Comparisons of Catalytic Efficiency during the Reduction of Lignocellulosic Substrates between Free Enzyme and Enzyme bound to Mobile Enzyme Sequestration Platforms (MESP)

Abstract

The cellulolytic bacterium Clostridium thermocellum employs a flexible cellulosome that can bind a variety of enzymes to breakdown cellulosic biomass. A key feature of the cellulosome is a cohesin (Type 1) – dockerin (Type 1) interaction that binds dockerin-endowed enzymes to the cohesin-ladened scaffoldin backbone. Multiple enzymes of various enzyme classes (e.g., exocellulases, endocellulases, xylanases) can bind to the cellulosome structure for synergistic deconstruction of cellulosic substrates. Several “artificial cellulosomes” have been engineered in an attempt to mimic this system for abiotic decomposition of lignocellulosic substrates. One such engineered system is called a mobile enzyme sequestration platform (MESP). The prototype of this platform was called a “rosettasome”. MESPs are chimera of an archaeal group II chaperonin permutant fused to C. thermocellum cohesin via a 9-12 amino acid linker. The archaeal chaperonin complex is thermotolerant. In prior work our lab demonstrated that binding cellulolytic enzymes to MESPs enhances lignocellulose breakdown efficiency over free enzyme in solution (sans platform). Interestingly, our prior data indicated that MESP systems may also be acidotolerant since substrate was treated with mild sulfuric acid. In this study, we tested the acidotolerance of MESP technology as well as the efficiency of carbohydrate reduction on other feedstock (e.g., rice straw). We also tested our MESPs against the prototype rosettasome to determine if this next-generation platform outperforms the original system. Here we present the results of our comparisons. Understanding the limits of MESP enhancement will direct future engineering of this system for industrial and/or agricultural applications.