Secretion of chimeric
cellulase from Clostridium thermocellum and Clostridium cellulolyticum by Bacillus
subtilis
Nguyen Hoang Ngoc Phuong, Phan Thi Phuong Trang, Tran Linh Thuoc and Nguyen Duc
Hoang
Res. J. Biotech.; Vol. 20(5); 182-190;
doi: https://doi.org/10.25303/205rjbt1820190; (2025)
Abstract
Lignocellulose recalcitrance poses a significant challenge in utilizing abundant
agricultural residues as renewable resources for high-value cellulose-based applications.
Some anaerobic bacteria can efficiently degrade lignocellulose using secreted protein
complexes known as cellulosomes. Chimeric cellulosomes are created by combining
genetic material from various sources to degrade different biomass substrates and
function well in extreme conditions. A variety of functional cellulases can be assembled
using scaffoldin which is a primary protein structure in a cellulosome through species-specific
interaction between a type-1 cohesin module from scaffoldin and a type-1 dockerin
module from cellulosomal cellulase.
This study focused on designing a chimeric cellulosomal cellulase which is a fusion
of the catalytic module of endoglucanase Cel5CCA from C. cellulolyticum (Ruminiclostridium
cellulolyticum) and a type-1 dockerin of endoglucanase Cel8A from C. thermocellum
(Acetivibrio thermocellus). The cellulase genes were cloned in Escherichia coli
and expressed extracellularly in Bacillus subtilis WB800N. They demonstrated carboxymethyl
cellulose hydrolysis (CMCase) in the Congo Red assay. Furthermore, they exhibited
protein bands around 50 kDa consistent with the theoretical molecular masses in
SDS-PAGE and Western blot analyses. Elisa analysis confirmed a species-specific
interaction between the type-1 dockerin of the cellulases and the type-1 cohesin
of the scaffoldin mini-CipA. Extracellular mixtures of the cellulases and mini-CipA
increased their synergistic CMCase activity by approximately 30% to 50% compared
to mixtures of the free cellulases without mini-CipA.
