Insights into cellulase-lignin non-specific binding revealed by computational redesign of the surface of green fluorescent protein

Carolyn N. Haarmeyer, Matthew D. Smith, Shishir P S Chundawat, Deanne Sammond, Timothy A. Whitehead

    Research output: Research - peer-reviewArticle

    • 3 Citations

    Abstract

    Biological-mediated conversion of pretreated lignocellulosic biomass to biofuels and biochemicals is a promising avenue toward energy sustainability. However, a critical impediment to the commercialization of cellulosic biofuel production is the high cost of cellulase enzymes needed to deconstruct biomass into fermentable sugars. One major factor driving cost is cellulase adsorption and inactivation in the presence of lignin, yet we currently have a poor understanding of the protein structure–function relationships driving this adsorption. In this work, we have systematically investigated the role of protein surface potential on lignin adsorption using a model monomeric fluorescent protein. We have designed and experimentally characterized 16 model protein variants spanning the physiological range of net charge (−24 to +16 total charges) and total charge density (0.28–0.40 charges per sequence length) typical for natural proteins. Protein designs were expressed, purified, and subjected to in silico and in vitro biophysical measurements to evaluate the relationship between protein surface potential and lignin adsorption properties. The designs were comparable to model fluorescent protein in terms of thermostability and heterologous expression yield, although the majority of the designs unexpectedly formed homodimers. Protein adsorption to lignin was studied at two different temperatures using Quartz Crystal Microbalance with Dissipation Monitoring and a subtractive mass balance assay. We found a weak correlation between protein net charge and protein-binding capacity to lignin. No other single characteristic, including apparent melting temperature and 2nd virial coefficient, showed correlation with lignin binding. Analysis of an unrelated cellulase dataset with mutations localized to a family I carbohydrate-binding module showed a similar correlation between net charge and lignin binding capacity. Overall, our study provides strategies to identify highly active, low lignin-binding cellulases by either rational design or by computational screening genomic databases. Biotechnol. Bioeng. 2017;114: 740–750.

    LanguageEnglish (US)
    Pages740-750
    Number of pages11
    JournalBiotechnology and Bioengineering
    Volume114
    Issue number4
    DOIs
    StatePublished - Apr 1 2017

    Profile

    Cellulase
    Lignin
    Green Fluorescent Proteins
    Proteins
    Adsorption
    Biofuels
    Biomass
    Membrane Proteins
    Costs and Cost Analysis
    Temperature
    Costs
    Surface potential
    Quartz Crystal Microbalance Techniques
    Cellulases
    Protein Binding
    Computer Simulation
    Freezing
    Carbohydrates
    Databases
    Mutation

    Keywords

    • biomass deconstruction
    • cellulase
    • computational protein design
    • lignin
    • protein engineering

    ASJC Scopus subject areas

    • Biotechnology
    • Bioengineering
    • Applied Microbiology and Biotechnology

    Cite this

    Insights into cellulase-lignin non-specific binding revealed by computational redesign of the surface of green fluorescent protein. / Haarmeyer, Carolyn N.; Smith, Matthew D.; Chundawat, Shishir P S; Sammond, Deanne; Whitehead, Timothy A.

    In: Biotechnology and Bioengineering, Vol. 114, No. 4, 01.04.2017, p. 740-750.

    Research output: Research - peer-reviewArticle

    Haarmeyer, Carolyn N. ; Smith, Matthew D. ; Chundawat, Shishir P S ; Sammond, Deanne ; Whitehead, Timothy A./ Insights into cellulase-lignin non-specific binding revealed by computational redesign of the surface of green fluorescent protein. In: Biotechnology and Bioengineering. 2017 ; Vol. 114, No. 4. pp. 740-750
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    abstract = "Biological-mediated conversion of pretreated lignocellulosic biomass to biofuels and biochemicals is a promising avenue toward energy sustainability. However, a critical impediment to the commercialization of cellulosic biofuel production is the high cost of cellulase enzymes needed to deconstruct biomass into fermentable sugars. One major factor driving cost is cellulase adsorption and inactivation in the presence of lignin, yet we currently have a poor understanding of the protein structure–function relationships driving this adsorption. In this work, we have systematically investigated the role of protein surface potential on lignin adsorption using a model monomeric fluorescent protein. We have designed and experimentally characterized 16 model protein variants spanning the physiological range of net charge (−24 to +16 total charges) and total charge density (0.28–0.40 charges per sequence length) typical for natural proteins. Protein designs were expressed, purified, and subjected to in silico and in vitro biophysical measurements to evaluate the relationship between protein surface potential and lignin adsorption properties. The designs were comparable to model fluorescent protein in terms of thermostability and heterologous expression yield, although the majority of the designs unexpectedly formed homodimers. Protein adsorption to lignin was studied at two different temperatures using Quartz Crystal Microbalance with Dissipation Monitoring and a subtractive mass balance assay. We found a weak correlation between protein net charge and protein-binding capacity to lignin. No other single characteristic, including apparent melting temperature and 2nd virial coefficient, showed correlation with lignin binding. Analysis of an unrelated cellulase dataset with mutations localized to a family I carbohydrate-binding module showed a similar correlation between net charge and lignin binding capacity. Overall, our study provides strategies to identify highly active, low lignin-binding cellulases by either rational design or by computational screening genomic databases. Biotechnol. Bioeng. 2017;114: 740–750.",
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