Abstract
BackgroundThe concerted action of three complementary cellulases from Clostridium thermocellum, engineered to be stable at elevated temperatures, was examined on a cellulosic substrate and compared to that of the wild-type enzymes. Exoglucanase Cel48S and endoglucanase Cel8A, both key elements of the natural cellulosome from this bacterium, were engineered previously for increased thermostability, either by SCHEMA, a structure-guided, site-directed protein recombination method, or by consensus-guided mutagenesis combined with random mutagenesis using error-prone PCR, respectively. A thermostable β-glucosidase BglA mutant was also selected from a library generated by error-prone PCR that will assist the two cellulases in their methodic deconstruction of crystalline cellulose. The effects of a thermostable scaffoldin versus those of a largely mesophilic scaffoldin were also examined. By improving the stability of the enzyme subunits and the structural component, we aimed to improve cellulosome-mediated deconstruction of cellulosic substrates.ResultsThe results demonstrate that the combination of thermostable enzymes as free enzymes and a thermostable scaffoldin was more active on the cellulosic substrate than the wild-type enzymes. Significantly, “thermostable” designer cellulosomes exhibited a 1.7-fold enhancement in cellulose degradation compared to the action of conventional designer cellulosomes that contain the respective wild-type enzymes. For designer cellulosome formats, the use of the thermostabilized scaffoldin proved critical for enhanced enzymatic performance under conditions of high temperatures.ConclusionsSimple improvement in the activity of a given enzyme does not guarantee its suitability for use in an enzyme cocktail or as a designer cellulosome component. The true merit of improvement resides in its ultimate contribution to synergistic action, which can only be determined experimentally. The relevance of the mutated thermostable enzymes employed in this study as components in multienzyme systems has thus been confirmed using designer cellulosome technology. Enzyme integration via a thermostable scaffoldin is critical to the ultimate stability of the complex at higher temperatures. Engineering of thermostable cellulases and additional lignocellulosic enzymes may prove a determinant parameter for development of state-of-the-art designer cellulosomes for their employment in the conversion of cellulosic biomass to soluble sugars.Graphical abstractConversion of conventional designer cellulosomes into thermophilic designer cellulosomesElectronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0577-z) contains supplementary material, which is available to authorized users.
Highlights
The concerted action of three complementary cellulases from Clostridium thermocellum, engineered to be stable at elevated temperatures, was examined on a cellulosic substrate and compared to that of the wild-type enzymes
In Clostridium thermocellum, the most extensively studied cellulosome producer, the non-catalytic primary scaffoldin subunit comprises a string of 9 repeating cohesin modules, plus a single carbohydrate-binding module (CBM) and an X-dockerin modular dyad that interacts with an anchoring scaffoldin at the cell surface [4]
Selection of thermostable Cel48S mutants Five potent mutants of Cel48S from C. thermocellum that were reported to be more thermostable than the wild-type enzyme were selected from the list of 60 mutants described in a previous study by Arnold and colleagues [27]
Summary
The concerted action of three complementary cellulases from Clostridium thermocellum, engineered to be stable at elevated temperatures, was examined on a cellulosic substrate and compared to that of the wild-type enzymes. We have employed the designer cellulosome strategy as a conceptual platform for promoting synergistic action among enzyme components [5,6,7,8] This strategy involves the use of recombinant chimaeric scaffoldins composed of cohesin modules originating from different bacterial species, whereby each cohesin binds to the matching dockerin of the same species, harbored by the different enzymes. These artificial nanodevices allow precision control of the composition and architecture of the cellulosome assembly and have proven to be efficient in their cellulolytic capacity [9,10,11,12]
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