Lactobacillus amylovorus α-amylase is an endoenzyme with a peculiar starch-binding domain containing five identical family 26 carbohydrate-binding modules(CBM26).To investigate the impact of CBMs on catalytic activit...Lactobacillus amylovorus α-amylase is an endoenzyme with a peculiar starch-binding domain containing five identical family 26 carbohydrate-binding modules(CBM26).To investigate the impact of CBMs on catalytic activity,C-terminally truncated derivatives were constructed.The catalytic domain alone shows low affinity for the substrate and a very slow reaction rate,highlighting the importance of CBMs in maintaining the enzyme’s optimal conformation and dynamics for efficient catalysis.CBMs enhance enzyme performance,as indicated by improved catalytic efficiency(kcat/Km).Interestingly,the enzyme variant LaCD3CBM,with three CBMs,exhibits the best catalytic efficiency on soluble starch,outperforming the wild-type amylase,with five CBMs.In the case of insoluble starch,the catalytic domain alone could not hydrolyze it and even adding a CBM,the release of reducing sugars was very inefficient.However,this efficiency was significantly improved by two orders of magnitude for the three-CBM variant and the wild-type amylase.CBMs also played a crucial role in protein thermostability,contributing to a higher melting temperature of the catalytic domain with just a single CBM.Notably,thermostability did not increase with the number of CBMs.In conclusion,spatial arrangement and interactions between the catalytic domain and CBMs significantly influenced enzymatic efficiency with both soluble and insoluble substrates.These interactions optimize enzymatic activity and improve thermostability.展开更多
Transitory starch is an important carbon source in leaves,and its biosynthesis and metabolism are closely related to grain quality and yield.The molecular mechanisms controlling leaf transitory starch biosynthesis and...Transitory starch is an important carbon source in leaves,and its biosynthesis and metabolism are closely related to grain quality and yield.The molecular mechanisms controlling leaf transitory starch biosynthesis and degradation and their effects on rice(Oryza sativa)quality and yield remain unclear.Here,we show that OsLESV and OsESV1,the rice orthologs of AtLESV and AtESV1,are associated with transitory starch biosynthesis in rice.The total starch and amylose contents in leaves and endosperms are significantly reduced,and the final grain quality and yield are compromised in oslesv and osesv1 single and oslesv esv1 double mutants.Furthermore,we found that OsLESV and OsESV1 bind to starch,and this binding depends on a highly conserved C-terminal tryptophan-rich region that acts as a starch-binding domain.Importantly,OsLESV and OsESV1 also interact with the key enzymes of starch biosynthesis,granule-bound starch synthase I(GBSSI),GBSSII,and pyruvate orthophosphote dikiase(PPDKB),to maintain their protein stability and activity.OsLESV and OsESV1 also facilitate the targeting of GBSSI and GBSSII from plastid stroma to starch granules.Overexpression of GBSSI,GBSSII,and PPDKB can partly rescue the phenotypic defects of the oslesv and osesv1 mutants.Thus,we demonstrate that OsLESV and OsESV1 play a key role in regulating the biosynthesis of both leaf transitory starch and endosperm storage starch in rice.These findings deepen our understanding of the molecular mechanisms underlying transitory starch biosynthesis in rice leaves and reveal how the transitory starch metabolism affects rice grain quality and yield,providing useful information for the genetic improvement of rice grain quality and yield.展开更多
Sour liquid fermentation is a traditional and unique method used in China to produce starch that has favorable sensory evaluation,especially a pleasant mouthfeel.However,the complexity of the natural microbial flora u...Sour liquid fermentation is a traditional and unique method used in China to produce starch that has favorable sensory evaluation,especially a pleasant mouthfeel.However,the complexity of the natural microbial flora used in the method often caused severe fluctuations in production efficiency and product quality.Hence,it is important to explore the performance and impact of each starch-flocculating bacteria on starch structure.In this study,five previously isolated starch-flocculating bacterial strains were used to ferment sweet potato starch individually.Through analyzing fermentation parameters,Leuconostoc citreum SJ-48 was identified as the strain with the highest starch flocculation ability.A more dispersed particle size distribution of starch after fermentation was observed,possibly due to the clumping of starch granules by the bacteria.It was found that the amylose content of fermented starch increased by 27.57%-42.28%,and the relative crystallinity of starch increased significantly after fermentation.Finally,the starch-binding proteins of the bacterial cell were characterized using LC-ESI-MS/MS,and it was revealed that the strong starch flocculation ability of L.citreum SJ-48 was linked to the glycoside hydrolase family 70 on its surface.This study demonstrated that the discrepancies in the flocculation ability of the different bacterial strains and subsequent changes in the fermented starch properties were mainly influenced by the different proteins on their surfaces.展开更多
基金supported by DGAPA,UNAM grant IN216722Conahcyt to complete their master's degrees in Biochemical Sciences at UNAM.
文摘Lactobacillus amylovorus α-amylase is an endoenzyme with a peculiar starch-binding domain containing five identical family 26 carbohydrate-binding modules(CBM26).To investigate the impact of CBMs on catalytic activity,C-terminally truncated derivatives were constructed.The catalytic domain alone shows low affinity for the substrate and a very slow reaction rate,highlighting the importance of CBMs in maintaining the enzyme’s optimal conformation and dynamics for efficient catalysis.CBMs enhance enzyme performance,as indicated by improved catalytic efficiency(kcat/Km).Interestingly,the enzyme variant LaCD3CBM,with three CBMs,exhibits the best catalytic efficiency on soluble starch,outperforming the wild-type amylase,with five CBMs.In the case of insoluble starch,the catalytic domain alone could not hydrolyze it and even adding a CBM,the release of reducing sugars was very inefficient.However,this efficiency was significantly improved by two orders of magnitude for the three-CBM variant and the wild-type amylase.CBMs also played a crucial role in protein thermostability,contributing to a higher melting temperature of the catalytic domain with just a single CBM.Notably,thermostability did not increase with the number of CBMs.In conclusion,spatial arrangement and interactions between the catalytic domain and CBMs significantly influenced enzymatic efficiency with both soluble and insoluble substrates.These interactions optimize enzymatic activity and improve thermostability.
基金supported by the"STI2030-Major Project"of China(2023ZD04072)the National Major Agriculture Science and Technolohy Project of China(NK2022050102)+1 种基金the National Natural Science Foundation of China(32372099 and 32188102)the Innovation Program of Chinese Academy of Agricultural Sciences(CAAS-CACB-202402).
文摘Transitory starch is an important carbon source in leaves,and its biosynthesis and metabolism are closely related to grain quality and yield.The molecular mechanisms controlling leaf transitory starch biosynthesis and degradation and their effects on rice(Oryza sativa)quality and yield remain unclear.Here,we show that OsLESV and OsESV1,the rice orthologs of AtLESV and AtESV1,are associated with transitory starch biosynthesis in rice.The total starch and amylose contents in leaves and endosperms are significantly reduced,and the final grain quality and yield are compromised in oslesv and osesv1 single and oslesv esv1 double mutants.Furthermore,we found that OsLESV and OsESV1 bind to starch,and this binding depends on a highly conserved C-terminal tryptophan-rich region that acts as a starch-binding domain.Importantly,OsLESV and OsESV1 also interact with the key enzymes of starch biosynthesis,granule-bound starch synthase I(GBSSI),GBSSII,and pyruvate orthophosphote dikiase(PPDKB),to maintain their protein stability and activity.OsLESV and OsESV1 also facilitate the targeting of GBSSI and GBSSII from plastid stroma to starch granules.Overexpression of GBSSI,GBSSII,and PPDKB can partly rescue the phenotypic defects of the oslesv and osesv1 mutants.Thus,we demonstrate that OsLESV and OsESV1 play a key role in regulating the biosynthesis of both leaf transitory starch and endosperm storage starch in rice.These findings deepen our understanding of the molecular mechanisms underlying transitory starch biosynthesis in rice leaves and reveal how the transitory starch metabolism affects rice grain quality and yield,providing useful information for the genetic improvement of rice grain quality and yield.
基金supported by the Natural Science Foundation of Sichuan Province 2023NSFSC0181the 2115 Talent Development Program of China Agricultural University.
文摘Sour liquid fermentation is a traditional and unique method used in China to produce starch that has favorable sensory evaluation,especially a pleasant mouthfeel.However,the complexity of the natural microbial flora used in the method often caused severe fluctuations in production efficiency and product quality.Hence,it is important to explore the performance and impact of each starch-flocculating bacteria on starch structure.In this study,five previously isolated starch-flocculating bacterial strains were used to ferment sweet potato starch individually.Through analyzing fermentation parameters,Leuconostoc citreum SJ-48 was identified as the strain with the highest starch flocculation ability.A more dispersed particle size distribution of starch after fermentation was observed,possibly due to the clumping of starch granules by the bacteria.It was found that the amylose content of fermented starch increased by 27.57%-42.28%,and the relative crystallinity of starch increased significantly after fermentation.Finally,the starch-binding proteins of the bacterial cell were characterized using LC-ESI-MS/MS,and it was revealed that the strong starch flocculation ability of L.citreum SJ-48 was linked to the glycoside hydrolase family 70 on its surface.This study demonstrated that the discrepancies in the flocculation ability of the different bacterial strains and subsequent changes in the fermented starch properties were mainly influenced by the different proteins on their surfaces.
