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.
A mixture of protein-encoding gene fragments was ligated into linearized expression vectors in one pot at a time (iPaT) to simultaneously generate each protein expression vector. This high-throughput iPaT ligation method was stable and highly efficient for the simultaneous generation of more than 10 different expression vectors.
Molecular evolution was used to generate capping molecules that selectively bound to the noncellulose components in cellulosic biomass and facilitated access of cellulolytic enzymes to the substrate components. The peptides, which were selected by means of a phage-display method, strongly promoted the enzymatic degradation of cellulose components in the biomass.Scheme 1 Phage-displayed peptide library method used in this study. † Electronic supplementary information (ESI) available: Experimental procedures, adsorption isotherms against cellulose (Fig. S1), and peptide properties for identified biomass-binding peptides (Table S1). See
The fluorescence in situ hybridization (FISH)-based padlock probe and rolling circle amplification (RCA) method allows for the detection of point mutations. However, it requires multiple reaction steps and solution exchanges, making it costly, labor-intensive, and time-consuming. In this study, we aimed to improve the efficiency of padlock/RCA by determining the effects of microchannel shape and ultrasonic solution mixing. Using a circular-shaped microchamber and ultrasonic mixing, the efficiency of microfluidic padlock/RCA was improved, and the consumption of the expensive probe solution was reduced from 10 µL to approximately 3.5 µL. Moreover, the fluorescent probe hybridization time was reduced to 5 min, which is four times faster than that of the standard protocol. We used this method to successfully detect mitochondrial DNA and transcripts of β-actin and K-ras proto-oncogene codon 12 in cells. Our method offers improvements over current padlock/RCA methods and will be helpful in optimizing other microfluidics-based FISH-related analyses.
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