title: A facile method for controlling the reaction equilibrium of sphingolipid ceramide N-deacylase for lyso-glycosphingolipid production.
abstract: Lyso-glycosphingolipids (lyso-GSLs), the N-deacylated forms of glycosphingolipids (GSLs), are important synthetic intermediates for the preparation of GSL analogs. Although lyso-GSLs can be produced by hydrolyzing natural GSLs using sphingolipid ceramide N-deacylase (SCDase), the yield for this reaction is usually low because SCDase also catalyzes the reverse reaction, ultimately establishing an equilibrium between hydrolysis and synthesis. In the present study, we developed an efficient method for controlling the reaction equilibrium by introducing divalent metal cation and detergent in the enzymatic reaction system. In the presence of both Ca(2+) and taurodeoxycholate hydrate, the generated fatty acids were precipitated by the formation of insoluble stearate salts and pushing the reaction equilibrium toward hydrolysis. The yield of GM1 hydrolysis can be achieved as high as 96%, with an improvement up to 45% compared with the nonoptimized condition. In preparative scale, 75 mg of lyso-GM1 was obtained from 100 mg of GM1 with a 90% yield, which is the highest reported yield to date. The method can also be used for the efficient hydrolysis of a variety of GSLs and sphingomyelin. Thus, this method should serve as a facile, easily scalable, and general tool for lyso-GSL production to facilitate further GSL research.
authors.affiliation: State Key Laboratory of Microbial Metabolism and Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
authors.forename: Feng-Tao
authors.initials: FT
authors.lastname: Huang
authors.affiliation: State Key Laboratory of Microbial Metabolism and Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
authors.forename: Yun-Bin
authors.initials: YB
authors.lastname: Han
authors.affiliation: State Key Laboratory of Microbial Metabolism and Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Feng
authors.affiliation: State Key Laboratory of Microbial Metabolism and Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China Shanghai Collaborative Innovation Center for Biomanufacturing (SCICB), East China University of Science and Technology, Shanghai 200237, China.
authors.forename: Guang-Yu
authors.initials: GY
authors.lastname: Yang
mesh_terms: D000581:Amidohydrolases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D002384:Catalysis; D004705:Endocytosis; D004926:Escherichia coli / Q000235:genetics; D005677:G(M1) Ganglioside / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D015971:Gene Expression Regulation, Enzymologic; D006028:Glycosphingolipids / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry
chemical_list: D006028:Glycosphingolipids; C029465:lysoGM1 ganglioside; D005677:G(M1) Ganglioside; D000581:Amidohydrolases; C096410:sphingolipid ceramide N-deacylase
keywords: endocytosis; enzymology; gangliosides; lyso-GM1; mass spectrometry; sphingolipid engineering; sphingolipids