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

    37 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

    Keywords

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

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