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Rapid Evolution for High Performance Nano-Cellulosome By Module Library Approach

Authors: 
Nakazawa, H., Tohoku University
Okada, I., Tohoku University
Ishigaki, Y., Tohoku University
Kobayashi, E., Tohoku University
Umetsu, M., Tohoku University

An economical conversion from inedible cellulosic biomass materials to water-soluble sugar units is expected to produce food, feed, chemicals and fuels. Microbial cellulolytic enzymes, called cellulase is an enzyme which hydrolyzes cellulose to water-soluble sugar units with low energy and low environmental loads, and more than 1000 cellulases are reported. About 33% of these cellulases have carbohydrate-binding domain (CBD), fused to catalytic domain (CD), and the CBDs promotes effective degradation of cellulose by increasing substantial substrate concentration around CD. Further, in some rumen bacteria, CDs with different functions are clustered on a giant scaffold protein containing a CBD, via cohesin-dockerin interaction, called cellulosome, to efficiently degrade cellulose. We have proposed a new design of artificial cellulosome, called hybrid nano-cellulosome (HyNaC), by clustering recombinant biotinylated CDs and CBDs on streptavidin-modified CdSe quantum dot via biotin-avidin interaction.This HyNaC shows drastic enhancement of degradation for water insoluble cellulose.

We selected optimal CD and CBD from the library containing 50 kinds of CDs and 20 kinds of CBDs for screening high performance HyNaC. The HyNaC structure format produced more reducing sugar than the corresponding native module structure, and a replacement of the kind of CBD contributed considerably to the enhancement of the degradation of amorphous cellulose on HyNaC, whereas, chimera module enzyme with each CBD did not show. In addition, mixture experiment of HyNaC with different CBD shows the synergistic effect between CBDs observed on HyNaC with most active CD of E2 from Thermobifida fusca.

In this study, to understand the mechanism of the enhanced degradation on HyNaC by variety of CBD, we investigated the binding affinity and the saturated binding amount of CBD library to amorphous and crystalline cellulose and extracted a rule to enhance the cellulolytic activity of HyNaC based on CBD.