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Characterization of a novel enzyme - Starmerella bombicola lactone esterase (SBLE) - responsible for sophorolipid lactonization

Katarzyna Ciesielska (UGent) , Sophie Roelants (UGent) , Inge Van Bogaert (UGent) , Stijn De Waele (UGent) , Isabel Vandenberghe (UGent) , Sara Groeneboer (UGent) , Wim Soetaert (UGent) and Bart Devreese (UGent)
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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 K (m) . 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 A degrees C. Additionally, we generated a rSBLE mutant through site-directed mutagenesis of Ser(194) in 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.
Keywords
Starmerella (Candida) bombicola, Sophorolipids, Lactonization, Lactone esterase, Natural product synthesis, Enzyme, ANTARCTICA LIPASE-A, CANDIDA-ANTARCTICA, PICHIA-PASTORIS, INTERFACIAL ACTIVATION, EXPRESSION, GENE, BIOSURFACTANTS, PURIFICATION, CLONING, LIPIDS

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Chicago
Ciesielska, Katarzyna, Sophie Roelants, Inge Van Bogaert, Stijn De Waele, Isabel Vandenberghe, Sara Groeneboer, Wim Soetaert, and Bart Devreese. 2016. “Characterization of a Novel Enzyme - Starmerella Bombicola Lactone Esterase (SBLE) - Responsible for Sophorolipid Lactonization.” Applied Microbiology and Biotechnology 100 (22): 9529–9541.
APA
Ciesielska, K., Roelants, S., Van Bogaert, I., De Waele, S., Vandenberghe, I., Groeneboer, S., Soetaert, W., et al. (2016). Characterization of a novel enzyme - Starmerella bombicola lactone esterase (SBLE) - responsible for sophorolipid lactonization. APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 100(22), 9529–9541.
Vancouver
1.
Ciesielska K, Roelants S, Van Bogaert I, De Waele S, Vandenberghe I, Groeneboer S, et al. Characterization of a novel enzyme - Starmerella bombicola lactone esterase (SBLE) - responsible for sophorolipid lactonization. APPLIED MICROBIOLOGY AND BIOTECHNOLOGY. 2016;100(22):9529–41.
MLA
Ciesielska, Katarzyna, Sophie Roelants, Inge Van Bogaert, et al. “Characterization of a Novel Enzyme - Starmerella Bombicola Lactone Esterase (SBLE) - Responsible for Sophorolipid Lactonization.” APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 100.22 (2016): 9529–9541. Print.
@article{8510825,
  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 K (m) . 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 A degrees C. Additionally, we generated a rSBLE mutant through site-directed mutagenesis of Ser(194) in 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.},
  author       = {Ciesielska, Katarzyna and Roelants, Sophie and Van Bogaert, Inge and De Waele, Stijn and Vandenberghe, Isabel and Groeneboer, Sara and Soetaert, Wim and Devreese, Bart},
  issn         = {0175-7598},
  journal      = {APPLIED MICROBIOLOGY AND BIOTECHNOLOGY},
  language     = {eng},
  number       = {22},
  pages        = {9529--9541},
  title        = {Characterization of a novel enzyme - Starmerella bombicola lactone esterase (SBLE) - responsible for sophorolipid lactonization},
  url          = {http://dx.doi.org/10.1007/s00253-016-7633-2},
  volume       = {100},
  year         = {2016},
}

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