We describe two novel approaches for the determination of glucosamine(GlcN).The first approach is based on the chemical derivatization of GlcN with the non-fluorophor 1,3-diphenyl-1,3-propanedione(DPPD),which results ...We describe two novel approaches for the determination of glucosamine(GlcN).The first approach is based on the chemical derivatization of GlcN with the non-fluorophor 1,3-diphenyl-1,3-propanedione(DPPD),which results in a condensation product with interesting fluorescent properties.The obtained compound was isolated by silica-gel chromatography and its structure elucidated by NMR and mass spectrometry.The second approach is based on a previously undescribed sensitivity of the enzyme glucosamine-6-phosphate deaminase(GPDA)towards GlcN,which resulted in the precipitation of the enzyme.Using a rational enzyme engineering approach and both liquid-based and plate-based screening methods,mutational GPDA variants with significantly improved precipitation properties were identified and characterized.These novel glucosamine detection methods may be a useful addition to the repertoire of currently available glucosamine detection sensors.展开更多
The posttranslational modification of proteins with complex carbohydrate moieties(glycosylation)regulates the process of fruit ripening.Exoglycosidases are enzymes that can trim this protein glycosylation and are ther...The posttranslational modification of proteins with complex carbohydrate moieties(glycosylation)regulates the process of fruit ripening.Exoglycosidases are enzymes that can trim this protein glycosylation and are therefore considered to be important targets in the control of fruit ripening and softening.Melting-type peaches are popular seasonal fruits in many Asian regions,but the extremely short shelf-life of the peach fruits significantly hampers their economic value.To investigate the effect of the protein glycosylation and exoglycosidase activities on the development and ripening of the peach fruit,the fruit flesh of the melting peach cultivar'Xia hui 6'at five different maturity stages were analyzed.The N-glycan profile of each sample was characterized and quantified by HILIC-UPLC and MALDI-TOF mass spectrometry,revealing two characteristic N-glycan structures(MMXF and GnGnMXF)which were strongly affected by the state of maturity.Furthermore,it was shown that one of the endogenous exoglycosidase activities analyzed(β-N-acetylhexosaminidase,β-Hex)correlated with the MMXF and GnGnMXF N-glycan structures(p<0.05)in an obverse manner.These findings lay the foundation for further elucidation of the physiological functions of protein glycosylation in peach fruit development and ripening.展开更多
基金supported in parts by the National Natural Science Foundation of China (grant numbers 31471703,31671854,and 31871754)the 100 Foreign Talents Plan (grant number JSB2014012).
文摘We describe two novel approaches for the determination of glucosamine(GlcN).The first approach is based on the chemical derivatization of GlcN with the non-fluorophor 1,3-diphenyl-1,3-propanedione(DPPD),which results in a condensation product with interesting fluorescent properties.The obtained compound was isolated by silica-gel chromatography and its structure elucidated by NMR and mass spectrometry.The second approach is based on a previously undescribed sensitivity of the enzyme glucosamine-6-phosphate deaminase(GPDA)towards GlcN,which resulted in the precipitation of the enzyme.Using a rational enzyme engineering approach and both liquid-based and plate-based screening methods,mutational GPDA variants with significantly improved precipitation properties were identified and characterized.These novel glucosamine detection methods may be a useful addition to the repertoire of currently available glucosamine detection sensors.
基金supported by the Priority Academic Program Development of Jiangsu Higher Education Institutions (grant KYZ201824 to Ting Wang)the National Natural Science Foundation of China (NSFC grants 31471703,31671854,31871793 and 31871754 to Josef Voglmeir and Li Liu).
文摘The posttranslational modification of proteins with complex carbohydrate moieties(glycosylation)regulates the process of fruit ripening.Exoglycosidases are enzymes that can trim this protein glycosylation and are therefore considered to be important targets in the control of fruit ripening and softening.Melting-type peaches are popular seasonal fruits in many Asian regions,but the extremely short shelf-life of the peach fruits significantly hampers their economic value.To investigate the effect of the protein glycosylation and exoglycosidase activities on the development and ripening of the peach fruit,the fruit flesh of the melting peach cultivar'Xia hui 6'at five different maturity stages were analyzed.The N-glycan profile of each sample was characterized and quantified by HILIC-UPLC and MALDI-TOF mass spectrometry,revealing two characteristic N-glycan structures(MMXF and GnGnMXF)which were strongly affected by the state of maturity.Furthermore,it was shown that one of the endogenous exoglycosidase activities analyzed(β-N-acetylhexosaminidase,β-Hex)correlated with the MMXF and GnGnMXF N-glycan structures(p<0.05)in an obverse manner.These findings lay the foundation for further elucidation of the physiological functions of protein glycosylation in peach fruit development and ripening.
