Characterization of a Thermophilic Monosaccharide Stimulated β-Giucosidase from Acidothermus cellulolyticus

The gene（ABK51908） from Acidothermus cellulolyncus encodes a mature protein of 484 residues with a calculated molecular mass of 53.0 kDa. Sequence analysis revealed that the protein had 59% identity to the β-glucosidases CAA82733, which belongs to glycoside hydrolase family I（GH1）. We cloned and expressed the gene in Escherichia coli BL21-Gold（DE3）. The recombinant protein（AcBg） had an optimal pH and temperature of 7.0 and 70℃, respectively. The specific activities of AeBg under optimal conditions were 290 and 33 U/rag for p-nitrophenyl-fl-D-glucopyranoside（pNPG） and cellobiose, respectively. AcBg hydrolyzed the oligosaccharide sequentially from the non-reducing end to produce glucose units according to the results of HPLC analysis. AcBg showed high salt tolerance and monosaccharide-stimulation properties. Its activity rose more than 2-fold when 5 mol/L NaC1/KC1 were added. The activity of the β-glucosidase was remarkably enhanced in the presence of 0.2 mol/L D-glucose（increased more than 1.9-fold）, 0.1 mol/L a-methyl-D-glucose（increased more than 1.4-fold） and 1.0 mol/L D-xylose（increased more than 1.9-fold）. The catalysis kinetics and structural changes in various concentra- tions of glucose were determined. The results indicate that glucose reduces substrate affinity and causes conforma- tional changes.

Biofuel production from lignocellulose via thermophile-based consolidated bioprocessing

The depletion of fossil fuels and their impact on the environment have led to efforts to develop alternative sustainable fuels.While biofuel derived from lignocellulose is considered a sustainable,renewable,and green energy source,enhancing biofuel production and achieving a cost-effective bioconversion of lignocellulose at existing bio-refineries remains a challenge.Consolidated bioprocessing(CBP)using thermophiles can simplify this operation by integrating multiple processes,such as hydrolytic enzyme production,lignocellulose degrada-tion,biofuel fermentation,and product distillation.This paper reviews recent developments in the conversion of lignocellulose to biofuel using thermophile-based CBP.First,advances in thermostable enzyme and thermophilic lignocellulolytic microorganism discovery and development for lignocellulosic biorefinery use are outlined.Then,several thermophilic CBP candidates and thermophilic microbes engineered to drive CBP of lignocellulose are reviewed.CRISPR/Cas-based genome editing tools developed for thermophiles are also highlighted.The poten-tial applications of the Design-Build-Test-Learn(DBTL)synthetic biology strategy for designing and constructing thermophilic CBP hosts are also discussed in detail.Overall,this review illustrates how to develop highly sophis-ticated thermophilic CBP hosts for use in lignocellulosic biorefinery applications.

Conversion of D-glucose to L-lactate via pyruvate by an optimized cell-free enzymatic biosystem containing minimized reactions

Cell-free synthetic enzymatic biosystem is emerging to expand the traditional biotechnological mode by utilizing a number of purified/partially purified enzymes and coenzymes in a single reaction vessel for the production of desired products from low-cost substrates.Here,a cell-free synthetic biosystem containing minimized number of reactions was designed for the conversion of D-glucose to L-lactate via pyruvate.This NADH-balanced biosystem was comprised of only 5 thermophilic enzymes without ATP supplementation.After optimization of enzyme loading amounts,buffer concentration and cofactor concentration,D-glucose was converted to L-lactate with a product yield of∼90%.Our study has provided an emerging platform with potentials in producing pyruvatederived chemicals,and may promote the development of cell-free synthetic enzymatic biosystems for biomanufacturing.