Patent number 8557742 is assigned to
The following quote was obtained by the news editors from the background information supplied by the inventors: "Cellulosic biomass is the most abundant renewable natural resource. Generated at a rate of .about.100 billion dry tons/year by the biosphere, cellulosic biomass has the potential to replace the world's demand for diminishing fossil fuels. However, according to Zhang, Y. H. P. 'One of the most important and difficult technological challenges is to overcome the recalcitrance of natural lignocellulosic materials, which must be enzymatically hydrolyzed to produce fermentable sugars.' See, (Zhang, Y. H. P., et al., 'Outlook for cellulase improvement: Screening and selection strategies.' Biotechnol. Adv., 2006, 24: 452-481).
"Cellulose is a polysaccharide consisting of 100 to 20,000.beta.-1-4 linked glucose units. Cellulases, the class of enzymes that hydrolyze cellulose, have attracted immense interest for their ability to degrade cellulosic biomass into glucose for biofuel production. Three cellulase sub-classes (endoglucanase, exoglucanase, .beta.-glucosidase) work synergistically to hydrolyze cellulose. Endoglucanases hydrolyze intramolecular .beta.-1-4-glucosidic bonds in insoluble cellulosic material to produce new chain ends. Exoglucanases progressively hydrolyzed the chain ends liberating small water-soluble oligosaccharide products. The soluble products are finally hydrolyzed by .beta.-glucosidase into glucose (Schulein, M., 'Protein engineering of cellulases.' Biochim. Biophys. Acta-Protein Struct. Molec. Enzym., 2000, 1543(2): 239-252)."
In addition to the background information obtained for this patent, NewsRx journalists also obtained the inventors' summary information for this patent: "The invention includes a system for improving enzyme activity toward a solid substrate. In one aspect, the invention includes a method of selecting for enhanced cellulase activity by means of in vitro compartmentalization in which a cellulosic microparticle functions both as the targeted solid substrate and as a structure for negative selection. In another aspect, the microparticle may be composed of lignin or other insoluble substrate.
"Various implementations of the invention include a system to generate a polynucleotide library. The library encodes enzyme variants having different activity toward the targeted solid substrate. The system further includes a means of linking individual or clonal copies of individual gene variants to a microparticle composed of the targeted solid substrate. The system also includes means for compartmentalizing individual microparticles with linked genes in an emulsion containing an in vitro transcription/translation reaction. Additionally, the system includes means for expressing each linked gene variant in order to produce enzyme in each emulsion compartment having different activity toward the microparticle. Microparticles within emulsion compartments containing highly active enzyme variants are degraded, thus liberating the linked gene variant from microparticle. Furthermore, the system includes means for breaking the emulsion compartments and selectively recovering the liberated gene variants that encode enzymes with enhanced enzyme activity toward the microparticle substrate. Further enzyme activity enhancement can be achieved by generating a new polynucleotide library derived from the recovered gene variants and repeating the steps above.
"Other implementations of the invention may include the use of a cleavable linker between the gene variant and a non-reactive carrier microparticle. The linker may, for example, be composed of cellulose, hemicellulose, or lignin. Within emulsion compartments containing highly active enzyme variants, the linker is degraded, thus liberating the linked gene variant from the carrier microparticle.
"The invention can include one or more of the following advantages. The invention utilizes a novel IVC-based, selection-mode approach to optimizing enzymatic activity of cellulases on insoluble cellulose substrates, and is extensible to optimizing enzymatic activity on any insoluble substrate. Enzyme selection is performed on natural insoluble cellulosic material. Therefore, enzyme activity is tailored to the actual substrate of commercial interest, not a surrogate soluble or fluorogenic substrate. Populations of 10.sup.10-10.sup.12 gene variants can be surveyed, a vast improvement over screening-based approaches that are usually limited to 10,000 variants. Enzyme selection is performed completely in vitro, eliminating the organismal metabolic and genomic background that leads to off-target optimization.
"These and other features and advantages of the present invention will be presented in more detail in the following specification of the invention and the accompanying figures, which illustrate, by way of example, the principles of the invention."
URL and more information on this patent, see: Blazej, Robert G.; Paegel, Brian M.. Method of Enhancing Enzyme Activity. U.S. Patent Number 8557742, filed
Keywords for this news article include: Cellulases, Glucosidases,
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