Glycosidases play a pivotal role in the biocatalytic production of high-value glycosides,but their industrial applications are often limited by poor thermo-pH tolerance and incompatible reaction conditions in multienz...Glycosidases play a pivotal role in the biocatalytic production of high-value glycosides,but their industrial applications are often limited by poor thermo-pH tolerance and incompatible reaction conditions in multienzyme systems.To address these universal challenges,we developed a rational N-terminal truncation strategy usingβ-glucosidase B6 as a model.By removing 33 hydrophilic residues from the terminus,the engineered enzyme(33aa)exhibited significantly improved properties:a 10◦C higher optimal temperature(50◦C),enhanced acid tolerance(>50%activity at pH 5.0),and stronger substrate affinity(K_(m)=0.8517 mmol/L).Structural analysis revealed that the truncation reduced solvent-accessible surface area(SASA)and increasedα-helix density,explaining its superior stability.To demonstrate industrial application potential,we paired 33aa with G4 glucosidase-a thermophilic enzyme with complementary regioselectivity-creating an efficient cascade system.The optimized conditions(57.5◦C,pH 6.0,5:1 activity ratio)achieved a 3.32-fold higher product yield compared to single-enzyme systems in ginsenoside C-K production.This study establishes N-terminal truncation as a generalizable protein engineering approach to synchronize reaction conditions and enhance catalytic efficiency in glycosidase-based bioconversions,offering a versatile solution for industrial glycoside production.展开更多
基金funded by Shandong Provincial Technological Innovation Guidance Plan(YDZX2024106)the National Key Research and Development Program of China(2021YFC2102800)the National Natural Science Foundation of China(22078014).
文摘Glycosidases play a pivotal role in the biocatalytic production of high-value glycosides,but their industrial applications are often limited by poor thermo-pH tolerance and incompatible reaction conditions in multienzyme systems.To address these universal challenges,we developed a rational N-terminal truncation strategy usingβ-glucosidase B6 as a model.By removing 33 hydrophilic residues from the terminus,the engineered enzyme(33aa)exhibited significantly improved properties:a 10◦C higher optimal temperature(50◦C),enhanced acid tolerance(>50%activity at pH 5.0),and stronger substrate affinity(K_(m)=0.8517 mmol/L).Structural analysis revealed that the truncation reduced solvent-accessible surface area(SASA)and increasedα-helix density,explaining its superior stability.To demonstrate industrial application potential,we paired 33aa with G4 glucosidase-a thermophilic enzyme with complementary regioselectivity-creating an efficient cascade system.The optimized conditions(57.5◦C,pH 6.0,5:1 activity ratio)achieved a 3.32-fold higher product yield compared to single-enzyme systems in ginsenoside C-K production.This study establishes N-terminal truncation as a generalizable protein engineering approach to synchronize reaction conditions and enhance catalytic efficiency in glycosidase-based bioconversions,offering a versatile solution for industrial glycoside production.
