belonging to the glycosyl hydrolase family 7; MU, 4-Methylumbelliferone; MULac, 4-Methylumbelliferyl-β-d-la
First Page Preview
Heterologous expression of Melanocarpus albomyces cellobiohydrolase Cel7B, and random mutagenesis to improve its thermostability
Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 41, Issue 3, 2 August 2007, Pages 234–243
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering