Theα-amylase(AoAmy)from Aspergillus oryzae,a food-grade enzyme,has great potential in retarding bread staling.However,its industrial application is limited by low productivity and poor stability.In this study,a C-ter...Theα-amylase(AoAmy)from Aspergillus oryzae,a food-grade enzyme,has great potential in retarding bread staling.However,its industrial application is limited by low productivity and poor stability.In this study,a C-terminal truncated variant AoAmyB1 was constructed based on rational structure-function design,and efficiently expressed in the Pichia pastoris system.In comparison with the wild-type AoAmy,the optimal temperature of AoAmyB1 decreased to 55℃ from 60℃ without compromising its thermal stability,and its optimal pH remained unchanged at 6.0.Both its expression level and enzymatic activity of AoAmyB1 in culture supernatant increased more than 2-fold compared to wild-type AoAmy.The catalytic efficiency(Kcat/Km)of AoAmyB1 was 1.45-fold higher than that of the AoAmy,and the proportion of maltotriose(G3)in its hydrolysis products enhanced 2.7 folds.Molecular docking analysis revealed that C-terminal truncation optimized the substrate binding mode of AoAmyB1 with the binding energy decreasing from7.4 to8.9 kcal/mol.The application of AoAmyB1 in bread baking significantly improved bread quality.Compared to AoAmy,bread supplemented with AoAmyB1 showed an 18.4%higher specific volume,a 53%reduction in hardness after 5 days of storage,a 63%higher springiness retention rate,and superior sensory scores,indicating the promising application of AoAmyB1 in retarding bread staling.This study provides a solid foundation for the industrial production of AoAmyB1 and its application in the baking industry.展开更多
基金supported by the National Natural Science Foundation of China.China(grant number 22338013)Natural Science Foundation of Henan Province,China(grant number 242300421106)+1 种基金National Key Research and Development Plan of China(grant number 2023YFC2604903)Cultivation Project of Tuoxin Team in Henan University of Technology,China(grant number 2024TXTD13).
文摘Theα-amylase(AoAmy)from Aspergillus oryzae,a food-grade enzyme,has great potential in retarding bread staling.However,its industrial application is limited by low productivity and poor stability.In this study,a C-terminal truncated variant AoAmyB1 was constructed based on rational structure-function design,and efficiently expressed in the Pichia pastoris system.In comparison with the wild-type AoAmy,the optimal temperature of AoAmyB1 decreased to 55℃ from 60℃ without compromising its thermal stability,and its optimal pH remained unchanged at 6.0.Both its expression level and enzymatic activity of AoAmyB1 in culture supernatant increased more than 2-fold compared to wild-type AoAmy.The catalytic efficiency(Kcat/Km)of AoAmyB1 was 1.45-fold higher than that of the AoAmy,and the proportion of maltotriose(G3)in its hydrolysis products enhanced 2.7 folds.Molecular docking analysis revealed that C-terminal truncation optimized the substrate binding mode of AoAmyB1 with the binding energy decreasing from7.4 to8.9 kcal/mol.The application of AoAmyB1 in bread baking significantly improved bread quality.Compared to AoAmy,bread supplemented with AoAmyB1 showed an 18.4%higher specific volume,a 53%reduction in hardness after 5 days of storage,a 63%higher springiness retention rate,and superior sensory scores,indicating the promising application of AoAmyB1 in retarding bread staling.This study provides a solid foundation for the industrial production of AoAmyB1 and its application in the baking industry.
