Background: Non-productive adsorption to lignin hinders cellulase efficiency.Results: 31 alanine mutants of the carbohydrate-binding module (CBM) exhibit highly correlated cellulose and lignin affinity.Conclusion: The cellulose-binding face of CBM is responsible for the majority of lignin affinity, although four mutations were identified that selectivity decrease lignin adsorption.Significance: The mutations studied inform future engineering efforts of fungal CBMs.

Non-productive binding of cellulases to lignin inhibits enzymatic hydrolysis of biomass, increasing enzyme requirements and the cost of biofuels. This study used site-directed mutagenesis of the Trichoderma Cel7A carbohydrate binding module (CBM) and linker to investigate the mechanisms of adsorption to lignin and engineer a cellulase with increased binding specificity for cellulose. CBM mutations that added hydrophobic or positively charged residues decreased the specificity for cellulose, while mutations that added negatively charged residues increased the specificity. Linker mutations that altered predicted glycosylation patterns selectively impacted lignin affinity. Beneficial mutations were combined to generate a mutant with 2.5-fold less lignin affinity while fully retaining cellulose affinity. This mutant was uninhibited by added lignin during hydrolysis of Avicel and generated 40% more glucose than the wild-type enzyme from dilute acid-pretreated Miscanthus. Biotechnol. Bioeng. 2016;113: 1369-1374. © 2015 Wiley Periodicals, Inc.

Enzymes that degrade cellulose into glucose are one of the most expensive components of processes for converting cellulosic biomass to fuels and chemicals. Cellulase enzyme Cel7A is the most abundant enzyme naturally employed by fungi to depolymerize cellulose, and like other cellulases is inhibited by its product, cellobiose. There is thus great economic incentive for minimizing the detrimental effects of product inhibition on Cel7A. In this work, we experimentally generated 10 previously proposed site‐directed mutant Cel7A enzymes expected to have reduced cellobiose binding energies (the majority of mutations were to alanine). We then tested their resilience to cellobiose as well as their hydrolytic activities on microcrystalline cellulose. Although every mutation tested conferred reduced product inhibition (and abolished it for some), our results confirm a trade‐off between Cel7A tolerance to cellobiose and enzymatic activity: Reduced product inhibition was accompanied by lower overall enzymatic activity on crystalline cellulose for the mutants tested. The tempering effect of mutations on inhibition was nearly constant despite relatively large differences in activities of the mutants. Our work identifies an amino acid in the Cel7A product binding site of interest for further mutational studies, and highlights both the challenge and the opportunity of enzyme engineering toward improving product tolerance in Cel7A. Biotechnol. Bioeng. 2016;113: 330–338. © 2015 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

With the goal of generating new enzymes that can cleave custom sequences, this article describes a selection strategy for evolving proteases with desirable characteristics. Positive selection and counter-selection are combined to select for and against specified cleavage sequences simultaneously. Cleavage of the positive selection sequence permits E. coli growth, and cleavage of the counter-selection sequence slows growth. Growth occurs when cleavage of the positive selection sequence releases β-lactamase into the periplasm where it can confer antibiotic resistance. The counter-selection traps β-lactamase in the cytoplasm, preventing antibiotic resistance and growth. Thus, proteases with a preference for the positive selection sequence relative to the counter-selection sequence grow more rapidly. This system was used to select a tobacco etch virus (TEV) protease mutant with new substrate compatibility. Biotechnol. Bioeng. 2016;113: 1187-1193. © 2015 Wiley Periodicals, Inc.