Structural analysis of Candida antarctica lipase B (CALB) indicates that side chain of leucine at 278 site lies above the entrance of the catalytic pocket, which prognosticates its potential role on substrate specific...Structural analysis of Candida antarctica lipase B (CALB) indicates that side chain of leucine at 278 site lies above the entrance of the catalytic pocket, which prognosticates its potential role on substrate specificity of the enzyme. To verify this presumption, shortened side chain of glycine or proline was rational designed and mutants were constructed by site-directed mutagenesis method. The colorimetric assay using p-nitrophenyl esters of fatty acids with various chain-lengths was used to study the substrate preference of lipases. Results indicated that L278G or L278P mutations both induced the drift of substrate specificity of CALB from p-nitrophenyl caprylate (pNP-C8) to longer carbon chain length of p-nitrophenyl caprate (pNP-C10). Meanwhile, Vmax value of two mutants to pNP-C10 was both higher than that of wild-type. Docking results also indicated that shortened side chain of glycine or proline residues substitution at this site could get rid of the space block present above the catalytic pocket, and made longer chain substrate (pNP-C10) enter into the catalytic pocket easier. The modulation of specificity observed allowed for building substrate binding model and opened new possibilities for designing ligand specific lipases.展开更多
In China,especially in Xinjiang Region,mulch film remaining in the soil has severely jeopardized the safety of soil resources.To numerically simulate the residual film-soil-recovery implementation system,a virtual mul...In China,especially in Xinjiang Region,mulch film remaining in the soil has severely jeopardized the safety of soil resources.To numerically simulate the residual film-soil-recovery implementation system,a virtual mulch film model with consistent physical and mechanical properties with real mulch film needs to be established.In this study,a flexible deformable virtual mulch film model was constructed using YADE software based on the Minkowski Sum principle and the ball-ball force-displacement constitutive rule,as well as the contact failure rule were established.The deformation behaviors of cylinders and PFacet elements,such as stretching,bending,and torsion,were described.By splicing the basic PFacet elements,a virtual mulch model was established.The mechanical model of a virtual mulch film under tension was established and the axial tensile stiffness coefficient kn was determined to be 43.30 N·m.To verify the physical and mechanical properties of this virtual mulch film,both real and virtual stretching and tearing tests were conducted.The experimental results showed that:in the process of stretching and tearing of real and virtual films,the properties of morphological features of both are basically identical;however,they clearly differ in force-displacement.The viscoelastic constitutive model between balls and yield judgment conditions requires further study.展开更多
文摘Structural analysis of Candida antarctica lipase B (CALB) indicates that side chain of leucine at 278 site lies above the entrance of the catalytic pocket, which prognosticates its potential role on substrate specificity of the enzyme. To verify this presumption, shortened side chain of glycine or proline was rational designed and mutants were constructed by site-directed mutagenesis method. The colorimetric assay using p-nitrophenyl esters of fatty acids with various chain-lengths was used to study the substrate preference of lipases. Results indicated that L278G or L278P mutations both induced the drift of substrate specificity of CALB from p-nitrophenyl caprylate (pNP-C8) to longer carbon chain length of p-nitrophenyl caprate (pNP-C10). Meanwhile, Vmax value of two mutants to pNP-C10 was both higher than that of wild-type. Docking results also indicated that shortened side chain of glycine or proline residues substitution at this site could get rid of the space block present above the catalytic pocket, and made longer chain substrate (pNP-C10) enter into the catalytic pocket easier. The modulation of specificity observed allowed for building substrate binding model and opened new possibilities for designing ligand specific lipases.
基金This work was financially supported by the Xinjiang Production and Construction Corps Major Technology Project(2018AA001/03)the China 13th Five-Year Key Research and Development Plan(2017YFD0701102-1)+1 种基金the Corps Key R&D Projects(2019AB007)the China National Natural Science Foundation Project(11562019).
文摘In China,especially in Xinjiang Region,mulch film remaining in the soil has severely jeopardized the safety of soil resources.To numerically simulate the residual film-soil-recovery implementation system,a virtual mulch film model with consistent physical and mechanical properties with real mulch film needs to be established.In this study,a flexible deformable virtual mulch film model was constructed using YADE software based on the Minkowski Sum principle and the ball-ball force-displacement constitutive rule,as well as the contact failure rule were established.The deformation behaviors of cylinders and PFacet elements,such as stretching,bending,and torsion,were described.By splicing the basic PFacet elements,a virtual mulch model was established.The mechanical model of a virtual mulch film under tension was established and the axial tensile stiffness coefficient kn was determined to be 43.30 N·m.To verify the physical and mechanical properties of this virtual mulch film,both real and virtual stretching and tearing tests were conducted.The experimental results showed that:in the process of stretching and tearing of real and virtual films,the properties of morphological features of both are basically identical;however,they clearly differ in force-displacement.The viscoelastic constitutive model between balls and yield judgment conditions requires further study.
