Hybrid Nanocellulosome Design from Cellulase Modules on Nanoparticles: Synergistic Effect of Catalytically Divergent Cellulase Modules on Cellulose Degradation Activity

Abstract

Cellulosomes, which are assemblies of cellulases with various catalytic functions on a giant scaffoldin protein with a carbohydrate-binding module (CBM), efficiently degrade solid cellulosic biomass by means of synergistically coupled hydrolysis reactions. In this study, we constructed hybrid nanocellulosomes from the biotinylated catalytic domains (CDs) of two catalytically divergent cellulases (an endoglucanase and a processive endoglucanase) and biotinylated CBMs by clustering the domains and modules on streptavidin-conjugated nanoparticles. Nanocellulosomes constructed by separately clustering each type of CD with multiple CBMs on nanoparticles showed 5-fold enhancement in cellulase degradation activity relative to that of the corresponding free CDs, and mixtures of the two types of nanocellulosomes gradually and synergistically enhanced cellulase degradation activity as the CBM valency increased (finally, 2.5 times). Clustering the two types of CD together on the same nanoparticle resulted in a greater synergistic effect that was independent of CBM valency; consequently, nanocellulosomes composed of equal amounts of the endo and endoprocessive CDs clustered on a nanoparticle along with multiple CBMs (CD/CBM = 7:23) showed the best cellulose degradation activity, producing 6.5 and 2.4 times the amount of reducing sugars produced from amorphous and crystalline cellulose, respectively, by the native free CDs and CBMs in the same proportions. Our results demonstrate that hybrid nanocellulosomes constructed from the building blocks of cellulases and cellulosomes modules have the potential to serve as high-performance artificial cellulosomes.

Enzymatic ligation of an antibody and arginine 9 peptide for efficient and cell-specific siRNA delivery

“…An effective strategy for functional expression is to separate and express each functional domain and then fuse them in vitro 22 . This can effectively eliminate the complications related to structure and function as well as reduce undesirable non-expression and expression as an insoluble fraction.…”

Enzymatic Ligation of Antibody and Cell-Penetrating Peptide for Efficient and Cell-Specific siRNA Delivery

“…An effective strategy for functional expression is to separate and express each functional domain and then fuse them in vitro. 22 This can effectively eliminate the complications related to structure and function, reduce undesirable non-expression, and expression as an insoluble fraction. Reconstruction of each functional unit is generally carried out by bioconjugation using a polymer or particle as support; however, the production of well-de ned antibody conjugates is extremely challenging owing to the required selectivity, speci city, and reactivity under physiological conditions.…”

Salt-Switchable Artificial Cellulase Regulated by a DNA Aptamer

“…Phosphoric-acid swollen cellulose (PASC) was prepared from Avicel according to the previous report. 15 Oligonucleotides. All of the following DNAs were synthesized by Tsukuba Oligo Service (Tsukuba, Japan) and dissolved in nucleasefree water (Wako) stocked at −20 °C before use.…”

“…Synthesized genes were cloned into NdeI and XhoI sites of modified pET22 vector with DNA fragment coding IgA hinge linker (SPSTPPTPSPSTPP)-biotin acceptor peptide (AviTag: GGLNDIFEAQKIEWH)-polyhistidinetag (HHHHHH) at the C terminus. 15 To replace the Avitag fragment with HAtag (YPYDVPDYA)-linker (GGGS)-MTG reactive lysine-tag (MRHKGS) fragment, each expression vector fragment except for AviTag was prepared using inverse PCR. DNA fragment encoding HAtag-linker-MTG reactive Ktag fragment with 15 base flanking regions homologous to vector ends was purchased from Fasmac (Kanagawa, Japan).…”