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
n-3 polyunsaturated fatty acids(n-3 PUFA)have biologically important functions in human beings,but their contents in natural oils are usually low.This study investigated the concentration of n-3 PUFA-enriched glycerid...n-3 polyunsaturated fatty acids(n-3 PUFA)have biologically important functions in human beings,but their contents in natural oils are usually low.This study investigated the concentration of n-3 PUFA-enriched glycerides by Candida antarctica lipase A(CAL-A)-catalyzed selective methanolysis of algal oil.First,lipases and acyl acceptors were screened.Subsequently,the methanolysis conditions including methanol concentration in aqueous solution,molar ratio of methanol to oil,reaction temperature,lipase loading and reaction time,were optimized.The results indicated that CAL-A could effectively identify n-3 PUFA during methanolysis.Saturated fatty acids and monounsaturated fatty acids were released from algal oil by CAL-A-catalyzed methanolysis,causing the production of n-3 PUFA-enriched glycerides.Under optimal conditions(3:1 of molar ratio of methanol to algal oil,20%methanol aqueous solution,6%CAL-A dosage,25℃,18 h),the n-3 PUFA content increased from the initial 45.96%to 73.96%,with an n-3 PUFA yield of 81.76%.The reusability of CAL-A demonstrated that under optimal conditions the lipase could be used for 8 times.Therefore,this enzymatic process is efficient for synthesis of n-3 PUFA-enriched glycerides.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 study was financially supported by"National Natural Science Foundation of China(Grant No.:31972035).
文摘n-3 polyunsaturated fatty acids(n-3 PUFA)have biologically important functions in human beings,but their contents in natural oils are usually low.This study investigated the concentration of n-3 PUFA-enriched glycerides by Candida antarctica lipase A(CAL-A)-catalyzed selective methanolysis of algal oil.First,lipases and acyl acceptors were screened.Subsequently,the methanolysis conditions including methanol concentration in aqueous solution,molar ratio of methanol to oil,reaction temperature,lipase loading and reaction time,were optimized.The results indicated that CAL-A could effectively identify n-3 PUFA during methanolysis.Saturated fatty acids and monounsaturated fatty acids were released from algal oil by CAL-A-catalyzed methanolysis,causing the production of n-3 PUFA-enriched glycerides.Under optimal conditions(3:1 of molar ratio of methanol to algal oil,20%methanol aqueous solution,6%CAL-A dosage,25℃,18 h),the n-3 PUFA content increased from the initial 45.96%to 73.96%,with an n-3 PUFA yield of 81.76%.The reusability of CAL-A demonstrated that under optimal conditions the lipase could be used for 8 times.Therefore,this enzymatic process is efficient for synthesis of n-3 PUFA-enriched glycerides.
