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About the Authors:
Craig Blanchette
* E-mail: [email protected] (CB); [email protected] (MT)
Affiliation: Physical and Life Sciences, Lawrence Livermore National Laboratory, Livermore, California
Catherine I. Lacayo
Nicholas O. Fischer
Mona Hwang
Michael P. Thelen
* E-mail: [email protected] (CB); [email protected] (MT)
Affiliation: Physical and Life Sciences, Lawrence Livermore National Laboratory, Livermore, California
Introduction
The inefficient conversion of plant-derived cellulose to fermentative sugars has been identified as one of the limiting factors for widespread production of alternative fuels from lignocellulose feedstocks [1]. New methods to increase cellulase enzyme kinetics and stability are critical for the economic feasibility of biofuel production. Most current industrial processing of plant biomass involves harsh thermochemical pretreatment to open up the physical structure of intricately complexed polymers of lignin, cellulose and hemicellulose polysaccharides, followed by hydrolysis of the cellulose using microbial enzymes that are free in solution. In contrast, particulate enzymes that are highly effective in deconstructing untreated biomass are found in bacterial cellulosomes. These multiprotein complexes assemble different glycosyl hydrolases on a scaffold protein [1]–[3], promoting synergy and increased efficiency in cellulolytic action [4], [5]. This cooperation of physically associated enzymes has lead to the notion of using synthetic biology to engineer novel cellulosomes for lignocellulose breakdown [4]–[6]. However, bacterial assembly of the cellulosome has been shown to occur through a tightly controlled and complex process, and little is known about the essential factors in this process [7]. Thus, the production of engineered cellulosomes for use in biofuel processing remains elusive.
Since the key feature of the enzymatic efficiency of cellulosomes is the clustering of cellulases in a single macromolecular complex, we hypothesize that assembling an isolated cellulase onto a suitable synthetic nano-scale material could result in an increase in enzymatic efficiency. A robust nano-scale platform, such as polymeric nanoparticles, can serve as an analog to the cellulosomal scaffold that holds together individual enzymes in the multimeric complex. This approach can be used with cellulase enzymes that have already been purified and extensively characterized, therefore bypassing the difficulties in recombinant methods required to engineer cellulosomes. To test our idea, we conjugated a cellulase to spherical nanometer-size beads (nanospheres), and characterized the enzymatic activity of the cellulase-nanosphere complex (cellulase:NS) on different substrates: soluble carboxymethyl cellulose (CMC); insoluble microcrystalline cellulose; and cellulose thickenings in secondary...