title: Alleviating product inhibition in cellulase enzyme Cel7A.
abstract: 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.
authors: Department of Chemistry, Chemical Biology Graduate Program, University of California, Berkeley, 94720, California.|Energy Biosciences Institute, University of California, Berkeley, 94720, California.
authors: Meera E
authors: ME
authors: Atreya
authors: Energy Biosciences Institute, University of California, Berkeley, 94720, California.|Department of Chemical and Biomolecular Engineering, University of California, Berkeley, 94720, California.
authors: Kathryn L
authors: KL
authors: Strobel
authors: Energy Biosciences Institute, University of California, Berkeley, 94720, California. clark@berkeley.edu.|Department of Chemical and Biomolecular Engineering, University of California, Berkeley, 94720, California. clark@berkeley.edu.
authors: Douglas S
authors: DS
authors: Clark
mesh_terms: D001665:Binding Sites; D002475:Cellobiose / Q000378:metabolism*; D002480:Cellulase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism*; D005658:Fungi / Q000201:enzymology*; D006868:Hydrolysis; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation
chemical_list: D004791:Enzyme Inhibitors; D050505:Mutant Proteins; D002475:Cellobiose; D002482:Cellulose; D002480:Cellulase
keywords: Cel7A; cellobiohydrolase I (CBH1); cellobiose; cellulase; cellulosic biofuels; glycoside hydrolase family 7; product inhibition