Researchers engineer enzyme surfaces to bind less to lignin; potential cost reduction for cellulosic ethanol production

Researchers engineer enzyme surfaces to bind less to lignin; potential cost reduction for cellulosic ethanol production

6 July 2017

Researchers at Rutgers University-New Brunswick and Michigan State University have devised a way to reduce the amount of enzymes needed to convert biomass into biofuels by designing and genetically engineering enzyme surfaces so they bind less to the lignin in biomass. This potentially could reduce enzyme costs in biofuels production. A paper on their work is published in the journal ACS Sustainable Chemistry & Engineering.

Cellulases (enzymes) deconstruct lignocellulosic biomass for conversion to biofuels such as cellulosic ethanol and biochemicals. However, lignin, an organic polymer in biomass that binds to and strengthens plant fibers, inactivates the cellulase enzymes via non-productive binding interactions. This leads to high enzyme loading requirements—and therefore high deconstruction costs.

Typically, the enzymes used to help turn switchgrass, corn stover (corn stalks, leaves and other leftovers) and poplar into biofuels amount to about 20% of production costs, said Shishir P. S. Chundawat, senior author of the study and an assistant professor in the Department of Chemical and Biochemical Engineering at Rutgers University-New Brunswick. Enzymes cost about $0.50 per gallon of ethanol, so recycling or using fewer enzymes would make biofuels more inexpensive.

In the United States, gasoline typically contains up to 10% ethanol and corn grain is the primary feedstock of ethanol, according to the US Energy Information Administration (EIA). Biorefineries produce about 15 billion gallons of ethanol a year.

Methods to minimize lignin-mediated enzyme inactivation include lignin removal prior to deconstruction, chemically modifying lignin or adding excipients to prevent enzyme adsorption, or increasing pH and decreasing temperature. An alternative solution is to re-engineer the amino acid sequences of cellulases to prevent lignin adsorption and inactivation. The advantage of this redesign strategy is that there may be no additional process costs, and re-optimization of process conditions may be unnecessary.

—Whitehead et al.

The researchers used computational design was used to supercharge negatively the surfaces of cellulases. The resulting designs maintained the same expression yield, thermal stability, and nearly identical activity on soluble substrate as the wild-type enzymes.

Four designs showed complete lack of inhibition by lignin—but with lower cellulose conversion compared to original enzymes. In other words, while the designs removed lignin inhibition, the redesigned enzymes did not maintain activity sufficient to reduce the overall enzyme loading.


The researchers found that increasing salt concentrations could partially rescue the activity of the supercharged enzymes, supporting a mechanism of electrostatic repulsion between designs and cellulose.

Results showcase a protein engineering strategy to construct highly active cellulases that are resistant to lignin-mediated inactivation, although further work is needed to understand the relationship between negative protein surface potential and activity on insoluble polysaccharides.

—Whitehead et al.

The challenge is breaking down cellulose material, using enzymes, into sugars that can be fermented into ethanol. So any advances on making the enzyme processing step cheaper will make the cost of biofuel cheaper. This is a fairly intractable problem that requires you to attack it from various perspectives, so it does take time.

—Shishir Chundawat

The study’s lead author is Professor Timothy Whitehead of Michigan State University. Other authors are Chandra K. Bandi, a doctoral student in Rutgers’ Department of Chemical and Biochemical Engineering; Marissa Berger, an undergraduate student in Rutgers’ Department of Biomedical Engineering; and Jihyun Park, a former undergraduate student in Rutgers’ Department of Chemical and Biochemical Engineering. Professor Chundawat’s ongoing research on engineering enzymes for enabling low-cost biofuel production is supported primarily by the National Science Foundation (NSF Awards Nº 1236120 qnd Nº 1604421).


  • Timothy A. Whitehead, Chandra K. Bandi, Marissa Berger, Jihyun Park, and Shishir P. S. Chundawat (2017) “Negatively Supercharging Cellulases Render Them Lignin-Resistant” ACS Sustainable Chemistry & Engineering 5 (7), 6247-6252
    doi: 10.1021/acssuschemeng.7b01202


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