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-glucos...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.展开更多
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 produc...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.展开更多
文摘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.
基金the Key Research Program of the Chinese Academy of Sciences(Grant No.ZDRW-ZS-2016-3)National Natural Science Foundation of China(Grant No.31600636).
文摘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.
