title: Characterization of a novel enzyme-Starmerella bombicola lactone esterase (SBLE)-responsible for sophorolipid lactonization.
abstract: We recently discovered a novel enzyme in the exoproteome of Starmerella bombicola, which is structurally related to Candida antarctica lipase A. A knockout strain for this enzyme does no longer produce lactonic sophorolipids, prompting us to believe that this protein is the missing S. bombicola lactone esterase (SBLE). SBLE catalyzes a rather unusual reaction, i.e., an intramolecular esterification (lactonization) of acidic sophorolipids in an aqueous environment, which raised questions about its activity and mode of action. Here, we report the heterologous production of this enzyme in Pichia pastoris and its purification in a two-step strategy. Purified recombinant SBLE (rSBLE) was used to perform HPLC and liquid chromatography mass spectrometry (LCMS)-based assays with different sophorolipid mixtures. We experimentally confirmed that SBLE is able to perform ring closure of acetylated acidic sophorolipids. This substrate was selected for rSBLE kinetic studies to estimate the apparent values of Km. We established that rSBLE displays optimal activity in the pH range of 3.5 to 6 and has an optimal temperature in the range of 20 to 50 °C. Additionally, we generated a rSBLE mutant through site-directed mutagenesis of Ser194in the predicted active site pocket and show that this mutant is lacking the ability to lactonize sophorolipids. We therefore propose that SBLE operates via the common serine hydrolase mechanism in which the catalytic serine residue is assisted by a His/Asp pair.
authors.affiliation: Laboratory for Protein Biochemistry and Biomolecular Engineering, Department of Biochemistry and Microbiology, Ghent University, K. Ledeganckstraat 35, 9000, Ghent, Belgium.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Ciesielska
authors.affiliation: Laboratory of Industrial Biotechnology and Biocatalysis, Ghent University, Coupure links 653, 9000, Ghent, Belgium.
authors.forename: Sophie L K W
authors.initials: SL
authors.lastname: Roelants
authors.affiliation: Laboratory of Industrial Biotechnology and Biocatalysis, Ghent University, Coupure links 653, 9000, Ghent, Belgium.
authors.forename: Inge N A
authors.initials: IN
authors.lastname: Van Bogaert
authors.affiliation: Laboratory for Protein Biochemistry and Biomolecular Engineering, Department of Biochemistry and Microbiology, Ghent University, K. Ledeganckstraat 35, 9000, Ghent, Belgium.
authors.forename: Stijn
authors.initials: S
authors.lastname: De Waele
authors.affiliation: Laboratory for Protein Biochemistry and Biomolecular Engineering, Department of Biochemistry and Microbiology, Ghent University, K. Ledeganckstraat 35, 9000, Ghent, Belgium.
authors.forename: Isabel
authors.initials: I
authors.lastname: Vandenberghe
authors.affiliation: Laboratory for Protein Biochemistry and Biomolecular Engineering, Department of Biochemistry and Microbiology, Ghent University, K. Ledeganckstraat 35, 9000, Ghent, Belgium.
authors.forename: Sara
authors.initials: S
authors.lastname: Groeneboer
authors.affiliation: Laboratory of Industrial Biotechnology and Biocatalysis, Ghent University, Coupure links 653, 9000, Ghent, Belgium.
authors.forename: Wim
authors.initials: W
authors.lastname: Soetaert
authors.affiliation: Laboratory for Protein Biochemistry and Biomolecular Engineering, Department of Biochemistry and Microbiology, Ghent University, K. Ledeganckstraat 35, 9000, Ghent, Belgium. Bart.devreese@ugent.be.
authors.forename: Bart
authors.initials: B
authors.lastname: Devreese
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002265:Carboxylic Ester Hydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020134:Catalytic Domain; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D017353:Gene Deletion; D015870:Gene Expression; D006017:Glycolipids / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007783:Lactones / Q000378:metabolism*; D013058:Mass Spectrometry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004718:Saccharomycetales / Q000201:enzymology* / Q000235:genetics; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D006017:Glycolipids; D007783:Lactones; D050505:Mutant Proteins; D011994:Recombinant Proteins; D002265:Carboxylic Ester Hydrolases
keywords: Enzyme; Lactone esterase; Lactonization; Natural product synthesis; Sophorolipids; Starmerella (Candida) bombicola